Biomedical subjects

K S Arnold

Publications and source records attributed to K S Arnold.

At least 19 recordsLinked to original sources

PubMed2000

Effect of arginine 172 on the binding of apolipoprotein E to the low density lipoprotein receptor.

The region of apolipoprotein E (apoE) that interacts directly with the low density lipoprotein (LDL) receptor lies in the vicinity of residues 136-150, where lysine and arginine residues are crucial for full binding activity. However, defective binding of carboxyl-terminal truncations of apoE3 has suggested that residues in the vicinity of 170-183 are also important. To characterize and define the role of this region in LDL receptor binding, we created either mutants of apoE in which this region was deleted or in which arginine residues within this region were sequentially changed to alanine. Deletion of residues 167-185 reduced binding activity (15% of apoE3), and elimination of arginines at positions 167, 172, 178, and 180 revealed that only position 172 affected binding activity (2% of apoE3). Substitution of lysine for Arg(172) reduced binding activity to 6%, indicating a specific requirement for arginine at this position. The higher binding activity of the Delta167-185 mutant relative to the Arg(172) mutant (15% versus 2%) is explained by the fact that arginine residues at positions 189 and 191 are shifted in the deletion mutant into positions equivalent to 170 and 172 in the intact protein. Mutation of these residues and modeling the region around these residues suggested that the influence of Arg(172) on receptor binding activity may be determined by its orientation at a lipid surface. Thus, the association of apoE with phospholipids allows Arg(172) to interact directly with the LDL receptor or with other residues in apoE to promote its receptor-active conformation.

Amino Acid Sequence↗

PubMed2000

Apolipoprotein E;-low density lipoprotein receptor interaction. Influences of basic residue and amphipathic alpha-helix organization in the ligand.

Conserved lysines and arginines within amino acids 140-150 of apolipoprotein (apo) E are crucial for the interaction between apoE and the low density lipoprotein receptor (LDLR). To explore the roles of amphipathic alpha-helix and basic residue organization in the binding process, we performed site-directed mutagenesis on the 22-kDa fragment of apoE (amino acids 1-191). Exchange of lysine and arginine at positions 143, 146, and 147 demonstrated that a positive charge rather than a specific basic residue is required at these positions. Consistent with this finding, substitution of neutral amino acids for the lysines at positions 143 and 146 reduced the binding affinity to about 30% of the wild-type value. This reduction corresponds to a decrease in free energy of binding of approximately 600 cal/mol, consistent with the elimination of a hydrogen-bonded ion pair (salt bridge) between a lysine on apoE and an acidic residue on the LDLR. Binding activity was similarly reduced when K143 and K146 were both mutated to arginine (K143R + K146R), indicating that more than the side-chain positive charge can be important.Exchanging lysines and leucines indicated that the amphipathic alpha-helical structure of amino acids 140-150 is critical for normal binding to the low density lipoprotein receptor.

Amino Acid Sequence↗

PubMed1999

Phylogenetic analysis of the apolipoprotein B mRNA-editing region. Evidence for a secondary structure between the mooring sequence and the 3' efficiency element.

Apolipoprotein (apo) B mRNA editing is the deamination of C(6666) to uridine, which changes the codon at position 2153 from a genomically encoded glutamine (CAA) to an in-frame stop codon (UAA). The apoB mRNA-editing enzyme complex recognizes the editing region of the apoB pre-mRNA with exquisite precision. Four sequence elements spanning 139 nucleotides (nt) on the apoB mRNA have been identified that specify this precision. In cooperation with the indispensable mooring sequence and spacer element, a 5' efficiency element and a 3' efficiency element enhance editing in vitro. A phylogenetic comparison of 32 species showed minor differences in the apoB mRNA sequence, and the apoB mRNA from 31 species was robustly edited in vitro. However, guinea pig mRNA was poorly edited. Compared with the consensus sequences of these 31 species, guinea pig apoB mRNA has three variations in the 3' efficiency element, and the conversion of these to the consensus sequence increased editing to the levels in the other species. From this information, a model for the secondary structure was formulated in which the mooring sequence and the 3' efficiency element form a double-stranded stem. Thirty-one mammalian apoB mRNA sequences are predicted to form this stem positioning C(6666) two nucleotides upstream of the stem. However, the guinea pig apoB mRNA has a mutation in the 3' efficiency element (C(6743) to U) that predicts an extension of the stem and hence the lower editing efficiency. A test of this model demonstrated that a single substitution at 6743 (U to C) in the guinea pig apoB mRNA, that should reduce the stem, enhanced editing, and mutations in the 3' efficiency element that extended the stem for three base pairs dramatically reduced editing. Furthermore, the addition of a 20-nucleotide 3' efficiency element RNA, to a 58-nucleotide guinea pig apoB mRNA lacking the 3' efficiency element more than doubled the in vitro editing activity. Based on these results, a model is proposed in which the mooring sequence and the 3' efficiency element form a double-stranded stem, thus suggesting a mechanism of how the 3' efficiency element enhances editing.

Animals↗

PubMed1999

Functional characterization of apolipoprotein E isoforms overexpressed in Escherichia coli.

Apolipoprotein (apo) E plays an important role in lipid metabolism, and the major isoforms of apoE (apoE2, apoE3, and apoE4) have significantly different metabolic effects. Apolipoprotein E4 is associated with a higher risk of both heart disease and Alzheimer's disease (AD). Patients homozygous for apolipoprotein E2 are predisposed to type III hyperlipoproteinemia, and apoE2 may be protective against AD. Structure/function studies have proved to be a useful tool in understanding how the different apoE isoforms result in different pathological consequences. As these studies continue, it is essential to have a reliable method to produce large quantities of apoE and mutants of apoE. We describe here a method of apoE production in Escherichia coli strain BL21(DE3). The cDNA from apoE isoforms was inserted into a pET32a vector with a T7 promoter and a fusion partner (thioredoxin). The T7 promoter results in high expression of an easily purified His-tagged fusion protein. A thrombin recognition site was positioned in the expression vector so that only two novel amino acids (Gly-Ser) are added to the amino terminus of apoE following the removal of thioredoxin. Approximately 20 mg of apoE is obtained from a 1-liter culture. The major isoforms of apoE produced with this system were extensively characterized for their ability to bind the low-density lipoprotein (LDL) receptor, for their characteristic lipid association preferences, and for their stability as measured by guanidine denaturation. The recombinant proteins behaved identically to plasma-derived apoE isoforms.

Amino Acid Sequence↗

PubMed1998

The carboxyl terminus in apolipoprotein E2 and the seven amino acid repeat in apolipoprotein E-Leiden: role in receptor-binding activity.

Both apolipoprotein (apo) E2 and apoE-Leiden (tandem repeat of amino acids 121-127) are associated with type III hyperlipoproteinemia and bind defectively to low density lipoprotein receptors. Removing the carboxyl terminus of both variants (residues 192-299) increases receptor-binding activity, suggesting that the carboxyl terminus modulates activity. To identify the region(s) that modulated binding activity, we produced carboxyl-terminal truncations in apoE2 and apoE-Leiden (terminating at positions 191, 223, 244, and 272) and in apoE3 (terminating at positions 191, 223, and 244) and compared their receptor-binding activities as dimyristoylphosphatidylcholine (DMPC) discs. The results suggest that the entire carboxyl terminus up to residue 272, not a discrete smaller segment, is responsible for the modulation in apoE2. Intact apoE-Leiden and the 223 and 244 variants displayed similar activities (approximately 25% of apoE3's), but the 191 variant's activity was identical to that of intact apoE3. ApoE-Leiden and its truncated variants formed larger DMPC discs than did intact or truncated apoE3 or apoE2. These discs contained more apoE molecules than apoE3 discs, suggesting that the apparently normal binding activity of the apoE-Leiden 191 variant results from an increased number of apoE molecules and that the binding activity is actually defective. Direct comparison in a solid-phase assay revealed that the binding activity of the apoE-Leiden fragment was defective (51.4+/-9.4%). Thus, the defective binding of apoE-Leiden results from a direct effect of the seven amino acid repeat on receptor-binding activity rather than from an indirect effect operating through the carboxyl terminus as previously believed.

Apolipoprotein E2↗

PubMed1997

Human low density lipoprotein receptor fragment. Successful refolding of a functionally active ligand-binding domain produced in Escherichia coli.

The low density lipoprotein (LDL) receptor plays a key role in cholesterol homeostasis, mediating cellular uptake of lipoprotein particles by high affinity binding to its ligands, apolipoprotein (apo) B-100 and apoE. The ligand-binding domain of the LDL receptor contains 7 cysteine-rich repeats of approximately 40 amino acids; each repeat contains 6 cysteines, which form 3 intra-repeat disulfide bonds. As a first step toward determining the structure of the LDL receptor, both free and bound to its ligands, we produced in Escherichia coli a soluble fragment containing the ligand-binding domain (residues 1-292) as a thrombin-cleavable, heat-stable thioredoxin fusion. Modest amounts (5 mg/liter) of partially purified but inactive fragment were obtained after cell lysis, heat treatment, thrombin cleavage, and gel filtration under denaturing conditions. We were able to refold the receptor fragment to an active conformation with approximately 10% efficiency. The active fragment was isolated and purified with an LDL affinity column. The refolded receptor fragment was homogeneous, as determined by sodium dodecyl sulfate or non-denaturing polyacrylamide gel electrophoresis and isoelectric focusing. The purified fragment did not react with fluorescein-5-maleimide, indicating that all 42 cysteines were disulfide linked. In addition, the refolded fragment exhibited properties identical to those of the intact native receptor: Ca2+-dependent binding and isoform-dependent apoE binding (apoE2 binding <5% of apoE3). Furthermore, antibodies to the fragment recognized native receptors and inhibited the binding of 125I-LDL to fibroblast LDL receptors. We conclude that we have produced a properly folded and fully active receptor fragment that can be used for further structural studies.

Apolipoprotein E2↗

PubMed1997

A novel translational repressor mRNA is edited extensively in livers containing tumors caused by the transgene expression of the apoB mRNA-editing enzyme.

Transgene expression of the apolipoprotein B mRNA-editing enzyme (APOBEC-1) causes dysplasia and carcinoma in mouse and rabbit livers. Using a modified differential display technique, we identified a novel mRNA (NAT1 for novel APOBEC-1 target no. 1) that is extensively edited at multiple sites in these livers. The aberrant editing alters encoded amino acids, creates stop codons, and results in markedly reduced levels of the NAT1 protein in transgenic mouse livers. NAT1 is expressed ubiquitously and is extraordinarily conserved among species. It has homology to the carboxy-terminal portion of the eukaryotic translation initiation factor (eIF) 4G that binds eIF4A and eIF4E to form eIF4F. NAT1 binds eIF4A but not eIF4E and inhibits both cap-dependent and cap-independent translation. NAT1 is likely to be a fundamental translational repressor, and its aberrant editing could contribute to the potent oncogenesis induced by overexpression of APOBEC-1.

APOBEC-1 Deaminase↗

PubMed1996

Novel mechanism for defective receptor binding of apolipoprotein E2 in type III hyperlipoproteinemia.

The defective binding of apolipoprotein (apo) E2 to lipoprotein receptors, an underlying cause of type III hyperlipoproteinemia, results from replacement of Arg 158 with Cys, disrupting the naturally occurring salt bridge between Asp 154 and Arg 158. A new bond between Asp 154 and Arg 150 is formed, shifting Arg 150 out of the receptor binding region. Elimination of the 154-150 salt bridge by site-directed mutagenesis of Asp 154 to Ala restored the receptor binding activity to near normal levels. The X-ray crystal structure of apoE2 Ala 154 demonstrated that Arg 150 was relocated within the receptor binding region. Our results demonstrate that defective binding of apoE2 occurs by a novel mechanism of the replacement of one salt bridge with another.

Apolipoprotein E2↗

PubMed1996

Differences in receptor binding of LDL subfractions.

Differences in low density lipoprotein (LDL) receptor-binding affinity among LDL particles of different size were examined in competitive binding assays in human skin fibroblasts and LDL (d = 1.020 to 1.050 g/mL) from subjects with a predominance of large (> or = 272 A), medium (259 to 271 A), and small (< or = 257 A) LDL. Among 57 normolipidemic subjects with LDL cholesterol (-C) levels < 160 mg/dL, binding affinity was reduced by 16% in those with predominantly large LDL and by 14% in those with small LDL compared with most subjects who had a predominance of medium-size LDL and in all LDL size subgroups in 66 subjects with LDL-C > or = 160 mg/dL. Differences in LDL receptor-binding affinity were further investigated by using LDL density subfractions (I, d = 1.026 to 1.032 g/mL; II, d = 1.032 to 1.038 g/mL; and III, d = 1.038 to 1.050 g/mL) from three subjects with predominantly large (pattern A) and small (pattern B) LDL particles. The binding affinity (Kd) of LDL-II was similar for patterns A and B (9.2 +/- 1.4 and 9.4 +/- 0.7, respectively) and 30% lower in LDL-III from both groups (P < .05). The binding affinity of LDL-I in pattern A (12.6 +/- 1.5 micrograms/mg) was lower (P < .05) than that in LDL-II and LDL-I from pattern B (8.0 +/- 2.4 micrograms/mg). After incubation with a monoclonal antibody that specifically blocked the LDL receptor-binding domain of apoE, LDL-I from two pattern B subjects showed substantially lower binding affinity (Kd = 20.0 and 19.2 micrograms/mg) than in pattern A (Kd = 13.2 and 14.2 micrograms/mg), a result consistent with our finding of a higher apoE content in pattern B LDL-I (P < .001). Thus, factors associated with variations in particle size and apoE content in LDL subclasses in normolipidemic subjects contribute to the differences in LDL receptor binding that may result in differing metabolic behavior in vivo.

Adult↗

PubMed1995

Apolipoprotein B mRNA-editing protein induces hepatocellular carcinoma and dysplasia in transgenic animals.

Apolipoprotein (apo-) B mRNA editing is the deamination of cytidine that creates a new termination codon and produces a truncated version of apo-B (apo-B48). The cytidine deaminase catalytic subunit [apo-B mRNA-editing enzyme catalytic polypeptide 1 (APOBEC-1)] of the multiprotein editing complex has been identified. We generated transgenic rabbits and mice expressing rabbit APOBEC-1 in their livers to determine whether hepatic expression would lower low density lipoprotein cholesterol concentrations. The apo-B mRNA from the livers of the transgenic mice and rabbit was extensively edited, and the transgenic animals had reduced concentrations of apo-B100 and low density lipoproteins compared with control animals. Unexpectedly, all of the transgenic mice and a transgenic rabbit had liver dysplasia, and many transgenic mice developed hepatocellular carcinomas. Many of the mouse livers were hyperplastic and filled with lipid. Other hepatic mRNAs with sequence motifs similar to apo-B mRNA were examined for this type of editing (i.e., cytidine deamination). One of these, tyrosine kinase, was edited in livers of transgenic mice but not of controls. This result demonstrates that other mRNAs can be edited by the overexpressed editing enzyme and suggests that aberrant editing of hepatic mRNAs involved in cell growth and regulation is the cause of the tumorigenesis. Finally, these findings compromise the potential use of APOBEC-1 for gene therapy to lower plasma levels of low density lipoproteins.

APOBEC-1 Deaminase↗

PubMed1994

Isolation and characterization of a platelet-derived macrophage-binding proteoglycan.

A macromolecule in human platelet secretory products was demonstrated previously to inhibit the binding and uptake of acetoacetylated (AcAc) low density lipoproteins (LDL) by scavenger receptors on mouse peritoneal macrophages. In the current study, this macromolecule was purified to apparent homogeneity by DEAE-Sephacel chromatography, Sephacryl S-300 chromatography, and sucrose gradient centrifugation. SDS-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of approximately 120 kDa. Chemical analysis indicated that the macromolecule (designated platelet-derived macrophage-binding proteoglycan (PDMBP)) was a chondroitin 4-sulfate proteoglycan with an approximately 32-kDa core protein. A polyclonal antibody produced against this proteoglycan identified only PDMBP on Western blots of platelet secretory products and removed all ability of these products to inhibit the binding of AcAc LDL to macrophages. Treatment of purified PDMBP with protease or chondroitinase AC or ABC abolished the ability of the proteoglycan to inhibit the binding of AcAc LDL to macrophages. Binding studies using radiolabeled PDMBP demonstrated that the proteoglycan bound directly to the macrophage cell surface and was competitively inhibited by AcAc LDL, acetyl-LDL, fucoidin, and unlabeled PDMBP. PDMBP inhibited binding of 125I-labeled AcAc LDL to macrophages but had no effect on binding to endothelial cells. The finding that PDMBP binds to the scavenger receptor on macrophages suggests a mechanism for the inhibition of foam cell formation and suggests that the receptor could be involved in the plasma clearance of chondroitin sulfate proteoglycans.

Amino Acids↗

PubMed1994

Isolation of allele-specific, receptor-binding-defective low density lipoproteins from familial defective apolipoprotein B-100 subjects.

Familial defective apolipoprotein B-100 (FDB) is a genetic disorder apparently caused by a single amino acid substitution (Arg3500-->Gln) that disrupts the binding of low density lipoproteins (LDL) to the LDL receptor. The plasma of FDB heterozygotes contains a mixture of normal LDL and LDL that is defective in binding to the LDL receptor. In this study, the monoclonal antibody MB19 (which recognizes an immunogenetic polymorphism in apolipoprotein B-100) was used to determine the percentage of defective LDL in the plasma of FDB heterozygotes and to isolate allele-specific receptor-binding-defective LDL. Several FDB heterozygotes were identified who were heterozygous for the MB19 polymorphism: one apolipoprotein B allotype in each of these individuals bound with low affinity to MB19 and possessed the Arg3500-->Gln mutation, whereas the other apolipoprotein B allotype bound with high affinity to MB19 and normally to the LDL receptor. Using MB19 radio-immunoassay, we determined that an average of 73% (range 65-87) of the total LDL from FDB heterozygotes contained the Arg3500-->Gln mutation. Antibody MB19-Sepharose immuno-affinity chromatography was used to separate the receptor-binding-defective LDL from the normal LDL. The isolated LDL contained primarily the Arg3500-->Gln mutation and had only about 9% of normal LDL receptor-binding ability. Finally, the MB19-Sepharose chromatography procedure may be useful for isolating other allele-specific LDL that have functionally significant mutations.

Alleles↗

PubMed1991

13C NMR evidence that substitution of glutamine for arginine 3500 in familial defective apolipoprotein B-100 disrupts the conformation of the receptor-binding domain.

Familial defective apoB-100 is a genetic mutation that is characterized by abnormal low density lipoprotein (LDL) and moderate hypercholesterolemia. Heterozygotes for this disorder possess two populations of LDL. One has normal receptor binding, and the other, which can be isolated by monoclonal antibody 19 immunoaffinity chromatography, has almost no binding activity. The mutation that disrupts binding is a Gln for Arg substitution of apoB-100 residue 3500. NMR spectra of LDL containing (13CH3)2Lys residues show that chemically modified Lys exist in two microenvironments. In normal human LDL, there are about 50 Lys with pK 8.9 and 170 Lys with pK 10.5; an upper limit of 10 pK 8.9 Lys may be particularly involved in binding to the LDL receptor. Examination of the mixture of normal LDL and mutant LDL from five patients shows that the latter have fewer pK 8.9 Lys. In purified defective LDL at least seven Lys are redistributed from the active to normal pool. The CD spectra of mutant and normal LDL are identical. Therefore, substitution of Gln for Arg at position 3500 induces a change in local conformation which disrupts the receptor-binding domain of apoB-100.

Apolipoprotein B-100↗

PubMed1988

Familial defective apolipoprotein B-100: enhanced binding of monoclonal antibody MB47 to abnormal low density lipoproteins.

Familial defective apolipoprotein (apo) B-100 is a recently described genetic disorder that appears to result from a mutation in the apoB-100 gene. This disorder is characterized by hypercholesterolemia resulting from elevated plasma concentrations of low density lipoprotein LDL. The disorder was first detected in three members of one family. The LDL from affected subjects binds defectively (approximately 30% of normal) to LDL receptors, retarding the clearance of LDL from plasma. In the present study, two other members of the affected family were found to possess abnormal LDL. In addition, abnormal LDL with a similar binding defect were found in a second, unrelated family. In both families, the defect is transmitted over three generations as an autosomal codominant trait and all affected members are heterozygotes. Since there is only one apoB-100 molecule per LDL particle, the abnormal LDL in heterozygous subjects is made up of two populations of particles: one that has normal binding activity to receptors and one that binds defectively. To localize the mutation in apoB-100, the binding of five apoB-100-specific monoclonal antibodies to abnormal LDL was assessed in a solid-phase RIA. Only antibody MB47, whose epitope is between residues 3350 and 3506, distinguished abnormal LDL from normal LDL isolated from control subjects with normal lipid levels; MB47 bound with a higher affinity (by approximately 60%) to abnormal LDL. In every individual with abnormal LDL, the MB47 antibody bound with a higher affinity. The convenience of this assay will facilitate screening of large populations to determine the frequency of this disorder.

Adult↗

PubMed1987

Familial defective apolipoprotein B-100: low density lipoproteins with abnormal receptor binding.

Previous in vivo turnover studies suggested that retarded clearance of low density lipoproteins (LDL) from the plasma of some hypercholesterolemic patients is due to LDL with defective receptor binding. The present study examined this postulate directly by receptor binding experiments. The LDL from a hypercholesterolemic patient (G.R.) displayed a reduced ability to bind to the LDL receptors on normal human fibroblasts. The G.R. LDL possessed 32% of normal receptor binding activity (approximately equal to 9.3 micrograms of G.R. LDL per ml were required to displace 50% of 125I-labeled normal LDL, vs. approximately equal to 3.0 micrograms of normal LDL per ml). Likewise, the G.R. LDL were much less effective than normal LDL in competing with 125I-labeled normal LDL for cellular uptake and degradation and in stimulating intracellular cholesteryl ester synthesis. The defect in LDL binding appears to be due to a genetic abnormality of apolipoprotein B-100: two brothers of the proband possess LDL defective in receptor binding, whereas a third brother and the proband's son have normally binding LDL. Further, the defect in receptor binding does not appear to be associated with an abnormal lipid composition or structure of the LDL: the chemical and physical properties of the particles were normal, and partial delipidation of the LDL did not alter receptor binding activity. Normal and abnormal LDL subpopulations were partially separated from plasma of two subjects by density-gradient ultracentrifugation, a finding consistent with the presence of a normal and a mutant allele. The affected family members appear to be heterozygous for this disorder, which has been designated familial defective apolipoprotein B-100. These studies indicate that the defective receptor binding results in inefficient clearance of LDL and the hypercholesterolemia observed in these patients.

Apolipoprotein B-100↗

PubMed1987

Receptor binding activity of lipid recombinants of apolipoprotein B-100 thrombolytic fragments.

Apolipoprotein (apo) B-100, the protein constituent of low density lipoproteins (LDL), is the determinant responsible for LDL binding to the apoB,E(LDL) receptor on cells. The current study was designed to identify the region(s) of apoB-100 that interact with the apoB,E(LDL) receptor. Apolipoprotein B-100 was fragmented by thrombin digestion, and the isolated fragments (T2, T3, T4) were recombined with cholesterol-induced canine high density lipoproteins (HDLc). Before the recombination, the receptor binding activity of apoE of the HDLc was abolished by reductive methylation and extensive trypsin treatment. This treatment permitted almost complete replacement of the small residual apoE fragments by the large apoB fragments. Recombinant apoB particles were isolated by ultracentrifugation and tested for binding to receptors on cultured human fibroblasts. The recombinant particles had chemical and physical properties similar to those of native HDLc. Recombinants of both the whole thrombolytic digest and of isolated fragments displayed specific binding to the apoB,E (LDL) receptor. Anti-apoB,E(LDL) receptor antibodies abolished 90% of the binding, and there was almost no specific binding to receptor-negative fibroblasts or to cells in which the receptors had been down-regulated. The binding of apoB-100 recombinants to the receptor also demonstrated calcium dependency; in addition, the surface binding of the recombinants was released by polyanionic compounds. All these recombinants had binding affinities comparable to one another but less than that of native LDL. Although T2, T3 and T4 recombinants can all bind specifically to the apoB,E(LDL) receptor, it remains to be established whether their activity represents physiologically relevant binding. Nevertheless, the present findings illustrate the potential of the recombinant method using HDLc lipids to reconstitute biological activity.

Apolipoprotein B-100↗

PubMed1986

Correlation of the presence of seminal white blood cells and the prevalence of separated spermatozoal heads with subclinical Brucella ovis infection in rams.

A breeding soundness evaluation was conducted on 824 Colorado range rams. These rams were determined to be free from epididymitis via testicular palpation. Semen evaluation included microscopic observation for the presence of WBC. Of the 824 rams, 15.5% failed the breeding soundness evaluation on the basis of the semen evaluation: 10.6% had WBC in the semen and 4.9% had poor sperm morphology. The prevalence of Brucella ovis isolation varied from 0% to 16.2% within flocks. The prevalence of subclinical B ovis infection was 10% in the control flocks. Brucella ovis was isolated from 71.9% of the rams that had WBC in their semen. From this study, it appeared that palpation and vaccination may be inadequate for control of ram epididymitis.

Animals↗

PubMed1984

Normalization of receptor binding of apolipoprotein E2. Evidence for modulation of the binding site conformation.

Apolipoprotein (apo-) E3, when combined with the phospholipid dimyristoylphosphatidylcholine (DMPC), binds avidly to apo-B,E (low density lipoprotein) receptors on human fibroblasts. Apolipoprotein E2 isolated from type III hyperlipoproteinemic subjects, which differs from apo-E3 by the presence of cysteine instead of arginine at residue 158, possesses only about 1% of the receptor binding activity of apo-E3. Modification of apo-E2 with cysteamine, which converts the cysteine at position 158 to a positively charged lysine analogue, activates receptor binding approximately 13-fold. In the present experiments, thrombin was used to cleave apo-E2 into two fragments (Mr = 22,000 and Mr = 10,000). The larger fragment, which has been shown to possess the receptor binding domain, displayed binding activity up to 12-fold greater than intact apo-E2 or equivalent to apo-E2 treated with cysteamine. When the Mr = 22,000 fragment was modified with cysteamine and combined with DMPC, receptor binding was further enhanced, attaining the level of activity of normal apo-E3 X DMPC, a 100-fold increase over apo-E2 X DMPC binding. When the cysteamine modification was reversed by incubation with beta-mercaptoethanol, the Mr = 22,000 fragment retained most of its binding activity. However, when the same sample was tested 24 h later, the level of binding activity dropped significantly. The receptor binding of apo-E2-containing beta-very low density lipoproteins could also be activated by cysteamine treatment, with the same retention of enhanced binding activity occurring after the reversal of the modification. These results indicate that apo-E2 can attain full binding activity by the removal of the carboxyl-terminal one-third of the molecule and the addition of a positive charge at residue 158 of the molecule. The retention of enhanced binding after the reversal of the cysteamine modification indicates that the enhanced binding is probably due to conformational changes induced in the binding domain (and maintained by the phospholipid) and not merely to the presence of the positive charge at residue 158.

Apolipoprotein E2↗