Two previously undescribed cholestanol saponins from the rhizomes of Paris fargesii var. petiolata.

Two previously undescribed cholestanol saponins, parpetiosides F - G (1-2), and six known analogs (3-8) were isolated from the rhizomes of Paris fargesii var. petiolata. Their structures were elucidated by extensive spectroscopic data analysis and chemical methods. Compound 1 was a rare 6/6/6/5/5 fused-rings cholestanol saponin with disaccharide moiety linked at C-26 of aglycone which was hardly seen in genus Paris. All of these compounds were discovered in this plant for the first time. In addition, the cytotoxicities of saponins (1-8) against three human cancer cell lines (U87, HepG2 and SGC-7901) were evaluated by CCK-8 method, and saponins 5-8 displayed certain cytotoxicities. The strong interactions between saponins 5-8 and SCUBE3, an oncogene for glioma cells, were displayed by molecular docking.

Effect of cholestanol and cholesterol-loaded cyclodextrin on stallion sperm function and capacitation post-cryopreservation.

Cryopreservation of stallion semen is less efficient than other species such as bovine. This is mainly because of the greater susceptibility of stallion sperm to the freezing damage that generates oxidative stress and plasma membrane injury, resulting in DNA fragmentation and cell death. These data suggest the need to develop new strategies of sperm cryopreservation that can improve the efficiency of this technique in stallions by reducing or preventing membrane damage and cell death. The present study aimed to evaluate the effect of adding membrane stabilizers to the freezing medium and assess the quality and in vitro capacitation of stallion sperm after thawing. Semen samples from three stallions frozen with membrane stabilizers (cholesterol-loaded cyclodextrin and cholestanol-loaded cyclodextrin) were evaluated in two experiments: i) sperm quality and functional analysis after thawing, and ii) sperm quality and functional analysis after 4 h of post-thaw incubation in capacitating conditions. Plasma membrane integrity, mitochondrial membrane potential, membrane lipid disorder, intracellular Ca2+, tyrosine phosphorylation, acrosome reaction, DNA damage, sperm motility, and binding to the zona pellucida were assessed. The results showed that cholesterol-loaded cyclodextrin was the stabilizer that most efficiently reduced the membrane disruption and post-thaw cell damage. In addition, this stabilizer made it possible to obtain in vitro capacitated sperm showing higher plasma membrane integrity, mitochondrial membrane potential, sperm motility, binding to the zona pellucida and better response to in vitro capacitating conditions.

A novel synergistic anticancer effect of fungal cholestanol glucoside and paclitaxel: Apoptosis induced by an intrinsic pathway through ROS generation in cervical cancer cell line (HeLa).

In the search for efficient therapeutics with economically viable for cancer treatment, combination therapy has developed as a keystone in the pursuit of novel approaches for drug discovery. In this regard, we confirmed the presence of cholestanol glucoside (CG) in Lasiodiplodia theobromae culture filtrate and its production was estimated to be 20.01 mg/l. The purified fungal CG was obtained with a molecular mass of 550.18 m/z. The combination of CG and paclitaxel (PTX) was found to have potent cytotoxicity against HeLa cells. We revealed that the synergistic effect of CG and PTX induced apoptosis through the formation of nuclear fragments, DNA fragmentation and sub G1 cell cycle arrest. Further, it was proven that apoptosis took place by loss of the mitochondrial membrane potential (MMP) through reactive oxygen species (ROS) production and caspase 3/7 activity. Moreover, the data suggests that the synergistic effect of CG and PTX played a role in a mitochondrial intrinsic pathway through the apoptotic gene expression of Bax, caspase-9 and caspase-3. In addition, the down-regulation of Bcl-2 strongly described the induced apoptosis through an intrinsic pathway using the Western blot analysis. The conclusion of this study is that a combination of CG and PTX has synergistic apoptotic effects in HeLa cells, which provides a possible therapeutic strategy for cancer therapy in the future.

A lipid-anchored neurokinin 1 receptor antagonist prolongs pain relief by a three-pronged mechanism of action targeting the receptor at the plasma membrane and in endosomes.

G-protein-coupled receptors (GPCRs) are traditionally known for signaling at the plasma membrane, but they can also signal from endosomes after internalization to control important pathophysiological processes. In spinal neurons, sustained endosomal signaling of the neurokinin 1 receptor (NK1R) mediates nociception, as demonstrated in models of acute and neuropathic pain. An NK1R antagonist, Spantide I (Span), conjugated to cholestanol (Span-Chol), accumulates in endosomes, inhibits endosomal NK1R signaling, and causes prolonged antinociception. However, the extent to which the Chol-anchor influences long-term location and activity is poorly understood. Herein, we used fluorescent correlation spectroscopy and targeted biosensors to characterize Span-Chol over time. The Chol-anchor increased local concentration of probe at the plasma membrane. Over time we observed an increase in NK1R-binding affinity and more potent inhibition of NK1R-mediated calcium signaling. Span-Chol, but not Span, caused a persistent decrease in NK1R recruitment of ß-arrestin and receptor internalization to early endosomes. Using targeted biosensors, we mapped the relative inhibition of NK1R signaling as the receptor moved into the cell. Span selectively inhibited cell surface signaling, whereas Span-Chol partitioned into endosomal membranes and blocked endosomal signaling. In a preclinical model of pain, Span-Chol caused prolonged antinociception (>9 h), which is attributable to a three-pronged mechanism of action: increased local concentration at membranes, a prolonged decrease in NK1R endocytosis, and persistent inhibition of signaling from endosomes. Identifying the mechanisms that contribute to the increased preclinical efficacy of lipid-anchored NK1R antagonists is an important step toward understanding how we can effectively target intracellular GPCRs in disease.

Isolation, Purification and Characterization of a Novel Steroidal Saponin Cholestanol Glucoside from Lasiodiplodia theobromae that Induces Apoptosis in A549 Cells.

Search for novel anticancer lead molecules continues to be a major focus of cancer research due to the limitations of existing drugs such as lack of tumor selectivity, narrow therapeutic index and multidrug resistance of cancer types. Natural molecules often possess better pharmacokinetic traits compared to synthetic molecules as they continually evolve by natural selection process to interact with biological macromolecules. Microbial metabolites constitute nearly half of the pharmaceuticals in market today. Endophytic fungi, owing to its rich chemical diversity, are viewed as attractive sources of novel bioactive compounds. In the present study, we report the purification and characterization of a novel steroidal saponin, cholestanol glucoside (CG) from Saraca asoca endophytic fungus Lasiodiplodia theobromae. The compound was assessed for its cytotoxic potentialities in six human cancer cell lines, A549, PC3, HepG2, U251, MCF7 and OVCAR3. CG exhibited significant cytotoxicities towards A549, PC3 and HepG2 among which A549 cells were most vulnerable to CG treatment. However, CG treatment exhibited negligible cytotoxicity in non malignant human lung fibroblast cell line (WI-38). Induction of cell death by CG treatment in A549 cells was further investigated. CG induced the generation of reactive oxygen species (ROS) and mitochondrial membrane permeability loss followed by apoptotic cell death. Mitochondrial membrane depolarization and apoptotic cell death in CG treated A549 cells were completely blocked in presence of an antioxidant, N-acetyl cysteine (NAC). Hence it could be concluded that CG initiates apoptosis in cancer cells by augmenting the basal oxidative stress and that the generation of intracellular ROS is crucial for the induction of apoptosis.

Competitive Solubilization of Cholesterol/Cholesteryl Oleate and Seven Species of Sterol/Stanol in Model Intestinal Solution System.

The addition of plant sterols/stanols (sterols or stanols) can reduce the solubilization of cholesterol in a model intestinal solution system. We studied the molecular structure of seven different sterols/stanols and the effect they had on the solubilization of cholesterol or cholesterol ester in a model intestinal solution. The differences in the molecular structures of the sterol/stanol species influenced their abilities to reduce the solubility of cholesterol in the competitive solubilization experiments. Cholestanol whose molecular structure resembled cholesterol was the most effective at reducing the solubilization of cholesterol and cholesterol ester, with the solubilities of cholesterol and cholesteryl oleate being 41% and 39% respectively of the values observed for the single solubilizate systems. ß-Sitosterol was also able to reduce the solubilities of cholesterol and cholesteryl oleate to 43% and 45% of those observed in a single solubilizate system. Both, stigmasterol and brassicasterol have an unsaturated double bond in a steroid side chain and did not exhibit major cholesterol-lowering effects. These results were reflected by the Gibbs free energy change values (ΔG(0)) for solubilization, where the sterol/stanol species with cholesterol-lowering effects had similar or larger negative ΔG(0) values than those observed for cholesterol.

Cholestanol-loaded-cyclodextrin improves the quality of stallion spermatozoa after cryopreservation.

This study was to compare the effect of adding cholesterol or cholestanol loaded cyclodextrins in stallion sperm prior to cryopreservation to optimize sperm cryosurvival. Ejaculates from each of eight stallions were diluted to 120 million cells in a S-MEDIUM diluent. The diluted sperm were sub-divided into three treatments: no additive (control); 0.75mg of cyclodextrin pre-loaded with cholesterol (CLC)/120 million sperm (positive control); 1.5mg CLC/120 million sperm; 0.75mg of cyclodextrin pre-loaded with cholestanol (CnLC)/120 million sperm; and 1.5mg CnLC/120 million sperm. To set the experiments, the treated sperm were incubated for 15min at 22°C to allow for the incorporation of cholesterol or cholestanol. In each experiment, treated sperm incubated for 15min at 22°C to allow for incorporation of cholesterol or cholestanol. The samples were then diluted 1:5 (v/v) with Lactose-Egg Yolk diluent and cooled to 5°C over a 2h period. Loaded into 0.25ml polyvinylchloride straws, frozen in liquid nitrogen vapor for 10min, and then plunged into liquid nitrogen until further use. Higher percentages of motile sperm and viable cells were achieved after thawing for stallion sperm treated with CLC and CnLC compared to control (P<0.05). Addition of CnLC also resulted in more number sperm binding to chicken egg perivitelline membrane (CEPM) after cryopreservation than cholestanol and control sperm (P<0.05). In conclusion, CnLC and CLC improved the percentage of post-thaw motility of equine sperm and CnLC provided greater binding efficiency.

Synthesis, spectroscopy, theoretical and biological studies of new gramine-steroids salts and conjugates.

New gramine connections with bile acids (lithocholic, deoxycholic, cholic) and sterols (cholesterol, cholestanol) were synthesized. The structures of products were confirmed by spectral (NMR, FT-IR) analysis, mass spectrometry (ESI-MS) as well as PM5 semiempirical methods. Unexpectedly, the products of the reaction of gramine with cholesterol and cholestanol were symmetrical compounds consisting of two molecules of sterols connected by N(CH3)2 group. All new synthesized compounds interact in vitro with the human erythrocyte membrane and alter discoid erythrocyte shape inducing stomatocytosis or echinocytosis. Increase in the incorporation of the fluorescent dye merocyanine 540 (MC540) into the erythrocyte membrane indicates that new compounds at sublytic concentrations are capable of disturbing membrane phospholipids asymmetry and loosening the molecular packing of phospholipids in the bilayer. Gramine significantly decreases the membrane partitioning properties as well as haemolytic activity of lithocholic acid in its new salt. Moreover, both deoxycholic and cholic acids completely lost their membrane perturbing activities in the gramine salts. On the other hand, the capacity of new gramine-sterols connections to alter the erythrocyte membrane structure and its permeability is much higher in comparison with sterols alone. The dual effect of gramine on the bile acid and sterols cell membrane partitioning activity observed in our study should not be neglected in vivo.

Phallusiasterols A and B: two new sulfated sterols from the Mediterranean tunicate Phallusia fumigata and their effects as modulators of the PXR receptor.

Purification of the apolar extracts of the marine ascidian Phallusia fumigata, afforded two new sulfated sterols, phallusiasterols A (1) and B (2). The structures of the new compounds have been elucidated using mass spectrometry and NMR experiments. The effects of phallusiasterols A and B as modulators of pregnane-X-receptor (PXR) have been investigated. These studies revealed that phallusiasterol A induces PXR transactivation in HepG2 cells and stimulates the expression of the PXR target genes CYP3A4 and MDR1 in the same cell line. Molecular docking calculations suggested the theoretical binding mode of phallusiasterol A with hPXR and revealed that phallusiasterol A fitted well in the LBD of PXR.

Ultrastructural alterations induced by Δ(24(25))-sterol methyltransferase inhibitors on Trichomonas vaginalis.

Trichomonas vaginalis is an important human parasite that causes trichomoniasis, a cosmopolitan sexually transmitted disease. Currently, the treatment of choice for T. vaginalis infections is metronidazole. The increase in metronidazole-resistant parasites and undesirable side effects of this drug make the search for alternative chemotherapeutic approaches a priority for the management of trichomoniasis. Here, the antiproliferative and ultrastructural effects of sterol biosynthesis inhibitors against T. vaginalis were investigated. It was found that 22,26-azasterol (5 µM) and 24(R,S),25-epiminolanosterol (10 µM), known inhibitors of Δ(24(25))-sterol methyltransferase, exhibited antiproliferative effects on T. vaginalis trophozoites cultured in vitro. Morphological analyses showed that azasterols induced changes in the ultrastructure of T. vaginalis. The most significant alterations were (1) membrane blebbing and disruption, (2) wrinkled cells and (3) the formation of cell clusters. In addition, autophagic vacuoles, Golgi duplication arrest, an abnormal Golgi enlargement and damaged hydrogenosomes were also observed. Nonspecific cytotoxicity assays using the cultured mammalian cell lines Madin-Darby canine kidney cells showed no effect of the azasterols on the viability and proliferation of these cells at a concentration that significantly inhibited the proliferation of T. vaginalis, indicating a selective antiparasitic action. Taken together, these results suggest that azasterols could be important compounds in the development of novel chemotherapeutic approaches against T. vaginalis.

Micron-scale phase segregation in lipid monolayers induced by myelin basic protein in the presence of a cholesterol analog.

It was previously shown that myelin basic protein (MBP) can induce phase segregation in whole myelin monolayers and myelin lipid films, which leads to the accumulation of proteins into a separate phase, segregated from a cholesterol-enriched lipid phase. In this work we investigated some factors regulating the phase segregation induced by MBP using fluorescent microscopy of monolayers formed with binary and ternary lipid mixtures of dihydrocholesterol (a less-oxidable cholesterol analog) and phospholipids. The influence of the addition of salts to the subphase and of varying the lipid composition was analyzed. Our results show that MBP can induce a dihydrocholesterol-dependent segregation of phases that can be further regulated by the electrolyte concentration in the subphase and the composition (type and proportion) of non-sterol lipids. In this way, changes of the lipid composition of the film or the ionic strength in the aqueous media modify the local surface density of MBP and the properties (phase state and composition) of the protein environment.

Delta24(25)-sterol methenyltransferase: intracellular localization and azasterol sensitivity in Leishmania major promastigotes overexpressing the enzyme.

Trypanosomatids contain predominantly ergostane-based sterols, which differ from cholesterol, the main sterol in mammalian cells, in the presence of a methyl group in the 24 position. The methylation is initiated by S-adenosyl-L-methionine:Delta(24 (25))-sterol methenyltransferase, an enzyme present in protozoa, but absent in mammals. The importance of this enzyme is underscored by its potential as a drug target in the treatment of the leishmaniases. Here, we report studies concerning the intracellular distribution of sterol methenyltransferase in Leishmania major promastigotes and overexpressing cells using a specific antibody raised against highly purified recombinant protein. It was found by immunofluorescence and electron microscopy studies that in L. major wild-type cells sterol methenyltransferase was primarily associated to the endoplasmic reticulum. In addition to this location, the protein was incorporated into translucent vesicles presumably of the endocytic pathway. We also found in this study that cells overproducing the enzyme do not have increased resistance to the sterol methenyltransferase inhibitor 22, 26 azasterol.

On the mechanism of cerebral accumulation of cholestanol in patients with cerebrotendinous xanthomatosis.

The most serious consequence of sterol 27-hydroxylase deficiency in humans [cerebrotendinous xanthomatosis (CTX)] is the development of cholestanol-containing brain xanthomas. The cholestanol in the brain may be derived from the circulation or from 7alpha-hydroxylated intermediates in bile acid synthesis, present at 50- to 250-fold increased levels in plasma. Here, we demonstrate a transfer of 7alpha-hydroxy-4-cholesten-3-one across cultured porcine brain endothelial cells (a model for the blood-brain barrier) that is approximately 100-fold more efficient than the transfer of cholestanol. Furthermore, there was an efficient conversion of 7alpha-hydroxy-4-cholesten-3-one to cholestanol in cultured neuronal and glial cells as well as in monocyte-derived macrophages of human origin. It is concluded that the continuous intracellular production of cholestanol from a bile acid precursor capable of rapidly passing biomembranes, including the blood-brain barrier, is likely to be of major importance for the accumulation of cholestanol in patients with CTX. Such a mechanism also fits well with the observation that treatment with chenodeoxycholic acid, which normalizes the level of the bile acid precursor, results in a reduction of cholestanol-containing xanthomas even in the brain.

Effects of pharmaceutical products and municipal wastewaters on temperature-dependent mitochondrial electron transport activity in Elliptio complanata mussels.

The advent of global warming has given rise to the issue on how temperature impacts the susceptibility of ectothermic organisms to pollution. The purpose of this study was to examine the effects of pharmaceutical products and domestic wastewaters on temperature-dependent mitochondrial electron transport activity in the freshwater mussel Elliptio complanata. Mitochondria from mussels were freshly prepared and exposed to increasing concentrations of various pharmaceutical products known to be found in municipal effluents for 30 min at 4, 12 and 20 degrees C. Electron transport activity as well as lipid peroxidation and DNA strand breaks were determined in the mitochondria. Next, mussels were placed in the aeration lagoons of two municipal wastewater treatment plants for 30 days. Mitochondrial electron transport (MET), temperature-dependent MET (MET(T)) and lipid peroxidation in gonad were then determined. The results show that all products were able to increase MET activity, but at two different ranges of threshold concentration. Certain pharmaceutical products (i.e., ibuprofen, cotinine, fluoxetine, coprostanol and trimethoprim) increased MET(T) at a lower threshold concentration than observed at 20 degrees C. Products of lesser potency in reducing lipid peroxidation were those that produced more DNA strand breaks in mitochondria. Both MET and MET(T) were significantly increased in mussels exposed to aeration lagoon effluents. Lipid peroxidation was also increased in the gonad and was significantly correlated with MET and MET(T) activities. The data indicate that pharmaceutical products and municipal effluents increase respiration rates in isolated mitochondria, such that interaction with temperature could enhance the susceptibility of mitochondrial energy production and oxidative stress in environments contaminated by domestic wastewater.

Effects of sterol biosynthesis inhibitors on endosymbiont-bearing trypanosomatids.

Some protozoa of the Trypanosomatidae family have a close relationship with an endosymbiotic bacterium. As the prokaryote envelope has a controversial origin, a sterol 24-methyltransferase inhibitor (20-piperidin-2-yl-5alpha-pregnan-3beta,20-diol; 22,26-azasterol) was used as a tool to investigate lipid biosynthetic pathways in Crithidia deanei, an endosymbiont-bearing trypanosomatid. Apart from antiproliferative effects, this drug induced ultrastructural alterations, consisting of myelin-like figures in the cytoplasm and endosymbiont envelope vesiculation. Concurrently, a dramatic reduction of 24-alkyl sterols was observed after 22,26-azasterol treatment, both in whole cell homogenates, as well as in isolated mitochondria. These effects were associated with changes of phospholipid composition, in particular a reduction of the phosphatidylcholine content and a concomitant increase in phosphatidylethanolamine levels. Lipid analyses of purified endosymbionts indicated a complete absence of sterols, and their phospholipid composition was different from that of mitochondria or whole protozoa, being similar to eubacteria closely associated with eukaryotes.

Antiproliferative synergism of azasterols and antifolates against Toxoplasma gondii.

The antiproliferative effects of two azasterols, 22,26-azasterol (20-piperidin-2-yl-5alpha-pregnan-3beta-20(R,S)-diol, AZA) and 24,25(R,S)-epiminolanosterol (EIL), in combination with sulphadiazine (SDZ) and pyrimethamine (PYR) were studied against Toxoplasma gondii tachyzoites growing in cultured mammalian cells. We had previously shown that AZA and EIL, two known inhibitors of Delta24(25)sterol methyl transferase in fungi and protozoa, have a potent and selective anti-T. gondii activity, although no 24-alkyl sterols have been detected in this parasite. We now report that when these sterol analogues were used in combination with the conventional SDZ/PYR treatment, potent synergistic effects were observed, ranging from 10- to 100-fold reductions of the IC50 values in the presence of sub-optimal doses of azasterols. When exposed to these drug combinations, intracellular T. gondii parasites displayed diverse subcellular alterations, including mitochondrial swelling, the arrest of the endodiogeny process with fragmented nuclei and subsequent cell lysis. These results suggest a potential new approach for the treatment of toxoplasmosis, which could significantly lower the required levels of antifolates and thus their adverse side effects.

Selective anti-Toxoplasma gondii activities of azasterols.

We report potent and selective inhibitory effects of 22,26-azasterol and 24,25-(R,S)-epiminolanosterol, known inhibitors of Delta24(25)-sterol methyltranferase (SMT) in fungi and protozoa, on the proliferation of Toxoplasma gondii in LLCMK2 cells. These compounds produced a dose-dependent reduction in parasite proliferation. 22,26-azasterol had an IC50 of 5.3 microM after 24 h and 4.5 microM after 48 h, while for 24,25-(R,S)-epiminolanosterol the IC50 values were 1 microM after 24 h and 0.12 microM after 48 h. The rapid reduction of parasite load suggested these compounds have selective cytotoxic effects against T. gondii. However, we were unable to detect 24-alkyl sterols in purified T. gondii tachyzoites using highly sensitive gas-liquid chromatography/mass spectrometry methods, a fact which indicated that the anti-proliferative effects of these azasterols were not mediated by inhibition of SMT. Transmission electron microscopy showed that the mitochondrion was the major target of drugs. Ultrastructural effects on plasma membrane, apicoplast and the formation of autophagosomal structures were also observed.

Presence of phytosterol oxides in crude vegetable oils and their fate during refining.

The content of phytosterol oxidation products was determined in samples of crude vegetable oils: peanut, sunflower, maize, palm nut, and lampante olive oils that were intended for refining and not for direct consumption. The 7 alpha- and 7 beta-hydroxy derivatives of beta-sitosterol, stigmasterol, and campesterol and the 7-keto-beta-sitosterol were the principal phytosterol oxides found in almost all of the oils analyzed. In some oils, the epoxy and dihydroxy derivatives of beta-sitosterol were also found at very low levels. The highest total concentrations of phytosterol oxides, ranging from 4.5 to 67.5 and from 4.1 to 60.1 ppm, were found in sunflower and maize oils, respectively. Lower concentrations were present in the peanut oils, 2.7-9.6 ppm, and in the palm nut oil, 5.5 ppm, whereas in the lampante olive oils, only three samples of the six analyzed contained a low concentration (1.5-2.5 ppm) of oxyphytosterols. No detectable levels of phytosterol oxides were found in the samples of palm and coconut oils. Bleaching experiments were carried out on a sample of sunflower oil at 80 degrees C for 1 h with 1 and 2% of both acidic and neutral earths. The bleaching caused a reduction of the hydroxyphytosterol with partial formation of steroidal hydrocarbons with three double bonds in the ring system at the 2-, 4-, and 6-positions (steratrienes). The same sunflower oil was deodorized at 180 degrees C under vacuum for 1 h, and no dehydration products were formed with a complete recovery of the hydroxyphytosterols. A bleaching test with acidic earths was carried out also with an extra virgin olive oil fortified with 7-keto-cholesterol, dihydroxycholesterol, and alpha-epoxy-cholesterol. There was no formation of steratrienes from these compounds, but dihydroxycholesterol underwent considerable decomposition and alpha-epoxycholesterol underwent ring opening with formation of the dihydroxy derivative, whereas 7-ketocholesterol was rather stable

Structural features of sterols required to inhibit human sperm capacitation.

Ejaculated mammalian sperm must undergo a final maturation (capacitation) before they can acrosome-react and fertilize eggs. Loss of cholesterol is an essential step in the capacitation of human sperm. Experimentally maintaining a high level of cholesterol inhibits capacitation, but the mechanism is unknown. The present study investigated the structural features that are required for cholesterol's inhibitory activity. Human sperm also contain much desmosterol, which is lost from sperm during capacitation. Preventing the loss of desmosterol inhibited capacitation (as assessed by acrosomal responsiveness), with an effectiveness approximately equal to cholesterol's inhibitory activity. Other structural analogs were added to the incubation medium to replace sperm cholesterol and desmosterol. Most inhibited capacitation, including those that lacked cholesterol's 3beta-OH group (cholesteryl methyl ether and epicholesterol) and those with modified C17 groups (ergosterol and diosgenin). Two steroids did not inhibit capacitation well. Coprostanol, which has a nonplanar steroid nucleus, had low inhibitory activity that could be explained by an elevated endogenous cholesterol concentration. Epicoprostanol, which has a nonplanar ring structure and a 3alpha-OH group, promoted rather than inhibited capacitation. The inhibitory activity of the analogs was correlated with their ability to promote order of egg phosphatidylcholine as measured by fluorescence anisotropy. In summary, a planar ring structure is required for sterol inhibitory activity, but a 3beta-OH group and a saturated cholesterol-like aliphatic tail on C17 are not required. The present results support the hypothesis that sperm sterols block capacitation by increasing order of phospholipids.

Synthesis and biochemical properties of a new photoactivatable cholesterol analog 7,7-azocholestanol and its linoleate ester in Chinese hamster ovary cell lines.

We report the chemical synthesis of a new photoactivatable cholesterol analog 7,7-azocholestanol (AC) and its linoleate ester (ACL). We also examined the biochemical properties of the sterol and its ester by employing several different mutant Chinese hamster ovary (CHO) cell lines with defined abnormalities in cholesterol metabolism as tools. AC mimics cholesterol in supporting the growth of a mutant cell line (M19) that requires cholesterol for growth. In normal cells, tritiated ACL present in low-density lipoprotein (LDL) was hydrolyzed and reesterified in a manner similar to tritiated cholesteryl linoleate (CL) in LDL. Also, in the mutant cell line (AC29) lacking the enzyme acyl-coenzyme A:cholesterol acyltransferase or in the mutant cell line (CT60) defective in the Niemann-Pick type C1 protein, the hydrolysis of ACL in LDL was normal, but the reesterification of the liberated AC was defective. Therefore, the metabolism of ACL in LDL is very similar to that of CL in LDL. Tritium-labeled AC delivered to intact CHO cells as a cyclodextrin complex was shown to photoaffinity label several discrete polypeptides, including caveolin-1. These results demonstrate AC as an effective reagent for studying cholesterol-protein interactions involved in intracellular cholesterol trafficking.