Oxidized derivatives of 7-dehydrocholesterol induce growth retardation in cultured rat embryos: a model for antenatal growth retardation in the Smith-Lemli-Opitz syndrome.
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7-Dehydrocholesterol accumulates in fetuses affected by the Smith-Lemli-Opitz syndrome as a result of a deficit in the ultimate step of cholesterol synthesis catalyzed by Delta7 reductase. Rat embryos explanted at gestation day 10 and cultured for 48 h in the presence of the Delta7 reductase inhibitor AY 9944 were used as a model to discriminate between the beneficial effect of supplementation with cholesterol and the deleterious effect of supplementation with 7-dehydrocholesterol. Cholesterol supplementation in the form of mixed cholesterol/lecithin liposomes added to serum serving as the culture medium restores the growth of embryos which is markedly decreased in the presence of the inhibitor. 7-Dehydrocholesterol under identical conditions does not restore growth and impairs the beneficial effect of cholesterol added simultaneously. UV-photooxidation of 7-dehydrocholesterol-supplemented culture medium enhances its embryotoxicity, which suggests uptake by the embryo of toxic by-products formed from 7-dehydrocholesterol. By contrast photooxidation of cholesterol-supplemented culture medium does not induce embryotoxicity. alpha-Tocopherol reduces the toxicity of photooxidized 7-dehydrocholesterol supplementing the culture medium. We conclude that 7-dehydrocholesterol does not fulfill the cholesterol requirement of the developing embryos and exerts an additional embryotoxic effect probably via oxidized by-products. This could explain the antenatal growth retardation of SLOS by a blockage of the maternal compensatory cholesterol influx. (+info)
The Opitz syndrome gene product, MID1, associates with microtubules.
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Opitz syndrome (OS) is a genetically heterogeneous disorder characterized by defects of the ventral midline, including hypertelorism, cleft lip and palate, heart defects, and mental retardation. We recently identified the gene responsible for X-linked OS. The ubiquitously expressed gene product, MID1, is a member of the RING finger family. These proteins are characterized by an N-terminal tripartite protein-protein interaction domain and a conserved C terminus of unknown function. Unlike other RING finger proteins for which diverse cellular functions have been proposed, the function of MID1 is as yet undefined. By using the green fluorescent protein as a tag, we show here that MID1 is a microtubule-associated protein that influences microtubule dynamics in MID1-overexpressing cells. We confirm this observation by demonstrating a colocalization of MID1 and tubulin in subcellular fractions and the association of endogenous MID1 with microtubules after in vitro assembly. Furthermore, overexpressed MID1 proteins harboring mutations described in OS patients lack the capability to associate with microtubules, forming cytoplasmic clumps instead. These data give an idea of the possible molecular pathomechanism underlying the OS phenotype. (+info)
Cholesterol biosynthesis from lanosterol. Molecular cloning, tissue distribution, expression, chromosomal localization, and regulation of rat 7-dehydrocholesterol reductase, a Smith-Lemli-Opitz syndrome-related protein.
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The cDNA encoding the 471-amino acid rat 7-dehydrocholesterol reductase (DHCR), an enzyme that has been implicated in both cholesterol biosynthesis and developmental abnormalities (e.g. Smith-Lemli-Opitz syndrome) in mammals, has been cloned and sequenced, and the primary structure of the enzyme has been deduced. The DHCR gene was mapped to chromosome 8q2.1 by fluorescence in situ hybridization. Rat DHCR, calculated molecular mass of 54.15-kDa polypeptide, shares a close amino acid identity with mouse and human DHCRs (96 and 87%, respectively) as compared with its other related proteins (e.g. fungal sterol Delta14-reductase) and exhibits high hydrophobicity (>68%) with 9 transmembrane domains. Five putative sterol-sensing domains were predicted to be localized in transmembrane domains 4-8, which are highly homologous to those found in 3-hydroxymethylglutaryl-CoA reductase, sterol regulatory element-binding protein cleavage-activating protein, and patched protein. The polypeptide encoded by DHCR cDNA was expressed in yeast as a 55.45-kDa myc-tagged fusion protein, which was recognized with anti-myc monoclonal antibody 9E10 and shown to possess full DHCR activity with respect to dependence on NADPH and sensitivity to DHCR inhibitors. Northern blot analysis indicates that the highest expression of DHCR mRNA was detected in liver, followed by kidney and brain. In rat brains, the highest level of mRNA encoding DHCR was detected in the midbrain, followed by the spinal cord and medulla. Feeding rats 5% cholestyramine plus 0.1% lovastatin in chow resulted in both approximately a 3-fold induction of DHCR mRNA and a 5-fold increase of the enzymic activity in the liver. When rats were fed 0.1% (w/w) AY-9944 (in chow) for 14-days, a complete inhibition of DHCR activity and a significant reduction in serum total cholesterol level were observed. However, the level of hepatic DHCR mRNA fell only slightly, suggesting that AY-9944 may act more rapidly at the protein level than at the level of transcription of the DHCR gene under these conditions. (+info)
Marked alteration of sterol metabolism and composition without compromising retinal development or function.
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PURPOSE: To evaluate the consequences of altering retinal sterol metabolism and composition on the development, histologic organization, and electrophysiological function of the retina, under conditions that mimic the biochemical hallmarks of the Smith-Lemli-Opitz (SLO) syndrome. METHODS: Pregnant Sprague-Dawley rats were fed cholesterol-free chow containing AY9944 (treated group), an inhibitor of 3beta-hydroxysterol delta7-reductase, from gestational day 6 through postnatal day (P)28. Control animals were fed the same chow, but without AY9944. In addition, progeny in the treated group were injected subcutaneously every other day from birth to P28 with an olive oil emulsion containing AY9944; control animals received olive oil emulsion alone. At various postnatal times, tissues from treated and control animals were harvested, and their sterol profiles were analyzed by reversed-phase high-performance liquid chromatography. Companion eyes from animals of both groups were examined histologically at P1. At P28, animals were evaluated by electroretinography; tissues were then harvested for biochemical analysis and companion eyes were subjected to histologic and ultrastructural analyses. RESULTS: Treatment of developing rats with AY9944 caused markedly abnormal accumulation of 7-dehydrosterols and severely reduced cholesterol levels in all tissues examined, relative to control animals. Despite this, treated animals exhibited normal retinal development and had no overt ocular defects or decrease in electroretinographic function, up to P28. CONCLUSIONS: These results were unexpected, given the known biophysical effects of such sterol alterations on membrane properties and the profound dysmorphic and cognitive abnormalities associated with genetic defects in 3beta-hydroxysterol delta7-reductase that have been linked to the SLO syndrome. The results suggest that 7-dehydrosterols can substitute functionally for cholesterol in the retina or perhaps can act synergistically with subthreshold levels of residual cholesterol to allow normal cellular structure and function to be achieved. (+info)
Functional characterization of the Opitz syndrome gene product (midin): evidence for homodimerization and association with microtubules throughout the cell cycle.
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Opitz syndrome (OS) is a multiple congenital anomaly manifested by abnormal closure of midline structures. The gene responsible for the X-linked form of this disease, MID1, encodes a protein (midin) that contains a RING, two B-boxes, a coiled-coil (the so-called tripartite motif) and an RFP-like domain. The tripartite motif is characteristic of a family of proteins, named the B-box family, involved in cell proliferation and development. Since the subcellular compartmentalization and the ability to form multiprotein structures both appear to be crucial for the function of this family of proteins, we have studied these properties on the wild-type and mutated forms of midin. We found that endogenous midin is associated with microtubules throughout the cell cycle, co-localizing with cytoplasmic fibres in interphase and with the mitotic spindle and midbodies during mitosis and cytokinesis. Immunoprecipitation experiments demonstrated the ability of the tripartite motif to mediate midin homodimerization, consistent with the evidence, obtained by gel filtration analysis, that midin exists in the form of large protein complexes. Functional characterization of altered forms of midin, resulting from mutations found in OS patients, revealed that association with microtubules is compromised, while the ability to homodimerize and form multiprotein complexes is retained. We suggest that midin is involved in the formation of multiprotein structures acting as anchor points to microtubules and that impaired association with these cytoskeletal structures causes OS developmental defects. (+info)
MID2, a homologue of the Opitz syndrome gene MID1: similarities in subcellular localization and differences in expression during development.
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The B-box family is an expanding new family of genes encoding proteins involved in diverse cellular functions such as developmental patterning and oncogenesis. A member of this protein family, MID1, is the gene responsible for the X-linked form of Opitz G/BBB syndrome, a developmental disorder characterized by defects of the midline structures. We now report the identification of MID2, a new transcript closely related to MID1. MID2 maps to Xq22 in human and to the syntenic region on the mouse X chromosome. The two X-linked genes share the same domains, the same exon-intron organization, a high degree of similarity at the protein level and the same subcellular localization, both being confined to the cytoplasm in association to micro-tubular structures. The expression pattern studied by RNA in situ hybridization in mouse revealed that Mid2 is expressed early in development and the highest level of expression is detected in the heart, unlike Mid1 for which no expression was detected in the developing heart. Together, these data suggest that midin and MID2 have a similar biochemical function but a different physiological role during development. (+info)
Bile acid synthesis in the Smith-Lemli-Opitz syndrome: effects of dehydrocholesterols on cholesterol 7alpha-hydroxylase and 27-hydroxylase activities in rat liver.
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The Smith-Lemli-Opitz syndrome (SLOS) is a congenital birth defect syndrome caused by a deficiency of 3beta-hydroxysterol Delta(7)-reductase, the final enzyme in the cholesterol biosynthetic pathway. The patients have reduced plasma and tissue cholesterol concentrations with the accumulation of 7-dehydrocholesterol and 8-dehydrocholesterol. Bile acid synthesis is reduced and unnatural cholenoic and cholestenoic acids have been identified in some SLOS patients. To explore the mechanism of the abnormal bile acid production, the activities of key enzymes in classic and alternative bile acid biosynthetic pathways (microsomal cholesterol 7alpha-hydroxylase and mitochondrial sterol 27-hydroxylase) were measured in liver biopsy specimens from two mildly affected SLOS patients. The effects of 7- and 8-dehydrocholesterols on these two enzyme activities were studied by using liver from SLOS model rats that were treated with the Delta(7)-reductase inhibitor (BM15.766) for 4 months and were comparable with more severe SLOS phenotype in plasma and hepatic sterol compositions. In the SLOS patients, cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase were not defective. In BM15.766-treated rats, both enzyme activities were lower than those in control rats and they were competitively inhibited by 7- and 8-dehydrocholesterols. Rat microsomal cholesterol 7alpha-hydroxylase did not transform 7-dehydrocholesterol or 8-dehydrocholesterol into 7alpha-hydroxylated sterols. In contrast, rat mitochondrial sterol 27-hydroxylase catalyzed 27-hydroxylation of 7- and 8-dehydrocholesterols, which were partially converted to 3beta-hydroxycholestadienoic acids. Addition of microsomes to the mitochondrial 27-hydroxylase assay mixture reduced 27-hydroxydehydrocholesterol concentrations, which suggested that 27-hydroxydehydrocholesterols were further metabolized by microsomal enzymes. These results suggest that reduced normal bile acid production is characteristic of severe SLOS phenotype and is caused not only by depletion of hepatic cholesterol but also by competitive inhibition of cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase activities by accumulated 7- and 8-dehydrocholesterols. Unnatural bile acids are synthesized mainly by the alternative pathway via mitochondrial sterol 27-hydroxylase in SLOS. (+info)
Smith-Lemli-Opitz syndrome: a treatable inherited error of metabolism causing mental retardation.
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Smith-Lemli-Opitz syndrome, a syndrome of multiple malformations and mental retardation that for years was relegated to the atlases of genetic esoterica, was recently found to be a relatively common inborn error of metabolism. The underlying defect is absent or deficient activity of 7-dehydrocholesterol- delta 7-reductase, the enzyme catalysing the final step of cholesterol synthesis. The discovery of the biochemical defect causing Smith-Lemli-Opitz syndrome has resulted in the development of a diagnostic test and a potentially beneficial treatment (dietary cholesterol supplementation). Infants and young children with the syndrome have shown marked improvement in growth, behaviour and general health after receiving cholesterol therapy; older children and adults have shown some improvement in development and intellectual functioning. Despite the excitement these developments have elicited among geneticists and biochemists, this syndrome remains relatively unknown to many primary care physicians. Increased awareness of Smith-Lemli-Opitz syndrome is needed to identify affected patients so that they and their families can benefit from appropriate treatment and genetic counselling. (+info)