Research Progress on Age Estimation Based on DNA Methylation.

ABSTRACT: Age estimation is of great significance in the fields of criminal investigation and forensic identification. It can provide the age information of individuals to judicial departments to facilitate the development of judicial work. In recent years, age estimation methods expanded from the morphological level to the molecular biology level. With the rapid development of epigenetics represented by DNA methylation, and the advancement of DNA methylation detection technology together with the detection platform, many age estimation methods based on DNA methylation biomarkers, or using several biological fluids, such as blood, blood stains, saliva, semen stains, etc. are developed. Currently, researches related to age estimation based on DNA methylation are relatively widely carried out. This paper summarizes the researches on age estimation based on DNA methylation, in order to provide references for related studies and forensic applications.

Type II deiodinase polymorphisms and serum thyroid hormone levels in patients with mild cognitive impairment.

We investigated type II deiodinase (DIO2) polymorphisms and serum thyroid hormone levels in subjects with mild cognitive impairment (MCI) in a Uygur population. We studied the DIO2 Thr92Ala (rs225014) and ORFa-Gly3Asp (rs12885300) polymorphisms of 129 unrelated MCI cases and 131 matched controls. All subjects were genotyped using SNaPshot SNP genotyping assays. Serum thyroid hormone levels were measured by radioimmunoassay. Levels of serum triiodothyronine and thyroxine in the MCI group were significantly lower than those in the control group. Genotype and allele frequencies in the DIO2 gene between the MCI and control groups were not significantly different. There was no association in genotype and allele frequencies of Thr92Ala between genders in both groups. ORFa-Gly3Asp genotype and allele frequencies were significantly different in patients and controls by gender. The Asp allele was less frequent among male MCI patients compared to controls (odds ratio = 0.471, 95% confidence interval = 0.261-0.848). However, female Asp carriers were more frequent among MCI patients than among controls (odds ratio = 2.842, 95% confidence interval = 1.326-6.09). Serum levels of triiodothyronine and thyroxine were lower in individuals of the Ala/Ala genotype than in those with the Thr/Thr or Thr/Ala genotype. Serum levels of triiodothyronine were lower in male Gly/Gly carriers than in Gly/Asp or Asp/Asp carriers. Decreased serum levels of triiodothyronine and thyroxine may influence the incidence of MCI in the Uygur population. DIO2 gene polymorphisms may play a role in the incidence of MCI in male patients.

Meta-analysis demonstrates no association between XRCC1 Arg399Gln polymorphism and bladder cancer risk.

We examined whether the X-ray repair cross-complementing group 1 (XRCC1) Arg399Gln polymorphism is a risk factor for bladder cancer by conducting a meta-analysis. We searched the Pubmed and Embase databases for study retrieval. This meta-analysis examined 16 case-control studies, including 892 prostate cancer cases and 1020 healthy controls. Meta-analysis results based on these studies showed no significant association between the XRCC1 Arg399Gln polymorphism and bladder cancer risk in comparisons of the glutamine (Gln) allele vs arginine (Arg) allele, Arg/Arg vs (Gln/Gln + Gln/Arg), Gln/Gln vs (Gln/Arg + Arg/Arg), Gln/Gln vs Arg/Arg, and Gln/Arg vs Arg/Arg [odds ratio (OR) = 0.96, 95% confidence interval (CI) = 0.80-1.16, P = 0.70; OR = 1.13, 95%CI = 0.70-1.82, P = 0.62; OR = 0.92, 95%CI = 0.79-1.07, P = 0.29; OR = 0.90, 95%CI = 0.69-1.16, P = 0.42; OR = 0.89, 95%CI = 0.75-1.05, P = 0.17, respectively]. In subgroup analysis by ethnicity, no association was observed between the XRCC1 Arg399Gln polymorphism and bladder cancer risk in Caucasian, Mongoloid, or black populations. We identified no association between the XRCC1 Arg399Gln polymorphism and bladder cancer risk.

Ron tyrosine kinase receptor synergises with EGFR to confer adverse features in head and neck squamous cell carcinoma.

BACKGROUND: Although EGFR inhibitors have shown some success in the treatment of head and neck squamous cell carcinomas (HNSCCs), the results are not dramatic. Additional molecular targets are urgently needed. We previously showed that the loss of Ron receptor activity significantly slowed squamous tumour growth and progression in a murine model. Based on these data, we hypothesised that Ron expression confers an aggressive phenotype in HNSCCs. We prospectively collected and evaluated 154 snap-frozen, primary HNSCCs for Ron and EGFR expression/phosphorylation. Biomarker correlation with clinical, pathological and outcome data was performed. The biological responses of HNSCC cell lines to Ron knockdown, its activation and the biochemical interaction between Ron and EGFR were examined. RESULTS: We discovered that 64.3% (99 out of 154) HNSCCs expressed Ron. The carcinomas expressed exclusively mature functional Ron, whereas the adjacent nonmalignant epithelium expressed predominantly nonfunctional Ron precursor. There was no significant association between Ron and sex, tumour differentiation, perineural/vascular invasion or staging. However, patients with Ron+HNSCC were significantly older and more likely to have oropharyngeal tumours. Ron+HNSCC also had significantly higher EGFR expression and correlated strongly with phosphorylated EGFR (pEGFR). Newly diagnosed HNSCC with either Ron/pEGFR or both had lower disease-free survival than those without Ron and pEGFR. Knocking down Ron in SCC9 cells significantly blunted their migratory response to not only the Ron ligand, MSP, but also EGF. Stimulation of Ron in SCC9 cells significantly augmented the growth effect of EGF; the synergistic effect of both growth factors in SCC9 cells was dependent on Ron expression. Activated Ron also interacted with and transactivated EGFR. CONCLUSION: Ron synergises with EGFR to confer certain adverse features in HNSCCs.

Meat-cooking mutagens and risk of renal cell carcinoma.

BACKGROUND: High-temperature cooked meat contains two families of carcinogens, heterocyclic amines (HCAs) and polycyclic aromatic hydrocarbons (PAHs). Given the kidneys' role in metabolism and urinary excretion of these compounds, we investigated meat-derived mutagens, as well as meat intake and cooking methods, in a population-based case-control study conducted in metropolitan Detroit and Chicago. METHODS: Newly diagnosed, histologically confirmed adenocarcinoma of the renal parenchyma (renal cell carcinoma (RCC)) cases (n=1192) were frequency matched on age, sex, and race to controls (n=1175). The interviewer-administered Diet History Questionnaire (DHQ) included queries for meat-cooking methods and doneness with photographic aids. Levels of meat mutagens were estimated using the DHQ in conjunction with the CHARRED database. RESULTS: The risk of RCC increased with intake of barbecued meat (P(trend)=0.04) and the PAH, benzo(a)pyrene (BaP) (multivariable-adjusted odds ratio and 95% confidence interval, highest vs lowest quartile: 1.50 (1.14, 1.95), P(trend)=0.001). With increasing BaP intake, the risk of RCC was more than twofold in African Americans and current smokers (P(interaction)<0.05). We found no association for HCAs or overall meat intake. CONCLUSION: BaP intake, a PAH in barbecued meat, was positively associated with RCC. These biologically plausible findings advocate further epidemiological investigation into dietary intake of BaP and risk of RCC.

Bcl-2 and c-myc expression, cell cycle kinetics and apoptosis during the progression of chronic myelogenous leukemia from diagnosis to blastic phase.

Chronic myelogenous leukemia (CML) has a progressive course but little is known about the biologic characteristics of disease progression. This study was designed to assess the changes in cell proliferative characteristics, apoptosis, the expression of the bcl-2 and c-myc genes between the time of initial diagnosis and entrance into the blastic phase of the disease. We observed that the rate of cell proliferation decreased and the cell death rate did not significantly change as the disease accelerated. The level of bcl-2 expression was significantly higher in accelerated/blastic phase cells than in the chronic phase cells in the population as a whole, however, the bcl-2 expression level did not change in blast cell subpopulation. c-myc Expression was significantly higher in the blast cell subpopulation of accelerated/blastic phase than in that of earlier phases of the disease. In conclusion, the characteristics of CML cells, namely proliferation rate, c-myc and bcl-2 change during the course of the disease. It is possible that the change in c-myc expression plays a causative role in evolution of the blastic phase from the chronic phase.

The effects of 13-cis retinoic acid and interferon-alpha in chronic myelogenous leukemia cells in vivo in patients.

The effects of the administration of a 3-day course of 13-cis retinoic acid in combination with interferon a [RA/IFN] on the leukemia cells was measured in vivo in 43 patients with chronic myelogenous leukemia. The administration of RA/IFN was associated with a significant fall in the white blood cell count of patients with chronic-phase disease and with a fall in the percentage S-phase cells in CML patients regardless of the stage of their leukemia. In two thirds of the patients studied the administration of RA/IFN was also associated with an increase in marrow apoptosis. The cytokine combination also suppressed bcl-2 and myc expression in a minority of patients and such expression appears to be associated with response to a treatment regimen which includes RA/IFN. These studies are the first to directly assess the effects of the combination of RA/IFN on chronic myelogenous leukemia cells in vivo in patients. These effects, if seen in other malignant diseases, could account for the therapeutic benefit which has been associated with the administration of this combination of biological agents to patients with malignant disease.

Apolipoprotein E polymorphism and coronary heart disease.

Serum lipoproteins and apolipoproteins were analyzed in 199 patients with acute myocardial infarction (CCU group), and in 211 normal healthy individuals (control group). It was shown that serum lipoprotein abnormalities, especially elevated low density lipoprotein (LDL) levels, are closely related to atherogenesis in relatively young patients and in subjects with severe coronary lesions. The frequency of apo E4 was higher and that of E2 was lower in the CCU group than in the control group. Apo E mutants, E7 and E5, were also frequent in the CCU group. Subjects with an E3/2 phenotype had reduced LDL and increased VLDL levels, and those with an E4/3 phenotype had increased LDL levels in serum. These results suggest that apo E4 is a positive risk factor and apo E2 a negative risk factor for atherosclerosis. The action of apo E isoproteins to increase or decrease serum LDL levels may be one of the mechanisms of atherosclerosis. Furthermore, the effect of apo E isoforms on serum lipoproteins was evident even in childhood. The mutant apo E binding to LDL receptors were investigated to clarify the metabolism of apo E5 and apo E7. The affinity of apo E5 was twice that of the wild form of apo E3. Apo E7, however, had a lower receptor affinity than apo E3. Therefore, the authors postulate that individuals with apo E5 have increased risk of developing hypercholesterolemia and subsequent atherosclerosis. From the binding data of apo E7, the action of apo E7 is considered similar to that of apo E2.

Human apolipoprotein E4 domain interaction. Arginine 61 and glutamic acid 255 interact to direct the preference for very low density lipoproteins.

Human apolipoprotein (apo) E contains an amino- and a carboxyl-terminal domain, which are connected by a hinge region (approximately residues 165 to 215). The interaction of the two domains has been suggested to be responsible for the apoE4-binding preference for very low density lipoproteins (VLDL). In the absence of this interaction in apoE3, the preference is for high density lipoproteins (HDL). To exclude the possibility that the interaction of apoE with other apolipoproteins on the native particles may contribute to the isoform-specific preferences, VLDL-like emulsion particles were incubated with apoE, and the lipid-bound apoE was separated from free apoE on a Superose 6 column. The apoE4 bound more effectively to these particles than did apoE3, indicating that the apoE4 preference for VLDL is due not to interactions with other apolipoproteins but to an intrinsic property of apoE4, likely related to domain interaction. Previously, arginine 61 was shown to be critical for the isoform preferences, suggesting that it interacted with an acidic residue(s) in the carboxyl terminus. Substitution of arginine 61 with lysine did not alter the preference of apoE4 for VLDL, demonstrating that a positive charge rather than a specific requirement for arginine is critical for domain interaction. To identify the acidic residue(s) in the carboxyl terminus interacting with arginine 61, the six acidic residues (244, 245, 255, 266, 270, and 271) in a region known to be important for both lipoprotein association and isoform-specific preferences were substituted individually with alanine in apoE4. Only substitution of glutamic acid 255 altered the preference of apoE4 from VLDL to HDL, indicating that this was the sole residue in the carboxyl terminus that interacts with arginine 61. The participation of the hinge region in domain interaction was examined with internal deletion mutants. Deletion of the residues 186-202 or 186-223, representing major portions of the hinge region, had no effect on the apoE4 preference for VLDL. This suggests that the hinge region may act as a spacer that connects the two domains. Further deletion into the carboxyl-terminal domain (to residue 244) results in a loss of apoE4 VLDL binding. These studies establish that interaction of arginine 61 and glutamic acid 255 mediates apoE4 domain interaction.

Site-directed mutagenesis of an apolipoprotein E mutant, apo E5(Glu3----Lys) and its binding to low density lipoprotein receptors.

Apo E5(Glu3----Lys) is a naturally occurring apolipoprotein E (apo E) mutant found in patients with hyperlipoproteinemia and atherosclerosis. It has been shown to have a high affinity for low density lipoprotein (LDL) receptors. In this study, mutant apo E5 was produced by Chinese hamster ovary cells by means of an in vitro site-directed mutagenesis technique, and its LDL receptor binding activity was assessed. The apo E5 obtained from gene expression bound more readily to the LDL receptor than did plasma apo E3. The concentrations required for 50% competitive binding of 125I-labeled LDL to the LDL receptors were 58.9 ng/ml for plasma apo E3 and 25.7 ng/ml for the expressed apo E5. The expressed apo E5 displayed 229% normal binding. This result is highly consistent with that obtained with plasma apo E5, which showed 217% normal binding. Although the experimental apo E isoproteins contained more sialic acid than plasma apo E, the extent of sialylation had no effect on the receptor binding of apo E.

Characterization of apolipoprotein E7 (Glu(244)-->Lys, Glu(245)--->Lys), a mutant apolipoprotein E associated with hyperlipidemia and atherosclerosis.

Previously, a mutant apolipoprotein (apo) E, apolipoprotein E7 (Glu(244)-->Lys, Glu(245)--->Lys), has been identified in association with hyperlipidemia and atherosclerosis. To investigate the effects of its structural changes on lipoprotein metabolisms and its correlation with atherosclerosis, we characterized this mutant apoE with respect to its receptor-binding, heparin-binding, and lipoprotein association. In a competitive binding assay, apoE7. dimyristoylphosphatidylcholine displayed a defective binding to the low density lipoprotein (LDL) receptor. The concentration of apoE7 required for 50% displacement of (125)I-labeled LDL was 0.223 microg/ml, while that for apoE3 was 0.048 microg/ml. ApoE7 possesses only 23% of normal binding activity. To investigate the lipoprotein preference of apoE7, we determined the relative amounts of apoE7 in plasma lipoprotein fractions obtained by ultracentrifugation or gel filtration. Like human apoE4, apoE7 was preferentially associated with the very low density lipoproteins (VLDL). For determination of heparin-binding activity, apoVLDL was applied to a heparin-Sepharose affinity column and the bound materials were eluted with a salt gradient. The apoE7 was eluted at a higher NaCl concentration (157 mm) than apoE3 (126 mm), indicating that this mutant has a higher affinity for heparin than does apoE3. While the reduced receptor-binding activity indicates delayed clearance of the triglyceride-rich lipoproteins, the preferential association of apoE7 with larger-size lipoproteins and the stronger interaction with heparin may compensate, to some extent, for the delayed clearance of triglyceride-rich lipoproteins. The strong interaction with proteoglycans in the arterial wall seems to be one of the possible explanations for the association of apoE7 with atherosclerosis.-Yamamura, T., L-M. Dong, and A. Yamamoto. Characterization of apolipoprotein E7 (Glu(244)-->Lys, Glu(245)--->Lys), a mutant apolipoprotein E associated with hyperlipidemia and atherosclerosis.

Enhanced binding activity of an apolipoprotein E mutant, APO E5, to LDL receptors on human fibroblasts.

We have previously demonstrated an apolipoprotein E (apo E) mutant, apo E5, associated with hyperlipidemia and atherosclerotic vascular diseases. To investigate the possible mechanism of apo E5 involvement in the development of hyperlipidemia, we have isolated the apo E isoprotein and determined its binding activity to LDL (low density lipoprotein) receptors. It was shown that the apo E5 isoprotein was two times more active for binding to LDL receptors than apo E3. The concentration of apo E, at which 50% of 125I-labeled LDL bound to the receptor was displaced, was 29 ng/ml for apo E5 and 63 ng/ml for apo E3. This result suggests that the high affinity of apo E5 to LDL receptors results in a high uptake of apo E5-containing lipoproteins by the liver and leads to a down-regulation of LDL receptors in the liver. We can postulate that individuals with apo E5 are susceptible to hypercholesterolemia and in consequence to atherosclerosis.

Spreading depression increases immunohistochemical staining of glial fibrillary acidic protein.

Reactive astrocytosis is a process by which astrocytes respond to brain injury by showing an increase in glial fibrillary acidic protein (GFAP) staining that is associated with hypertrophy and/or hyperplasia of these cells. Because spreading depression (SD) is a perturbation uncomplicated by neuronal necrosis and is seen in both in vivo and in vitro neural structures, we sought to determine whether SD was a sufficient stimulus to induce enhanced GFAP staining. SD was elicited in anesthetized rats by application of KCI to parietal cortex for 3 hr; equimolar NaCI was applied to contralateral cortex. SD was confirmed by monitoring DC potentials in frontal neocortices. Animals were allowed to recover for 48 hr, and their brains were processed for semiquantitative and computer-based analyses of GFAP staining intensity. Experimental GFAP staining was referenced to contralateral control levels. Neocortical SD (13-37 SDs) was associated with a significant (p less than 10(-4)), 43% increase in GFAP staining intensity, which remained statistically greater than normal for more than 2 weeks. If SD was inhibited by combined hyperoxia and hypercarbia, only a nonsignificant (p greater than 0.20), 7% increase in GFAP staining was seen. Thus, SD may be a useful physiologic process with which to begin to explore the cellular mechanisms that induce the transformation of normal astrocytes into reactive species.

Introduction of human apolipoprotein E4 "domain interaction" into mouse apolipoprotein E.

Human apolipoprotein E4 (apoE4) binds preferentially to lower density lipoproteins, including very low density lipoproteins, and is associated with increased risk of atherosclerosis and neurodegenerative disorders, including Alzheimer's disease. This binding preference is the result of the presence of Arg-112, which causes Arg-61 in the amino-terminal domain to interact with Glu-255 in the carboxyl-terminal domain. ApoE2 and apoE3, which have Cys-112, bind preferentially to high density lipoproteins (HDL) and do not display apoE4 domain interaction. Mouse apoE, like apoE4, contains the equivalent of Arg-112 and Glu-255, but lacks the critical Arg-61 equivalent (it contains Thr-61). Thus, mouse apoE does not display apoE4 domain interaction and, as a result, behaves like human apoE3, including preferential binding to HDL. To assess the potential role of apoE4 domain interaction in atherosclerosis and neurodegeneration, we sought to introduce apoE4 domain interaction into mouse apoE. Replacing Thr-61 in mouse apoE with arginine converted the binding preference from HDL to very low density lipoproteins in vitro, suggesting that apoE4 domain interaction could be introduced into mouse apoE in vivo. Using gene targeting in embryonic stem cells, we created mice expressing Arg-61 apoE. Heterozygous Arg-61/wild-type apoE mice displayed two phenotypes found in human apoE4/E3 heterozygotes: preferential binding to lower density lipoproteins and reduced abundance of Arg-61 apoE in the plasma, reflecting its more rapid catabolism. These findings demonstrate the successful introduction of apoE4 domain interaction into mouse apoE in vivo. The Arg-61 apoE mouse model will allow the effects of apoE4 domain interaction in lipoprotein metabolism, atherosclerosis, and neurodegeneration to be determined.

Stable expression and secretion of apolipoproteins E3 and E4 in mouse neuroblastoma cells produces differential effects on neurite outgrowth.

Previously, we demonstrated in cultured dorsal root ganglion neurons that, in the presence of beta-migrating very low density lipoproteins (beta-VLDL), apolipoprotein (apo) E4, but not apoE3, suppresses neurite outgrowth. In the current studies, murine neuroblastoma cells (Neuro-2a) were stably transfected with human apoE3 or apoE4 cDNA, and the effect on neurite outgrowth was examined. The stably transfected cells secreted nanogram quantities of apoE (44-89 ng/mg of cell protein in 48 h). In the absence of lipoproteins, neurite outgrowth was similar in the apoE3- and apoE4-secreting cells. The apoE4-secreting cells, when incubated with beta-VLDL, VLDL, cerebrospinal fluid lipoproteins (d < 1.21 g/ml), or with triglyceride/phospholipid (2.7:1 (w/w)) emulsions, showed a reduction in the number of neurites/cell, a decrease in neurite branching, and an inhibition of neurite extension, whereas in the apoE3-secreting cells in the presence of a lipid source, neurite extension was increased. Uptake of beta-VLDL occurred to a similar extent in both the apoE3- and apoE4-secreting cells. With low density lipoproteins or with dimyristoylphosphatidylcholine emulsions, either alone or complexed with cholesterol, no differential effect on neurite outgrowth was observed. A slight differential effect was observed with apoE-containing high density lipoproteins. The differential effect of apoE3 and apoE4 in the presence of beta-VLDL was blocked by incubation of the cells with heparinase and chlorate, with lactoferrin, or with receptor-associated protein, all of which prevent the uptake of lipoproteins by the low density lipoprotein receptor-related protein (LRP). The data suggest that the secreted and/or cell surface-bound apoE interact with the lipoproteins and facilitate their internalization via the heparan sulfate proteoglycan-LRP pathway. The mechanism by which apoE3 and apoE4 exert differential effects on neurite outgrowth remains speculative. However, the data suggest that apoE4, which has been shown to be associated with late onset familial and sporadic Alzheimer's disease, may inhibit neuronal remodeling and contribute to the progression of the disease.

Identification and characterization of a novel apolipoprotein E variant, apolipoprotein E3' (Arg136-->His): association with mild dyslipidemia and double pre-beta very low density lipoproteins.

Apolipoprotein (apo) E mediates the removal of chylomicron and VLDL remnants from plasma. In a proband with mild hyperlipidemia and a family history of premature coronary artery disease, we have identified a new mutant of apoE with an isoelectric point close to but distinct from that of apoE3. Sequencing of the apoE gene from this subject (JB) revealed that the subject was heterozygous for a G to A substitution in codon 136, resulting in the substitution of histidine for arginine; therefore, we have designated this isoform apoE3' (Arg136-->His). Examination of the proband's kindred revealed that the nine carriers (all heterozygotes) of the variant isoform displayed a twofold elevation in the concentration of very low density lipoprotein (VLDL) cholesterol (40 +/- 8 mg/dl) and triglyceride (109 +/- 19) compared to the nine noncarriers (19 +/- 3 and 55 +/- 13, respectively). In all carriers, the VLDL displayed an abnormal double pre-beta pattern upon electrophoresis. The low density lipoprotein receptor-binding activity of purified apoE3' (Arg136-->His) when complexed with DMPC was slightly defective (80% of the activity of normal apoE). The mutant apoE also displayed a reduced affinity for heparin compared to apoE3. As both of these biochemical parameters are known to be important in VLDL clearance, the defects associated with this variant are likely responsible for the increase in VLDL observed in carriers. None of the carriers displayed clinical features of type III hyperlipoproteinemia, suggesting that the relatively mild dyslipoproteinemic phenotype associated with this variant might be associated with recessive expression of this disorder. However, the abnormal VLDL phenotype appears to be dominantly expressed.

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.

Human apolipoprotein E. Role of arginine 61 in mediating the lipoprotein preferences of the E3 and E4 isoforms.

Human apolipoprotein (apo) E4 (arginine at residue 112) preferentially associates with very low density lipoproteins (VLDL), and apoE3 (cysteine at 112) associates with high density lipoproteins. It has been postulated that the amino-terminal domain, which contains residue 112, influences the lipoprotein preference by interacting with the carboxyl-terminal domain, which contains the lipid-binding region. To delineate the region in the carboxyl-terminal domain mediating lipoprotein binding and involved in isoform preference, we produced truncated apoE3 and apoE4 variants (terminating at residues 251, 260, 266, or 272) in Escherichia coli and assessed them for lipoprotein association. This analysis suggested that residues 260-272 contain important determinants for complete lipoprotein association and isoform preferences. To determine whether positive charge at residue 112 was an absolute requirement for the apoE4 VLDL preference, we compared the distributions of rabbit apoE (equivalent to apoE3, with cysteine at a position corresponding to 112), canine apoE (arginine at the corresponding site), and cysteamine-treated rabbit apoE (cysteine converted to a positively charged residue). Surprisingly, all distributed like human apoE3, suggesting that positive charge at a position corresponding to 112 was not directly responsible for the isoform preference and that other residues in the amino-terminal domain were involved. To determine which residues were involved, the structure of the apoE4 22-kDa fragment (the amino-terminal two-thirds of the molecule) was determined to 2.5 A by x-ray crystallography. Compared with the known four-helix bundle structure of apoE3, the only significant differences in the apoE4 structure were that glutamic acid 109 formed a salt bridge with arginine 112 and that the arginine 61 side chain was displaced to a new position. Site-directed mutagenesis of glutamic acid 109 in apoE3 and arginine 61 in apoE4 demonstrated that the position of the arginine 61 side chain in apoE4 was critical in determining apoE4 lipoprotein distribution, suggesting that arginine 61 interacted with the carboxyl-terminal domain to direct binding to VLDL.

Tissue expression studies on the mouse acyl-CoA: cholesterol acyltransferase gene (Acact): findings supporting the existence of multiple cholesterol esterification enzymes in mice.

Cholesterol esterification is involved in the regulation of cellular cholesterol content and has been hypothesized to play a role in important physiologic processes including intestinal cholesterol absorption, hepatic lipoprotein production, and macrophage foam cell formation in atherosclerotic lesions. Although initial studies of the mouse acyl CoA:cholesterol acyltransferase gene (Acact) suggested that its gene product was responsible for cholesterol esterification in most tissues, we observed recently that Acact-/- mice have only tissue-specific reductions in cholesterol esterification. To better understand the role of Acact in cholesterol esterification, we used in situ hybridization and immunoblotting to perform tissue expression studies in wild-type mice. We found high levels of Acact expression in steroidogenic tissues, sebaceous glands, and atherosclerotic lesions, but not in the liver or the small intestine. These data support the hypothesis that multiple cholesterol esterification enzymes exist in mammals and that another enzyme is likely to be responsible for cholesterol esterification activity in mouse liver and intestine.

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.