Antley-Bixler syndrome (ABS) represents a group of heterogeneous disorders characterized by skeletal, cardiac, and urogenital abnormalities that have frequently been associated with mutations in fibroblast growth factor receptor 2 or cytochrome P450 reductase genes. In some ABS patients, reduced activity of the cholesterogenic cytochrome P450 CYP51A1, an ortholog of the mouse CYP51, and accumulation of lanosterol and 24,25-dihydrolanosterol has been reported, but the role of CYP51A1 in the ABS etiology has remained obscure. To test whether Cyp51 could be involved in generating an ABS-like phenotype, a mouse knock-out model was developed that exhibited several prenatal ABS-like features leading to lethality at embryonic day 15. Cyp51؊/؊ mice had no functional Cyp51 mRNA and no immunodetectable CYP51 protein. The two CYP51 enzyme substrates (lanosterol and 24,25-dihydrolanosterol) were markedly accumulated. Cholesterol precursors downstream of the CYP51 enzymatic step were not detected, indicating that the targeting in this study blocked de novo cholesterol synthesis. This was reflected in the up-regulation of 10 cholesterol synthesis genes, with the exception of 7-dehydrocholesterol reductase. Lethality was ascribed to heart failure due to hypoplasia, ventricle septum, and epicardial and vasculogenesis defects, suggesting that Cyp51 deficiency was involved in heart development and coronary vessel formation. As the most likely downstream molecular mechanisms, alterations were identified in the sonic hedgehog and retinoic acid signaling pathways. Cyp51 knock-out mice provide evidence that Cyp51 is essential for embryogenesis and present a potential animal model for studying ABS syndrome in humans.

BackgroundCircadian rhythms have a profound effect on human health. Their disruption can lead to serious pathologies, such as cancer and obesity. Gene expression studies in these pathologies are often studied in different mouse strains by quantitative real time polymerase chain reaction (qPCR). Selection of reference genes is a crucial step of qPCR experiments. Recent studies show that reference gene stability can vary between species and tissues, but none has taken circadian experiments into consideration.ResultsIn the present study the expression of ten candidate reference genes (Actb, Eif2a, Gapdh, Hmbs, Hprt1, Ppib, Rn18s, Rplp0, Tbcc and Utp6c) was measured in 131 liver and 97 adrenal gland samples taken from three mouse strains (C57BL/6JOlaHsd, 129Pas plus C57BL/6J and Crem KO on 129Pas plus C57BL/6J background) every 4 h in a 24 h period. Expression stability was evaluated by geNorm and NormFinder programs. Differences in ranking of the most stable reference genes were observed both between individual mouse strains as well as between tissues within each mouse strain. We show that selection of reference gene (Actb) that is often used for analyses in individual mouse strains leads to errors if used for normalization when different mouse strains are compared. We identified alternative reference genes that are stable in these comparisons.ConclusionsGenetic background and circadian time influence the expression stability of reference genes. Differences between mouse strains and tissues should be taken into consideration to avoid false interpretations. We show that the use of a single reference gene can lead to false biological conclusions. This manuscript provides a useful reference point for researchers that search for stable reference genes in the field of circadian biology.

causes a suppressed synthesis, reduced absorption from the intestine, and increased rate of degradation into bile acids. The latter two effects are mediated by the nuclear liver X receptor (LXR) ( 2 ). Under in vitro conditions, sidechain oxidized oxysterols have a much higher capacity to downregulate cholesterol synthesis and to activate the LXR target genes than does cholesterol itself (as reviewed in 3 ). Because of this, oxysterols have been suggested to mediate, at least in part, the above effects of cholesterol. There are three different mechanisms by which the oxysterols may affect cholesterol-sensitive genes: 1 ) interaction with the sterol-regulatory element binding proteins (SREBP mechanism); 2 ) activation of the LXR mechanisms; and 3 ) effects on the degradation of specifi c enzymes, in particular HMG-CoA reductase (HMGCR).The major oxysterols in the circulation of man and mouse are 24S-hydroxycholesterol (24OH) and 27-hydroxycholesterol (27OH) ( 3 ). 24OH is formed by the enzyme cholesterol 24S-hydroxylase (CYP46A1). This enzyme is located almost exclusively in the brain. There is a constant fl ux of 24OH from the brain across the blood-brain barrier into the circulation. This oxysterol is then taken up by the liver and converted into bile acids and conjugates of unmetabolized or partially metabolized 24OH ( 4 ). 27OH is formed by the enzyme sterol 27-hydroxylase (CYP27A1). This enzyme is present in almost all cells in the body. There is a constant fl ux of 27OH and metabolites of this Abstract The two oxysterols, 27-hydroxycholesterol (27OH) and 24S-hydroxycholesterol (24OH), are both inhibitors of cholesterol synthesis and activators of the liver X receptor (LXR) in vitro. Their role as physiological regulators under in vivo conditions is controversial, however. In the present work, we utilized a previously described mouse model with overexpressed human sterol 27-hydroxylase (CYP27A1). The levels of 27OH were increased about 12-fold in the brain. The brain levels of HMG-CoA reductase mRNA and HMG-CoA synthase mRNA levels were increased. In accordance with increased cholesterol synthesis, most of the cholesterol precursors were also increased. The level of 24OH, the dominating oxysterol in the brain, was decreased by about 25%, most probably due to increased metabolism by CYP27A1. The LXR target genes were unaffected or slightly changed in a direction opposite to that expected for LXR activation. In the brain of Cyp27 Cholesterol has a remarkable capacity to regulate its own synthesis and metabolism ( 1 ). Abbreviations: CYP46A1, cholesterol 24S-hydroxylase; CYP27A1, sterol 27-hydroxylase; FF-MAS, follicular fl uid meiosis-activating sterol; HMGCR, HMG CoA reductase; LXR, liver X receptor; 24OH, 24S-hydroxycholesterol; 27OH, 27-hydroxycholesterol; 25OH, 25-hydroxycholesterol; SREBP, sterol-regulatory element binding protein; T-MAS, testis meiosis-activating sterol.1 According to a recent paper, the preferred nomenclature for 27-hydroxylation and 27-hydroxycholesterol should be (25R)26-hydroxylation...

Cholesterol synthesis is a ubiquitous and housekeeping metabolic pathway that leads to cholesterol, an essential structural component of mammalian cell membranes, required for proper membrane permeability and fluidity. The last part of the pathway involves steroidal triterpenes with cholestane ring structures. It starts by conversion of acyclic squalene into lanosterol, the first sterol intermediate of the pathway, followed by production of 20 structurally very similar steroidal triterpene molecules in over 11 complex enzyme reactions. Due to the structural similarities of sterol intermediates and the broad substrate specificity of the enzymes involved (especially sterol-Δ24-reductase; DHCR24) the exact sequence of the reactions between lanosterol and cholesterol remains undefined. This article reviews all hitherto known structures of post-squalene steroidal triterpenes of cholesterol synthesis, their biological roles and the enzymes responsible for their synthesis. Furthermore, it summarises kinetic parameters of enzymes (Vmax and Km) and sterol intermediate concentrations from various tissues. Due to the complexity of the post-squalene cholesterol synthesis pathway, future studies will require a comprehensive meta-analysis of the pathway to elucidate the exact reaction sequence in different tissues, physiological or disease conditions. A major reason for the standstill of detailed late cholesterol synthesis research was the lack of several steroidal triterpene standards. We aid to this efforts by summarizing commercial and laboratory standards, referring also to chemical syntheses of meiosis-activating sterols.