MD-2 Homologue Recognizes the White Spot Syndrome Virus Lipid Component and Induces Antiviral Molecule Expression in Shrimp.

The myeloid differentiation factor 2 (MD-2)-related lipid-recognition (ML) domain is found in multiple proteins, including MD-2, MD-1, Niemann-Pick disease type C2, and mite major allergen proteins. The significance of ML proteins in antibacterial signal transduction and in lipid metabolism has been well studied. However, their function in host-virus interaction remains poorly understood. In the current study, we found that the ML protein family is involved in resistance against white spot syndrome virus in kuruma shrimp, Marsupenaeus japonicus One member, which showed a high similarity to mammalian MD-2/MD-1 and was designated as ML1, participated in the antiviral response by recognizing cholesta-3,5-diene (CD), a lipid component of the white spot syndrome virus envelope. After recognizing CD, ML1 induced the translocation of Rel family NF-κB transcription factor Dorsal into the nucleus, resulting in the expression of Vago, an IFN-like antiviral cytokine in arthropods. Overall, this study revealed the significance of an MD-2 homologue as an immune recognition protein for virus lipids. The identification and characterization of CD-ML1-Dorsal-Vago signaling provided new insights into invertebrate antiviral immunity.

Sulfated steroid-amino acid conjugates from the Irish marine sponge Polymastia boletiformis.

Antifungal bioactivity-guided fractionation of the organic extract of the sponge Polymastia boletiformis, collected from the west coast of Ireland, led to the isolation of two new sulfated steroid-amino acid conjugates (1 and 2). Extensive 1D and 2D NMR analyses in combination with quantum mechanical calculations of the electronic circular dichroism (ECD) spectra, optical rotation, and 13C chemical shifts were used to establish the chemical structures of 1 and 2. Both compounds exhibited moderate antifungal activity against Cladosporium cucumerinum, while compound 2 was also active against Candida albicans. Marine natural products containing steroidal and amino acid constituents are extremely rare in nature.

A DSC and FTIR spectroscopic study of the effects of the epimeric 4,6-cholestadien-3-ols and 4,6-cholestadien-3-one on the thermotropic phase behaviour and organization of dipalmitoylphosphatidylcholine bilayer membranes.

We present the results of a comparative differential calorimetric and Fourier transform infrared spectroscopic study of the effect of cholesterol and five analogues on the thermotropic phase behaviour and organization of dipalmitoylphosphatidylcholine bilayer membranes. These sterols/steroids differ in both the nature and stereochemistry of the polar head group at C3 (ßOH, αOH or C=O) and in the position(s) of the double bond(s). In the Δ(5) sterols/steroid series, the concentration of these compounds required to abolish the DPPC pretransition, inversely related to their relative ability to disorder gel state DPPC bilayers, decreases markedly in the order ßOH>αOH>C=O. However, in the Δ(4,6) series, these concentrations are similar, regardless of polar head group chemical structure. Similarly, the residual enthalpy of the DPPC main phase transition at 50mol% sterol/steroid, which is inversely related to the miscibility of these compounds in the DPPC bilayer, also increases in the order ßOH>αOH>C=O, but this effect is attenuated in the Δ(4,6) series. In the two pairs of sterol epimers, the Δ(4,6) compounds exhibit a greater decrease in the temperature and enthalpy of both the pretransition and the main phase transition, whereas the opposite result is observed in the ketosteroid pair. Similarly, the ability of these compounds to order the DPPC hydrocarbon chains decreases in the order ßOH>αOH>C=O in both series of compounds, but in the two pairs of sterol epimers, hydrocarbon chain ordering is greater for the Δ(5) than the Δ(4,6) sterols, whereas the opposite is the case for the steroid pair. Thus, the characteristic effects of sterols/steroids on fluid lipid bilayers are optimal when an OH group rather than C=O group is present at C3, and when this OH group is in the equatorial orientation. We suggest that the presence of keto-enol tautomerism in the conjugated Δ(4,6) ketosteroid may provide additional H-bonding opportunities to adjacent DPPC molecules in the bilayer, which results in more cholesterol-like effects.

Docosahexaenoic acid supplementation fully restores fertility and spermatogenesis in male delta-6 desaturase-null mice.

Delta-6 desaturase-null mice ((-/-)) are unable to synthesize highly unsaturated fatty acids (HUFAs): arachidonic acid (AA), docosahexaenoic acid (DHA), and n6-docosapentaenoic acid (DPAn6). The (-/-) males exhibit infertility and arrest of spermatogenesis at late spermiogenesis. To determine which HUFA is essential for spermiogenesis, a diet supplemented with either 0.2% (w/w) AA or DHA was fed to wild-type ((+/+)) and (-/-) males at weaning until 16 weeks of age (n = 3-5). A breeding success rate of DHA-supplemented (-/-) was comparable to (+/+). DHA-fed (-/-) showed normal sperm counts and spermiogenesis. Dietary AA was less effective in restoring fertility, sperm count, and spermiogenesis than DHA. Testis fatty acid analysis showed restored DHA in DHA-fed (-/-), but DPAn6 remained depleted. In AA-fed (-/-), AA was restored at the (+/+) level, and 22:4n6, an AA elongated product, accumulated in testis. Cholesta-3,5-diene was present in testis of (+/+) and DHA-fed (-/-), whereas it diminished in (-/-) and AA-fed (-/-), suggesting impaired sterol metabolism in these groups. Expression of spermiogenesis marker genes was largely normal in all groups. In conclusion, DHA was capable of restoring all observed impairment in male reproduction, whereas 22:4n6 formed from dietary AA may act as an inferior substitute for DHA.

Cytotoxicity of 3-O-(beta-D-glucopyranosyl) etioline, a steroidal alkaloid from Solanum diphyllum L.

In continuation of our interest in phytochemical screening of the Egyptian flora for potential drugs, the reinvestigation of the methanolic extract of the roots of Solanum diphyllum, which grows naturally in the south of Egypt and is recorded as new to the Egyptian flora, afforded an interesting, highly cytotoxic compound, named 3-O-(beta-D-glucopyranosyl) etioline [(25S)-22,26-epimino-3beta-(beta-D-glucopyranosyloxy) cholesta-5,22(N)-dien-16alpha-ol]. The chemical structure of this compound was determined by comprehensive NMR studies, including DEPT, COSY, HMQC, and MS. The compound exhibited high cytotoxic effects against the cervical cancer cell line, Hela cells, with an IC50 value of 150 microg/mL.

A facile and efficient synthesis of some (6E)-hydroximino-4-en-3-one steroids, steroidal oximes from Cinachyrella spp. sponges.

Using beta-sitosterol as a starting material, (6E)-hydroximino-24-ethylcholest-4-en-3-one (1), a natural steroidal oxime from Cinachyrella alloclada and C. apion, was synthesized in four steps with a high overall yield. First, beta-sitosterol (5a) is transformed into the corresponding 24-ethylcholest-4-en-3,6-dione (6a) via oxidation with pyridinium chlorochromate (PCC). Selective reduction of 6a by NaBH(4) in the presence of CoCl(2) gives 24-ethylcholest- 4-en-3beta-ol-6-one (7a). The reaction of 7a with hydroxylamine hydrochloride offers the oxime 8a and the oxidation of 8a by Jones reagent gives the target steroid 1. (6E)-Hydroximinocholest-4-en-3-one (2) and (6E)-hydroximino-24-ethylcholest-4,22-dien-3-one (4) were synthesized by a similar method. The cytotoxicity of the synthesized compounds against sk-Hep-1 (human liver carcinoma cell line), H-292 (human lung carcinoma cell line), PC-3 (human prostate carcinoma cell line) and Hey-1B (human ovarian carcinoma cell line) cells were investigated. The presence of a cholesterol-type side chain appears to be necessary for the biological activity.

[Phytochemical investigation on Lysimachia fortunei].

OBJECTIVE: To investigate the chemical constituents in the ethyl acerate extract of Lysimachia fortunei. METHOD: The compounds were isolated by silica gel chromatography, and their structures were elucidated by NMR data and references. RESULT: Nine natural constituents were isolated, and their structures were identified as 9, 19-cyclolanost-24-en-3-one (1), 24-ethyl-5alpha-cholesta-7, 22(E)-dien-3-one (2), 1-pentatriacontanol (3), beta-stigmasterol (4), 24-ethyl-5alpha-cholesta-7, 22(E)-dien-3beta-ol (5), palmitic acid (6), isorhamnetin (7), kaempferol (8) and quercetin (9) respectively. CONCLUSION: All compounds mentioned above were isolated from this plant for the first time, and compound 1, 2 and 5 were obtained from the genus for the first time.

[Primary biliary cirrhosis].

Primary biliary cirrhosis (PBC) is a chronic cholestatic autoimmune liver disease that predominantly affects middle-aged women. It is characterized by slowly progressive destruction of the small intrahepatic bile ducts together with portal inflammation, and this initially leads to fibrosis and later to cirrhosis. It is currently accepted that the pathogenesis of PBC is multifactorial with genetic and environmental factors interplaying to determine the disease onset and progression. In addition to antimitochondrial antibody (AMA), which is the hallmark of PBC and is detected in at least 90% of the patients, other autoantibodies (antinuclear antibody, anti-smooth muscle antibody and rheumatoid factor, etc.) may also be found in the patients. There is no correlation between the titer of AMAs and the disease severity. Most patients are diagnosed either during the asymptomatic phase of PBC or after presenting with non-specific symptoms. Pruritus and fatigue are the most common symptoms of PBC. The prognosis of PBC has improved significantly during the last few decades. Patients are now diagnosed earlier in its clinical course, they are more likely to be asymptomatic at diagnosis and they are more likely to receive medical treatment. A wide variety of drugs have been assessed for the treatment of this condition: such immunosuppressive agents as corticosteroids, cyclosporine and azathioprine have a weak effect on the disease's natural history. Ursodeoxycholic acid (UDCA) is the only currently approved medical treatment. For PBC patients with end-stage liver disease or an unacceptable quality of life, liver transplantation is the only accepted therapeutic option. Early diagnosis and treatment of PBC are important because effective treatment with UDCA has been shown to delay disease progression and improve rate survival in the early stage.

Novel polyhydroxysterols from the Red Sea marine sponge Lamellodysidea herbacea.

Chemical investigation of the dichloromethane extract of the Red Sea marine sponge Lamellodysidea herbacea led to the isolation of four novel polyhydroxysteroids: cholesta-8-en-3beta,5alpha,6alpha,25-tetrol (1), cholesta-8(14)-en-3beta,5alpha,6alpha,25-tetrol (2), cholesta-8,24-dien-3beta,5alpha,6alpha-triol (3), and cholesta-8(14),24-dien-3beta,5alpha,6alpha-triol (4). Their structures were identified through 1D and 2D NMR studies. Relative stereochemistries were established by analysis of chemical shifts, coupling constants, and NOESY correlations. Compounds 3-4 showed antifungal activity against Candida tropicalis, with an inhibition diameter of 13 and 11 mm at 10 microg/disc, respectively.

[Oxysterol (3,5-cholestadien-7-one, 5 beta-cholestan-3-one, 5,24-cholestadien-3 beta-OL) induced cytotoxicity and apoptosis in gallbladder epithelial cells].

BACKGROUND/AIMS: Biliary epithelial cells are exposed to highly concentrated oxysterols. Therefore, oxysterols may play a role in pathogenesis of biliary tract diseases. We investigated the cytotoxic effect and apoptosis inducing effect of oxysterol on gallbladder epithelial cells. METHODS: We studied the cytotoxic effect of 3,5- cholestadien-7-one, 5 beta-cholestan-3-one and 5,24-cholestadien-3 beta-OL which are identified in human bile and pigment gallstones on dog gallbladder epithelial cells (DGBE) and mouse gallbladder epithelial cells (MGBE). We used model bile to dissolve oxysterols as in vitro experiment and also used MTT, cell count, Diff-Quick stain, and flow cytometry to investigate cytotoxicity and apoptosis. RESULTS: Oxysterols dissolved in model bile have cytotoxic effects in a dose dependent fashion. In oxysterol containing model bile, viable cells are 51% in 500 microM 5 beta-cholestan-3-one (cholesterol:oxysterol 50:50) and 47% in 5 mM 3,5-cholestadien-7-one (90:10) on MGBE, and are 129% and 38% in 500 microM (50:50) 3,5-cholestadien-7-one and 5 beta-cholestan-3-one on DGBE, and are 74% and 71.5% in 5 mM (90:10) 3,5-cholestadien-7-one and 5 beta-cholestan-3-one on DGBE, respectively. 500 microM (50:50) 3,5- cholestadien-7-one, 5 beta-cholestan-3-one, and 5,24-cholestadien-3 beta-OL treated on DGBE increase the apoptotic cell number as 22.0+/-8.8, 30.2+/-12.6, and 45.5+/-13.2%, respectively, compared with control (14.6+/-10.0%). 500 microM (50:50) 3,5-cholestadien-7-one, 5 beta-cholestan-3-one, and 5,24-cholestadien-3 beta-OL also affect the changes in cell cycles compared with the control. CONCLUSIONS: We concluded that oxysterol containing model bile is useful as an in vitro experiment as model to analyze the effects of oxysterols on biliary epithelial cells and that adequate concentration of oxysterols can induce the cytotoxic effect and the apoptosis on gallbladder epithelial cells.

CH(3)ReO(3)-catalyzed oxidation of cholesta-5,7-dien-3beta-yl acetate with the urea-hydrogen peroxide adduct under various conditions. Synthesis of the natural epoxy sterol 9alpha,11alpha-epoxy-5alpha-cholest-7-en-3beta,5,6beta-triol.

This article describes the oxidation of cholesta-5,7-dien-3beta-yl acetate (4) with the urea-hydrogen peroxide adduct (UHP) using methyltrioxorhenium (MTO) as catalyst, under various conditions. Specifically, the effects of using different solvents (CHCl(3) and ethers) and additives (EtOH and pyridine) on the course of the MTO-catalyzed oxidation of 4 were investigated. Some new steroids (6, 9, 10 and 11), obtained from this oxidation, were isolated and characterized on the basis of chemical evidence and interpretation of spectroscopic data including H-H COSY and HMBC experiments. The optimal solvent for the oxidation of 4 with MTO/UHP oxidizing system was diethyl ether. In this solvent the reaction is clean and gave as the main product 5,6beta-dihydroxy-5alpha-cholest-7-en-3beta-yl acetate (8, 65% yield), obtained with a more simple procedure and with a higher yield than that reported in literature. Sterol 8 is a key intermediate compound in the synthesis of many steroids of marine origin, biologically active, oxygenated at the B/C rings. In fact, starting from diol 8, we performed the synthesis of the natural cytotoxic epoxy sterol 9alpha,11alpha-epoxy-5alpha-cholest-7-en-3beta,5,6beta-triol (15, 21% yield) with an improvement in yield and number of steps over a synthesis of the same natural product previously reported. When the oxidation of 4 with the MTO/UHP system in diethyl ether was performed in the presence of pyridine as ligand, the unsaturated epoxide 5,6alpha-epoxy-5alpha-cholest-7-en-3beta-yl acetate (10, 90% yield) was obtained after only 5 min in good yield. In fact, pyridine, besides having beneficial effect on the reaction rate, shuts down the ring opening reactions, as reported in literature.

Cellular sterol accumulation stimulated by cholesterol 5 beta,6 beta-epoxide in J774 macrophages.

A significant accumulation of cellular free cholesterol and steryl esters is observed in J774 macrophages when cells are exposed to low-density lipoproteins (LDL) containing cholesterol 5 beta,6 beta-epoxide. This cellular sterol accumulation is mainly due to the formation of esterified cholesterol and desmosterol. Cellular steryl esters increased to 39.4 and 22.4 micrograms/mg cell protein with 0.8 microM of cholesterol 5 beta,6 beta-epoxide and 3,5-cholestadien-7-one, respectively, whereas hardly detectable levels were observed with the absence of oxysterols. The total cellular sterols increased 45% above the value of control with cholesterol 5 beta,6 beta-epoxide. The uptake of [3H] cholesteryl oleate-LDL was also enhanced by cholesterol 5 beta,6 beta-epoxide. The rapid displacement of desmosterol with cholesterol was observed when cells were treated with cholesterol 5 beta,6 beta-epoxide or 3,5-cholestadien-7-one in the presence of LDL. Cholesterol 5 beta,6 beta-epoxide became associated with LDL in the culture conditions, and its uptake into J774 cells and the cytotoxicity were reduced significantly by the association with LDL. The comparison of selected oxysterols for their ability to stimulate cellular sterol accumulation indicated that cholesterol 5 beta,6 beta-epoxide is the most potent. Cholesterol esterification was enhanced significantly by cholesterol 5 beta,6 beta-epoxide whereas cholesterol 5 alpha,6 alpha-epoxide and 3,5-cholestadien-7-one produced a modest response. In contrast, although cholestantriol, the metabolic hydrolysis product of cholesterol epoxides, also associated with LDL, it showed no stimulating effect on both cellular sterol content and sterol esterification. These results indicate that some oxysterols, such as cholesterol 5 beta,6 beta-epoxide and possibly 3,5-cholestadien-7-one, stimulate cellular sterol accumulation in J774 macrophages and may play an important role in atherogenesis.

Direct and carbonylative vinylation of steroidal triflates in the presence of homogeneous palladium catalysts.

The palladium-catalyzed vinylation of several steriod 2-enyl- and 3,5-dienyl-3 triflates and estrone-3-triflate was systematically examined using vinyltributylstannane as a vinylating agent. In carbon monoxide atmosphere the insertion of a CO molecule took place and unsaturated ketones were obtained. In this way new steroid derivatives containing unsaturated side chain were produced, which can serve as starting material for further functionalization of the steroid skeleton.

A sterol with an unusual side chain from Anoectochilus koshunensis.

A new sterol with a non-conventional side chain has been isolated from the whole plant of Anoectochilus koshunensis, together with four known sterols, a megastigmane glucoside and 2'-deoxyadenosine. The structure of the new sterol was elucidated as 26-methylstigmasta-5,22,25, (27)-trien-3 beta-ol based on chemical and detailed spectroscopic evidence.

Cholesterylene, a newly recognized tissue lipid, found at high levels in the cornea.

In the course of measuring the concentration of cholesterol in an opacified dog cornea by gas-chromatography, relatively large amounts of an unidentified non-saponifiable lipid were recognized. When the unknown lipid was subjected to gas chromatographic-mass spectral analysis it displayed a major ion at m/z 368 M+. and was identified as cholesta-3,5-diene, cholesterylene, by computer match with mass spectral-registry data. Cholesterylene was then shown to be present in the corneas of normal dogs, cows and humans, accounting for 20-25% of the total steroid-sterol in dog corneas and 5-10% in cow and human. Cholesterylene, which can be considered as an extremely nonpolar dehydration product of cholesterol, has not previously been recognized in animal tissues. Although the source of corneal cholesterylene is unknown, preliminary results suggest non-enzymatic formation from cholesterol.

Structures of 3 beta-tetrahydropyranyloxy-5 alpha-cholesta-20(21),24-diene and 3 beta-tetrahydropyranyloxy-21-nor-5 alpha-ergost-24-en-20-one.

3 beta-Tetrahydropyranyloxy-5 alpha-cholesta-20(21),24-diene: C32H52O2, Mr = 468.77, orthorhombic, P2(1)2(1)2(1), a = 6.710 (4), b = 11.361 (4), c = 37.812 (11) A, V = 2882 (2) A3, Z = 4, Dx = 1.08 g cm-3, lambda(Cu K alpha) = 1.54178 A, mu = 4.60 cm-1, F(000) = 1040, T = 224 K, final R = 0.088 for 1729 unique observed reflections. 3 beta-Tetrahydropyranyloxy-21-nor-5 alpha-ergost- 24-en-20-one: C32H52O3, Mr = 484.77, triclinic, P1, a = 6.640 (2), b = 9.589 (2), c = 12.202 (3) A, alpha = 111.33 (2), beta = 101.22, gamma = 90.27 (2) degrees, V = 707.4 (3) A3, Z = 1, Dx = 1.14 g cm-3, lambda(Mo K alpha) = 0.71069 A, mu = 0.8 cm-1, F(000) = 268, T = 225 K, final R = 0.058 for 2208 unique observed reflections. The configuration at C(17) of these synthetic sterol derivatives, which had been uncertain, is unambiguously established to be 'normal' (possessing a 17 alpha-H).

20-isosterols from the marine bivalve Macoma balthica.

A fraction of 20-isosterols has been isolated from the Baltic Sea bivalve Macoma balthica. The main component of this fraction, (20S)-cholest-5-en-3 beta-ol, has been characterized by 300 MHz nuclear magnetic resonance spectroscopy. 20-Isosterols in M balthica probably originate from sea-bottom sediments, an important component of a diet of the animal studied.

Quantification of high-density-lipoprotein cholesterol by precipitation with phosphotungstic acid/MgCl2.

We evaluated the use of a modified phosphotungstic acid/MgCl2 precipitation procedure for the precipitation of apolipoprotein B-containing lipoproteins. Precipitation of these lipoproteins [very-low- and low-density lipoproteins, and lipoprotein (a)] is complete, with negligible coprecipitation of high-density lipoprotein subfractions (HDL1, HDL2, HDL3), even in hypertriglyceridemic sera. In comparison with ultracentrifugation, the precipitation method yields, on the average, values that are 0.17 mmol/L lower for cholesterol values but almost identical for apolipoprotein A-I and phosphatidylcholine. Looking for delta 3,5-cholestadiene formed from cholesterol in the precipitation residue, we used "high-performance" liquid chromatography and "high-performance" thin-layer chromatography and found none.

The photobiogenesis and metabolism of vitamin D.

Provitamin D3 (7-dehydrocholesterol) is converted to previtamin D3 by the action of ultraviolet radiation on the skin. Previtamin D3 thermally isomerizes to vitamin D3 in the skin and the vitamin is then transported to the liver on the vitamin D-binding protein. Although there are extrahepatic vitamin D-25-hydroxylases, the liver is the major site for the 25-hydroxylation of vitamin D. In response to hypocalcemia and hypophosphatemia, 25-OH-D is metabolized by a renal-cytochrome. P450-dependent mixed function oxidase system is 1alpha,25(OH)2D. When calcium and phosphate homeostasis prevails the renal 25-OH-D-1alpha-hydroxylase activity is limited and instead a non-cytochrome P450 mixed function oxidase metabolizes 25-OH-D to 24R,25(OH)2D. Parathyroid hormone has clearly been shown to be a trophin for the renal synthesis of 1,25(OH)2D; however, the role and significance of the adrenal steroids, or gonadal and pituitary hormones, on the renal 25-OH-D-1alpha-hydroxylase is not well defined. The regulation of the photometabolism of provitamin D3 to vitamin D3, the role and significance of the side-chain metabolism of 1,25(OH)2D by the small intestine, and the metabolism of 25-OH-D to 24R,25(OH)2D by chondrocytes and its stimulation of protein synthesis in these cells are just a few issues that will require further investigation.