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Biomedical subjects

J M Taylor

Publications and source records attributed to J M Taylor.

At least 145 records · Page 8Linked to original sources
PubMed1995

Regulation of the myoblast-specific expression of the human beta-enolase gene.

The muscle-specific beta-enolase gene is expressed in proliferating adult myoblasts as well as in differentiated myotubes. Through deletion-transfection analysis, we identified a 79-base pair enhancer from the beta-enolase gene that leads to high level expression of a reporter gene in myoblasts, but not in fibroblasts. Following myoblast differentiation into myotubes, the activity of the enhancer declined, indicating that beta-enolase gene expression in myotubes is mediated by other regulators, possibly the myogenic helix-loop-helix family of transcription factors. Electrophoretic mobility shift assays indicated that proteins present in myoblast nuclear extracts specifically bind to the 3' half of the 79-base pair enhancer. This region contains an ets DNA-binding motif which is required not only for high level activity in myoblasts, but also for repressing activity in fibroblasts. Furthermore, the beta-enolase myoblast-specific enhancer shows limited similarity to the myoblast-specific enhancer associated with the human desmin gene, suggesting that gene expression in adult myoblasts may be coordinately regulated.

Animals↗
PubMed1995

Dose-response relationship for radiation therapy of subclinical disease.

PURPOSE: To determine the dose-response relationship for elective treatment of subclinical metastatic deposits and validate a model for metastatic tumor cell burden. METHODS AND MATERIALS: The incidence of overt metastases in electively irradiated potential sites of spread from carcinomas of the head and neck, breast, cervix, ovary, lung, and testis, and from melanomas and osteosarcomas, was compared with the incidence in patients not receiving elective irradiation. The reduction in incidence of metastases was analyzed as a function of radiation dose. RESULTS: The dose-response curve for control of subclinical metastases is linear and shallow, and extrapolates to a dose intercept not demonstrably different from zero. A small threshold may reflect growth of residual micrometastases between treatment for the primary and elective irradiation. The shallow linear dose response reflects interpatient heterogeneity in metastatic tumor cell burden, ranging from 1 to M cells, where M is the upper limit of clinical undetectability. While a dose of 50 Gy in 2 Gy fractions is necessary to achieve an overall 90% reduction in the incidence of metastases, the metastatic cell burden in a proportion of patients can be eliminated by low doses. Thus, worthwhile rates of control can still be achieved when "tolerance" dictates lower than optimal doses, evidenced by the linearity and lack of significant threshold in the dose-response curve. This is an important difference from treatment of gross disease. The biological effectiveness of elective treatment is measured directly by the percent reduction in failure rate. Although it depends upon the log cell kill, it relates only to that proportion of patients harboring subclinical disease, and, therefore, is not well described by the increase in the cure rate for the total patient population. The linear dose-response relationship for reduction in failure rate is independent of the "natural" (untreated) incidence of subclinical metastasis, and, therefore, of site, histology, growth rate, stage, or other characteristics of the tumor. Conversely, the clinical effectiveness of elective treatment is measured by increase in tumor control rate and depends upon the "natural" incidence of metastasis: the higher it is, the greater the absolute increase in cure rate from a constant biological effect (log cell kill). CONCLUSIONS: (a) High control rates for subclinical metastases require doses of about 50 Gy in 2 Gy fractions, but worthwhile benefits can be achieved by lower doses if necessitated by reduced tolerance; (b) elective treatment of subclinical metastases should be instituted close to the time of treatment of the primary; (c) the biological effectiveness of elective radiation (or chemotherapy) should be measured by the percentage decrease in metastasis, not by improvements in the rate of control; and (d) demonstration of success in clinical trials of adjuvant therapy is more likely the higher the incidence of metastases in untreated controls.

Dose-Response Relationship, Radiation↗
PubMed1995

The effect of interleukin-1 beta or transforming growth factor-beta on radiation-impaired murine skin wound healing.

The ability of exogenous interleukin-1 beta (IL-1 beta) or transforming growth factor-beta (TGF-beta) to reverse radiation-induced defective wound healing was investigated. Mice were irradiated with 8.5 or 11 Gy to total body (TB), 12 or 16 Gy to hemibody (HB), or 20 or 26 Gy to skin alone immediately prior to surgical wounding in order to determine the effect of hematopoietic system depletion on cytokine action. A single dose of IL-1 beta or TGF-beta or vehicle control was applied to each wound. All radiation doses and types resulted in a deficit in wound healing when measured on Days 11 and 14. IL-1 beta enhanced wound tensile strength (WTS) in TB-irradiated mice, while TGF-beta enhanced WTS in HB-irradiated mice. Neither cytokine was effective at enhancing WTS in unirradiated or skin-only irradiated animals. In addition IL-1 beta and TGF-beta showed distinct differences in their effects on the kinetics of healing with time after wounding. The effects of TGF-beta appeared to be transient with compromise of the gain in WTS. The differences in the effects of these two cytokines in affecting wound healing reflect their involvement in different cellular events in the wound healing process.

Animals↗
PubMed1995

Characterization of an upstream regulatory element of the human apolipoprotein E gene, and purification of its binding protein from the human placenta.

We have previously demonstrated that expression of the human apolipoprotein (apo) E gene is controlled by multiple regulatory elements in the promoter [Paik et al. (1988) J. Biol. Chem. 263, 13340-13349; Chang et al. (1990) J. Biol. Chem. 265, 9496-9504]. To extend these studies, we have characterized an element in the apoE gene promoter that spans nucleotides -101 to -89, upstream regulatory element 3 (URE3). Transcription of promoter/marker gene constructs in vitro showed that URE3 modulates gene expression. Gel mobility shift assays of URE3 using human placental nuclear extracts detected a specific binding protein whose activity can be modulated by micromolar amounts of divalent copper and zinc. Competitive binding and gel shift assays with mutant oligonucleotides revealed critical nucleotides within URE3 required for its specific nuclear protein-binding activity. Gel filtration and oligonucleotide affinity chromatography were employed to isolate a URE3-binding protein (URE3BP) from human placental nuclear extracts. Purified URE3BP appears to be a M(r) = 300,000 protein that is composed of four equally-sized basic subunits of M(r) = 67,000. These studies indicate that URE3 is an active regulatory component of the apoE gene.

Apolipoproteins E↗
PubMed1995

Hepatitis delta virus mutant: effect on RNA editing.

During the replication cycle of hepatitis delta virus (HDV), RNA editing occurs at position 1012 on the 1679-nucleotide RNA genome. This changes an A to G in the amber termination codon, UAG, of the small form of the delta antigen (delta Ag). The resultant UGG codon, tryptophan, allows the translation of a larger form of the delta Ag with a 19-amino-acid C-terminal extension. Using HDV cDNA-transfected cells, we examined the editing potential of HDV RNA mutated from G to A at 1011 on the antigenome, adjacent to normal editing site at 1012. Four procedures were used to study not only the editing of the A at 1012, but also that of the new A at 1011: (i) nucleotide sequencing, (ii) a PCR-based RNA-editing assay, (iii) immunoblot assays, and (iv) immunofluorescence. Five findings are reported. (i) Even after the mutation at 1011, editing still occurred at 1012. (ii) Site 1011 itself now acted as a novel RNA-editing site. (iii) Sites 1011 and 1012 were edited independently. (iv) At later times, both sites became edited, thereby allowing the synthesis of the large form of the delta Ag (delta Ag-L). (v) Via immunofluorescence, such double editing became apparent as a stochastic event, in that groups of cells arose in which the changes had taken place. Evaluation of these findings and of those from previous studies of the stability of the HDV genomic sequence (H.J. Netter et al., J. Virol. 69:1687-1692, 1995) supports both the recent reevaluation of HDV RNA editing as occurring on antigenomic RNA (Casey and Gerin, personal communication) and the interpretation that editing occurs via the RNA-modifying enzyme known as DRADA.

Amino Acid Sequence↗
PubMed1995

Intracellular cleavage and ligation of hepatitis delta virus genomic RNA: regulation of ribozyme activity by cis-acting sequences and host factors.

During replication, a ribozyme within the genomic RNA of hepatitis delta virus cleaves multimeric precursors to release a unit-length linear intermediate. Intramolecular ligation of this intermediate produces the circular genomic RNA. Although one copy of the ribozyme is reconstituted by such ligation, it does not subsequently cleave and destroy the circular conformation. We have identified cis-acting attenuator sequences that prevent self-cleavage of the circular product by base pairing with and inactivating the ribozyme. Furthermore, we have shown that during the initial processing of the multimeric precursor RNA, host-specific factors activate the ribozyme by preventing its association with the attenuator sequences. Thus, we demonstrate a novel switching mechanism that regulates ribozyme activity inside the cell.

Base Sequence↗
PubMed1995

Nucleotide sequence stability of the genome of hepatitis delta virus.

Cultured cells were cotransfected with a fully sequenced 1,679-base cDNA clone of human hepatitis delta virus (HDV) RNA genome and a cDNA for the genome of woodchuck hepatitis virus (WHV). The HDV particles released were able to infect a woodchuck that was chronically infected with WHV. The HDV so produced was passaged a total of six times in woodchucks in order to determine the stability of the HDV nucleotide sequence. During a final chronic infection with such virus, liver RNA was extracted, and the HDV nucleotide sequence for the 352-base region, positions 905 to 1256, was obtained. By means of PCR, we obtained double-stranded cDNA both for direct sequencing and also for molecular cloning followed by sequencing. By direct sequencing, we found that a consensus sequence existed and was identical to the original sequence. From the sequences of 31 clones, we found 32% (10 of 31) to be identical to the original single nucleotide sequence. For the remainder, there were neither insertions nor deletions but there was a small number of single-nucleotide changes. These changes were predominantly transitions rather than transversions. Furthermore, the transitions were largely of just two types, uridine to cytidine and adenosine to guanosine. Of the 40 changes detected on HDV, 35% (14 of 40) occurred within an eight-nucleotide region that included position 1012, previously shown to be a site of RNA editing. These findings may have significant implications regarding both the stability of the HDV RNA genome and the mechanism of RNA editing.

Animals↗
PubMed1995

Overexpression of human apolipoprotein B-100 in transgenic rabbits results in increased levels of LDL and decreased levels of HDL.

In this study, and 80-kb human genomic DNA fragment spanning the human apoB gene was used to generate transgenic New Zealand White rabbits that expressed human apoB-100. The concentration of human apoB in the plasma of the transgenic rabbits ranged between 5 and 100 mg/dL. The transgenic rabbits had nearly threefold elevations in the plasma levels of triglycerides and cholesterol compared with nontransgenic controls. Nearly all the cholesterol and human apoB in the plasma was in the LDL fraction. Pronounced triglyceride enrichment of the LDL fraction was a striking feature of human apoB overexpression in the transgenic rabbits, in which the LDL fraction contained more than 75% of the plasma triglycerides. The triglyceride-enriched LDL particles were smaller and more dense than the native rabbit LDL and contained markedly increased amounts of apoE and apoC-III. In the nontransgenic control animals most of the triglycerides were in the VLDL, and most of the apoE and apoC-III were in the VLDL and HDL fractions. In addition to increased LDL levels, overexpression of human apoB in rabbits resulted in lower plasma levels of HDL cholesterol and apoA-I. In our prior studies on transgenic mice expressing human apoB, we documented triglyceride-rich LDL and reduced levels of HDL cholesterol. These prior findings in mice, together with the present findings in transgenic rabbits, suggest that triglyceride-rich LDL and lowered levels of HDL cholesterol may be hallmark features of apoB overexpression.

Animals↗
PubMed1995

Macrophage-specific expression of human apolipoprotein E reduces atherosclerosis in hypercholesterolemic apolipoprotein E-null mice.

apoE deficiency causes hyperlipidemia and premature atherosclerosis. To determine if macrophage-specific expression of apoE would decrease the extent of atherosclerosis, we expressed human apoE in macrophages of apoE-null mice (apoE-/-) and assessed the effect on lipid accumulation in cells of the arterial wall. Macrophage-specific expression of human apoE in normal mice was obtained by use of the visna virus LTR. These animals were bred with apoE-/- mice to produce animals hemizygous for expression of human apoE in macrophages in the absence of murine apoE (apoE-/-,hTgE+/0). Low levels of human apoE mRNA were present in liver and spleen and high levels in lung and peritoneal macrophages. Human apoE was secreted by peritoneal macrophages and was detected in Kupffer cells of the liver. Human apoE in the plasma of apoE-/-,hTgE+/0 mice (n = 30) was inversely correlated (P < 0.005) with the plasma cholesterol concentration. After 15 wk on a normal chow diet, atherosclerosis was assessed in apoE-/-,hTgE+/0 animals and in apoE-/-,hTgE0/0 littermates matched for plasma cholesterol level (approximately 450 mg/dl) and lipoprotein profile. There was significantly less atherosclerosis in both the aortic sinus and in the proximal aorta (P < 0.0001) in the animals expressing the human apoE transgene. In apo-E-/-,hTgE+/0 animals, which had detectable atherosclerotic lesions, human apoE was detected in the secretory apparatus of macrophage-derived foam cells in the arterial wall. The data demonstrate that expression of apoE by macrophages is antiatherogenic even in the presence of high levels of atherogenic lipoproteins. The data suggest that apoE prevents atherosclerosis by promoting cholesterol efflux from cells of the arterial wall.

Animals↗
PubMed1995

Semi-parametric estimation in failure time mixture models.

A mixture model is an attractive approach for analyzing failure time data in which there are thought to be two groups of subjects, those who could eventually develop the endpoint and those who could not develop the endpoint. The proposed model is a semi-parametric generalization of the mixture model of Farewell (1982). A logistic regression model is proposed for the incidence part of the model, and a Kaplan-Meier type approach is used to estimate the latency part of the model. The estimator arises naturally out of the EM algorithm approach for fitting failure time mixture models as described by Larson and Dinse (1985). The procedure is applied to some experimental data from radiation biology and is evaluated in a Monte Carlo simulation study. The simulation study suggests the semi-parametric procedure is almost as efficient as the correct fully parametric procedure for estimating the regression coefficient in the incidence, but less efficient for estimating the latency distribution.

Animals↗
PubMed1995

The subcellular localization of defensins and myeloperoxidase in human neutrophils: immunocytochemical evidence for azurophil granule heterogeneity.

We used immunogold electron microscopy to examine the distribution of human defensins 1-3 (HNP1-3) and myeloperoxidase (MPO) in the cytoplasmic granules of thin-sectioned, normal human neutrophils. The presence of MPO, defensins, or both was noted in 388 (62.3%) of 623 granule profiles. Whereas MPO and defensins colocalized in 32.5% of such granules, 32.7% demonstrated only MPO and 34.8% demonstrated only defensins. The staining densities of MPO and HNP1-3 showed a low correlation, even when both molecules were present in the same granules (r = 0.09), indicating that they were not present in fixed relative proportions even when colocalized. The low partial correlation coefficient (r = -0.23) between MPO and HNP1-3 for the 388 labeled granules controlling for granule area suggested that the deposition of MPO and defensins might even be negatively correlated. The distribution of MPO and defensins within the cytoplasmic granules of human neutrophils followed a "non-colocalization and multi-compartmental" model that conflicts with the classical notion of azurophil granules as a homogeneous population of organelles marked by the presence of MPO.

Blood Proteins↗
PubMed1995

Annexin I concentration, phospholipase activity and thromboxane synthesis in irradiated rat lung.

Annexin I, a member of a family of Ca(2+)-dependent phospholipid-binding proteins (PLBP), has been suggested as a regulator of prostaglandin metabolism as a result of its inhibitory effect on phospholipase A2. Synthesis of prostaglandin is increased in irradiated tissue, but the mechanism underlying this increase has not been delineated. It is conceivable that a decrease in the level of annexin I resulting in increased phospholipase activity may be responsible for the enhanced synthesis of prostaglandin. Accordingly, we measured the level of a lung 36 kDa PLBP, which possesses characteristics of annexin I, as well as the activity of phospholipase and the synthesis of thromboxane A2 (TXA2) in irradiated rat lung. The right lung of rats was irradiated with 0, 15 or 30 Gy of X rays and the animals were sacrificed after 3 months. Phospholipid binding protein was assayed by its ability to transfer unilamellar liposomes to multilamellar liposomes and by immunoblotting against anti-36 kDa rabbit PLBP antisera. Production of TXA2 by minced lung tissue was determined by radioimmunoassay of its stable metabolite TXB2. Phospholipase activity was assayed by hydrolysis of [14C]dioleoylphosphatidylcholine. Our results showed that PLBP activity in the lungs irradiated with 30 Gy was lower than that in the lungs irradiated with 0 and 15 Gy (8.82 +/- 0.47 compared to 9.73 +/- 0.49 and 9.95 +/- 0.78 nmol phospholipid transferred/mg protein, respectively). Western blotting demonstrated a near total depletion of annexin I in the lungs irradiated with 30 Gy. Phospholipase activity was also lower in the lungs irradiated with 30 Gy compared to that in the lungs irradiated with 0 Gy (0.23 +/- 0.01 vs 0.32 +/- 0.01 nmol phosphatidylcholine liberated/mg protein/min, P < 0.001). Reduced phospholipase activity was observed not only in the cytosolic or soluble fraction of lung homogenate, but also in precipitates obtained after 21,000g and 100,000g centrifugation. Despite this decline in phospholipase activity, there was a 2.8-fold increase in the synthesis of thromboxane (367 +/- 65 compared to 1076 +/- 143 pg TXB2/mg tissue/10 min for lungs irradiated with 0 and 30 Gy, respectively). These results are not consistent with the hypothesis that increased synthesis of thromboxane A2 in irradiated rat lung is a direct result of elevated phospholipase activity. In fact, phospholipase activity is decreased in the irradiated lung, despite a decline in the concentration of annexin I, its putative inhibitor.

Animals↗
PubMed1994

Binding of an alpha 2 adrenergic receptor third intracellular loop peptide to G beta and the amino terminus of G alpha.

The structural basis of receptor-G protein interactions was examined using a photoaffinity derivative of a G protein-activating receptor-derived peptide (Q peptide) from the carboxyl-terminal region of the third intracellular loop of alpha 2 adrenergic receptor. A diazopyruvoyl photoaffinity derivative of this peptide (DAP-Q) was cross-linked to purified bovine brain Go. Specific, competable cross-linking of 750 nM DAP-Q to sites on both the alpha o and beta subunits was observed. No specific cross-linking was seen with non-target proteins or heat-denatured G protein subunits. 125I-DAP-Q labeled the 2-kDa amino-terminal fragment of alpha o as determined by protease digestion of the cross-linked G protein followed by gel electrophoresis or h igh pressure liquid chromatography purification and mass spectroscopy of the radiolabeled proteolysis fragment. The functional significance of incorporation into beta gamma subunit is supported by the absolute requirement of beta gamma subunit for DAP-Q stimulation of Go/Gi GTPase. Thus, specific interactions of G protein-coupled receptors with the beta subunit of G protein, in addition to those with the alpha subunit, appear to be important for receptor-G protein coupling.

Amino Acid Sequence↗
PubMed1994

The extent, time course, and fraction size dependence of mouse spinal cord recovery from radiation injury.

PURPOSE: This experiment was designed to assess: (a) the influence of fraction size and time interval between fractions on the tolerance of the spinal cord to high cumulative doses of radiation; and (b) the influence of the long-term recovery process on the tolerance of the spinal cord to reirradiation. METHODS AND MATERIALS: The T10-L2 level of the spinal cord of C3Hf mice was irradiated using a conventionally fractionated regimen of 2.0 Gy once daily, a prolonged fractionated regimen of 1.2 Gy once daily, a hyperfractionated regimen of 1.2 Gy twice daily, or a single dose of 12 Gy followed 0-190 days later by a second dose of 5-20 Gy. Mice in the multifractionated regimen groups were given a single 15 Gy top-up dose 24 h after reaching a cumulative fractionated dose of 24-70 Gy. Hind limb strength was measured weekly for 2 years after the completion of irradiation. RESULTS: Paralysis occurred in a bimodal time distribution, with peaks at 5-10 months and 15-23 months after the completion of irradiation. The cumulative radiation dose was directly associated with the incidence of paralysis in each radiation schedule (p < 0.0001) and inversely associated with the time to onset of paralysis in the 1.2 Gy b.i.d. (p = 0.0001) and 2.0 Gy q.d. schedules (p = 0.03). The median latency of paralysis in each group was inversely associated with the incidence of paralysis in that group (p < 0.001). Decreasing the fraction size from 2.0 to 1.2 Gy once daily markedly increased the radiation tolerance of the spinal cord (p < 0.0001), consistent with a very small alpha-beta value of -0.30 Gy (approximately 95% confidence interval -0.72, +0.18) in the linear-quadratic model. Decreasing the time interval from 24 h to alternating 8 and 16 h periods produced an offsetting diminuation in cord tolerance (p < 0.0001). The 1.2 Gy once daily schedule resulted in ED20 and ED50 values that were approximately double those of the 2.0 Gy once daily and the 1.2 Gy twice daily schedules and a relative risk of paralysis from a given dose that was 0.03 times the risk associated with the other two regimens (p < 0.0001). There was no significant difference between the 2.0 Gy once daily and the 1.2 Gy twice daily dose-paralysis curves (p = 0.86). The residual from a single 12 Gy radiation dose was 17% after 190 days, leaving the retreatment ED50 only 10% below the ED50 of previously unirradiated spinal cord. The relative risk of paralysis after 12 Gy plus a second radiation dose decreased from 1.00 with no time interval between doses to 0.51-0.73 with a 0.25, 1 or 3 day interval, 0.32 with a 7 day interval, 0.11 with a 30 day interval, and 0.06 with a 190 day interval. CONCLUSION: The increased radiation tolerance of the murine spinal cord produced by decreasing the fraction size from 2.0 to 1.2 Gy was offset by the diminished tolerance produced by decreasing the time interval between fractions from 24 to 8-16 h, resulting in no significant difference in the dose-paralysis curves of conventional and hyperfractionated radiation schedules. The rodent spinal cord eliminates the majority of the occult radiation injury produced by a radiation dose equal to half the ED50 during the months following irradiation. This permits retreatment of previously irradiated spinal cord to high doses without the induction of myelopathy.

Animals↗
PubMed1994

Overexpression of hepatic lipase in transgenic rabbits leads to a marked reduction of plasma high density lipoproteins and intermediate density lipoproteins.

To elucidate the precise metabolic roles of hepatic lipase (HL), a human HL cDNA in a liver-specific expression vector was used to generate transgenic lines in the rabbit, an animal that normally expresses low levels of this enzyme. HL was detected in the plasma of all rabbits only after the administration of heparin; HL activity in transgenic rabbits was found at levels up to 80-fold greater than that in nontransgenic littermates. This increase in enzyme activity was associated with as much as a 5-fold decrease in total plasma cholesterol levels. Expression of the transgene resulted in a dramatic reduction in the level of large high density lipoproteins (HDL1 and HDL2) as well as dense HDL3. A reduction in the quantity of intermediate density lipoproteins (IDL) was also observed. These results demonstrate that HL functions in the metabolism of HDL and IDL, thereby playing a key role in plasma cholesterol homeostasis.

Animals↗
PubMed1994

Smoothing grouped bivariate data to obtain the incubation period distribution of AIDS.

We use a penalized likelihood approach to obtain a smooth estimate of a bivariate distribution from grouped data where each observation consists of a region in a plane. The purpose of the analysis is to estimate the incubation period distribution of AIDS from the Multicenter AIDS Cohort Study, a prevalent cohort of homosexual men. In this article we illustrate the usefulness of the penalized likelihood approach. We also discuss the use of a cross-validation and a Bayesian scheme to choose the smoothing parameters and bootstrap samples to assess uncertainty.

AIDS Serodiagnosis↗
PubMed1994

Enhanced binding and uptake of remnant lipoproteins by hepatic lipase-secreting hepatoma cells in culture.

Rat hepatoma McA-RH7777 cells transfected with a human hepatic lipase (HL) cDNA synthesized and secreted 50-80 ng of human HL/mg of cell protein at 4 h, approximately 50% of which was bound to cell-surface heparan sulfate proteoglycans (HSPG). The newly synthesized HL possessed enzymatic activity. When rabbit beta-very low density lipoproteins (beta-VLDL) and canine chylomicrons or chylomicron remnants were incubated with HL-secreting cells, remnant binding and uptake were enhanced 3-fold compared with nontransfected cells. Furthermore, fluorescence microscopy showed enhanced uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled beta-VLDL by the HL-transfected cells. When 125I-beta-VLDL were added to conditioned medium from HL-secreting cells, the HL in the media enhanced the binding and uptake of the remnant lipoproteins by nontransfected cells about 3-fold. Likewise, surface-bound HL (without HL in the medium) also was able to mediate the enhanced binding of the remnants. This HL-enhanced binding was shown to be mediated by an interaction with cell-surface HSPG. Heparinase treatment to remove cell-surface HSPG or chlorate treatment to prevent HSPG sulfation of the HL-secreting cells abolished all the HL-mediated enhanced binding and uptake. Furthermore, heparinase pretreatment of nontransfected cells prevented the enhanced binding and uptake of beta-VLDL incubated with conditioned medium from HL-secreting cells. As binding was not enhanced in the absence of HSPG, an HL-HSPG initial interaction appears essential. Addition of apolipoprotein (apo) E to the beta-VLDL did not facilitate HL-mediated binding and uptake; in fact, beta-VLDL from apoE-null mice demonstrated a similar degree of enhanced binding as did rabbit beta-VLDL with or without added apoE. On the other hand, beta-VLDL from transgenic mice overexpressing binding-defective apoE(Arg142-->Cys) did not display any enhanced binding and uptake by the HL-secreting cells, and it appears that the apoE(Arg142-->Cys) actually inhibited the HL-mediated interaction. This mutant form of apoE is associated with a dominant mode of expression of type III hyperlipoproteinemia in contrast to the more commonly occurring recessive disorder. Impaired HL interaction with the apoE(Arg142-->Cys) beta-VLDL may contribute to remnant lipoprotein accumulation in the plasma of patients with this mutant form of apoE. Thus, HL contributes to the enhanced cell association of specific types of remnant lipoproteins by initiating their binding to cell-surface HSPG.

Animals↗