Yasuko Miyake

LDLR Database (second edition): New additions to the database and the software, and results of the first molecular analysis

Mutations in the LDL receptor gene (LDLR) cause familial hypercholesterolemia (FH), a common autosomal dominant disorder. The LDLR database is a computerized tool that has been developed to provide tools to analyse the numerous mutations that have been identified in the LDLR gene. The second version of the LDLR database contains 140 new entries and the software has been modified to accommodate four new routines. The analysis of the updated data (350 mutations) gives the following informations: (i) 63% of the mutations are missense, and only 20% occur in CpG dinucleotides; (ii) although the mutations are widely distributed throughout the gene, there is an excess of mutations in exons 4 and 9, and a deficit in exons 13 and 15; (iii) the analysis of the distribution of mutations located within the ligand-binding domain shows that 74% of the mutations in this domain affect a conserved amino-acid, and that they are mostly confined in the C-terminal region of the repeats. Conversely, the same analysis in the EGF-like domain shows that 64% of the mutations in this domain affect a non-conserved amino-acid, and, that they are mostly confined in the N-terminal half of the repeats. The database is now accessible on the World Wide Web at http://www.umd.necker.fr

Transformation of intramembrane particles of HVJ (Sendai virus) envelopes from an invisible to visible form on aging of virions.

Abstract When HVJ virions harvested from embryonated eggs 3 days after infection are freeze-fractured, large intramembrane particles, with a mode of 150-A diameter, are exposed on the E fracture faces but not on the P fracture faces. The intramembrane particles seem to be hydrophobic parts of viral spikes of HANA and F glycoproteins. No intramembrane particles are observed in young virions harvested 1 day after infection, although many spikes project from their envelopes. When young virions are incubated at 37° for 2 days in vitro , large intramembrane particles develop and are exposed on the E fracture face: These particles resemble those observed in old virions harvested from eggs 3 days after infection. Besides this difference in intramembrane particles, morphological differences are seen between young virions and old virions aged in ovo or in vitro : in young virions the nucleocapsid strand is regularly folded under the envelope and the virion structure is rigid; but in old virions, aged in ovo or in vitro , the nucleocapsid strand is irregularly folded and detached from the envelope, and the virions are no longer rigid but are easily distorted by external forces. From these findings, the possible correlation between the transformation of intramembrane particles from an invisible to visible form and the disruption of the regular arrangement of the nucleocapsid under the viral envelope is discussed.

Cells of a human monocytic leukemia cell line (THP-1) synthesize and secrete apolipoprotein E and lipoprotein lipase

A human cell line established from a patient of an acute monocytic leukemia (THP-1) retained an ability to synthesize and secrete plasma apolipoprotein E like protein. The protein was identified with monospecific antibody raised against human plasma apolipoprotein E. The cells also secreted lipoprotein lipase (EC 3.1.1.34). The enzyme was characterized as lipoprotein lipase on the basis of the requirement of apolipoprotein C-II as an activator and the inhibition of its activity by sodium chloride. The secretion of both apolipoprotein E and lipoprotein lipase was markedly enhanced in the process of differentiation into macrophage-like cells by the addition of 4 beta-phorbol 12 beta-myristate 13 alpha-acetate.

Common Mutations in the Low-Density-Lipoprotein–Receptor Gene Causing Familial Hypercholesterolemia in the Japanese Population

Familial hypercholesterolemia (FH) is a common genetic disorder caused by mutations of the LDL-receptor gene. In the present study, we investigated four Japanese FH homozygotes and identified five point mutations: a splice site mutation in intron 12 (the 1845 + 2 T-->C mutation), a missense mutation in exon 7 (the C317S mutation), a nonsense mutation in exon 17 (the K790X mutation), a missense mutation in exon 14 (the P664L mutation), and a missense mutation in exon 4 (the E119K mutation). We developed simple methods for detecting these mutations. When we examined the presence of these mutations in 24 unrelated FH homozygotes, the 1845 + 2 T-->C mutation was found in 7 of them, and the other four mutations were unique for each proband. We also screened 120 unrelated FH heterozygotes for these mutations and found that the frequencies of the 1845 + 2 T-->C, C317S, K790X, P664L, and E119K mutations were 13.3% (16/120), 6.7% (8/120), 6.7% (8/120), 3.3% (4/120), and 1.7% (2/120), respectively. These mutations were found in more than 30% of unrelated Japanese FH patients. By using the detection methods developed in this study, the diagnosis of more than 30% of the genetic bases of Japanese FH heterozygotes is expected.

Effects of cytochalasin D on fusion of cells by HVJ (Sendai virus).

Abstract Fusion of cells mediated by HVJ was inhibited completely with 5 μg/ml or more of cytochalasin D (CD). With cytochalasin, HVJ-cell interaction at 0 °C proceeded as well as without cytochalasin; HVJ was adsorbed to cell surfaces and the cells agglutinated together. Then the virus particles were enfolded with cell membranes, which resulted in the disappearance of hemadsorption activity on the cell surfaces. When the cell-virus complex was incubated at 37 °C, the early reactions proceeded as well as without cytochalasin; the hemadsorption activity reappeared on the cell surfaces, the viral envelopes fused with cell membranes at the same degree as without cytochalasin, and a stage sensitive to sodium azide appeared as in a control without cytochalasin. But cell-to-cell fusion did not occur in the presence of cytochalasin; cells were dissociated freely from the cell aggregates during incubation. This indicates that cell-to-cell fusion was inhibited but HVJ envelope to cell membrane interactions proceeded well on incubation at 37 °C. These findings suggest that viral envelope-cell membrane fusion and cell-cell fusion are separable, and participation of a cytoskeleton system including microfilaments in the cells is essential for cell-cell fusion.

Low density lipoprotein (LDL) binding affinity for the LDL receptor in hyperlipoproteinemia

We measured the binding affinity of low density lipoprotein (LDL) for the LDL receptor in patients with various types of hyperlipoproteinemia and investigated the effects of LDL lipid composition and particle size on receptor affinity. LDL (1.019 < d < 1.063) was isolated by sequential ultracentrifugation from the serum of normolipidemic controls and patients with hyperlipoproteinemia. Patients with type IIa hyperlipoproteinemia had LDL with a similar receptor affinity to that of normal LDL. However, patients with hypertriglyceridemia (type IIb and type IV hyperlipoproteinemia) had LDL with a low receptor affinity, and the degree of the reduction in affinity paralleled the severity of the hypertriglyceridemia. The LDL of hypertriglyceridemic patients was rich in protein and triglycerides, had a low content of cholesterol and phospholipids, and was smaller than normal, thus resembling the atherogenic lipoprotein known as small, dense LDL. These abnormalities were observed even in patients with mild hypertriglyceridemia regardless of their serum cholesterol levels. The degree of alteration in LDL lipid composition and particle size was strongly associated with the reduction of LDL receptor affinity. We also examined the effects of two lipid-lowering agents (bezafibrate and probucol) on the characteristics of LDL. LDL receptor affinity was only improved when the lipid composition and particle size were normalized by drug therapy. Although it has been reported that decreased cholesteryl ester transfer protein (CETP) activity results in the formation of small LDL, plasma CETP activity was normal in the hyperlipoproteinemic patients and the normalization of LDL characteristics by drug therapy was not accompanied by an increase of CETP activity. Our results suggested that an abnormal lipid composition and/or small particle size might cause a decrease in the receptor affinity of LDL. These structural and functional abnormalities were reversed by drug therapy, underlining the importance of treating hypertriglyceridemia for the prevention of atherosclerosis.

Update of Japanese common LDLR gene mutations and their phenotypes: Mild type mutation L547V might predominate in the Japanese population

We investigated the LDLR gene mutations in 205 unrelated Japanese FH (familial hypercholesterolemia) heterozygotes to see if there is a prevalence of common mutations in the Japanese population. A total of 53 different small mutations (<25bp) and 10 kinds of large deletions (>25bp) were identified. Among them there were eight relatively frequent mutations: C317S, c.1845+2T>C, K790X, L547V, P664L, D412H, c.2312-3C>A and V776M. The patients with these mutations comprised 32% of the total FH heterozygotes investigated. Comparison of clinical phenotypes of the eight frequent mutations disclosed that a missense mutation, L547V, manifested a milder phenotype than the other mutations. The mild clinical phenotype was shown to be based on the high level of receptor activity remaining on the patients cell surfaces. When we examined the presence of common mutations in a general population, the L547V mutation was detected with unexpectedly higher frequency than the other mutations, suggesting an underestimation of the frequency of this mutation in FH heterozygote patients. In conclusion, although there is a broad spectrum of LDLR gene mutations in the Japanese population, eight common mutations were observed. Among them, a mild phenotype mutation, L547V, might predominate in the Japanese population.

Uptake of oxidized low-density lipoprotein in a THP-1 cell line lacking scavenger receptor A☆

We previously isolated THP-1 subtype cells (sTHP-1), a cell line that expresses scanty amounts of scavenger receptor A (ScR-A) and does not undergo foam cell formation when incubated with acetylated low-density lipoprotein (Ac-LDL). In this study, we investigated the accumulation of esterified cholesterol in sTHP-1 cells incubated with oxidized LDL (Ox-LDL), a physiologically modified lipoprotein in human. While sTHP-1 cells incubated with Ac-LDL accumulated only small amounts of esterified cholesterol, those incubated with Ox-LDL accumulated amounts similar to those accumulated by parent THP-1 (pTHP-1) cells. sTHP-1 cells expressed CD36 in amounts similar to the amounts expressed by pTHP-1 cells, and Ox-LDL was internalized through this CD36. The amount of accumulated esterified cholesterol was 73-81% of that accumulated in pTHP-1 cells expressing ScR-A. The levels of 125I-Ox-LDL binding, association, and degradation in sTHP-1 cells were 64-70% of the corresponding levels in pTHP-1 cells. In our experiments utilizing ScR-A-deficient sTHP-1 cells and a specific antibody against human CD36, most of the Ox-LDL interacted with the CD36 receptor. In addition, a substantial amount of Ox-LDL (28-42%) was bound and degraded by sTHP-1 macrophages when both of the two major scavenger receptors, ScR-A and CD36, were deficient or blocked. These results indicate that CD36 in macrophages plays an important role in foam cell formation by Ox-LDL, while additional scavenger receptor(s) may take part in significant pathways of Ox-LDL uptake in macrophages.

Improved methods using HVJ(Sendai virus) for introducing substances into cells

Abstract When HVJ virions were sonicated in the presence of a given protein, about 0.2%–0.3% of total protein added was recovered in the virions. The protein molecules could be introduced effectively into cells. In this study, fragment A of diphtheria toxin was used as the test protein. More than 96% of L cells were killed after short exposure to the virus suspension containing fragment A diluted so as to contain only about 0.004 μg of fragment A per ml.

Histochemical characterization of low density lipoprotein receptors in internalization-defective familial hypercholesterolemia.

We studied the localization of low density lipoprotein (LDL) bound to the receptors on the cultured fibroblasts from a patient (MN) with homozygous familial hypercholesterolemia and a defect in internalization of LDL and compared the localization with normal fibroblasts and with those from another internalization-defective cell, GM2408A. Monolayers of cells were cultured with lipoprotein-deficient human serum, and the cells were incubated with 125I-or ferritin-labeled LDL. The LDL binding was observed by autoradiography or by an electron microscope. Autoradiographs of bound 125I-LDL confirmed that MNs cells could not internalize LDL inside the cell at 37° C. In these cells, ferritin LDL was found mainly in noncoated regions at 4° C; it was not found in endocytic vesicles after incubation at 37° C. Ferritin cores that bound on the surface of the cells from the normal subject, from GM2408A, from MN, and from MNs parents, were counted and quantitatively analyzed at 4° C. In normal cells, 62% of the ferritin cores were bound in the coated pits; in MNs cells, only 11% of the ferritin LDL was found in the coated pits; in the GM2408A cells, 12% of the ferritin LDL was found in the coated pits; in the cells from MNs parents, 40% of the ferritin LDL was found on the coated pits. The results indicate that the internalization defect in MNs cells is the same as that in the GM2408A cells; neither can localize the LDL and LDL receptor complex in coated pits.