Cholestane-3ß,5α,6ß-Triol Inhibits Acid-Sensing Ion Channels and Reduces Acidosis-Mediated Ischemic Brain Injury.

BACKGROUND: Activation of the acid-sensing ion channels (ASICs) by tissue acidosis, a common feature of brain ischemia, contributes to ischemic brain injury, while blockade of ASICs results in protection. Cholestane-3ß,5α,6ß-triol (Triol), a major cholesterol metabolite, has been demonstrated as an endogenous neuroprotectant; however, the mechanism underlying its neuroprotective activity remains elusive. In this study, we tested the hypothesis that inhibition of ASICs is a potential mechanism. METHODS: The whole-cell patch-clamp technique was used to examine the effect of Triol on ASICs heterogeneously expressed in Chinese hamster ovary cells and ASICs endogenously expressed in primary cultured mouse cortical neurons. Acid-induced injury of cultured mouse cortical neurons and middle cerebral artery occlusion-induced ischemic brain injury in wild-type and ASIC1 and ASIC2 knockout mice were studied to examine the protective effect of Triol. RESULTS: Triol inhibits ASICs in a subunit-dependent manner. In Chinese hamster ovary cells, it inhibits homomeric ASIC1a and ASIC3 without affecting ASIC1ß and ASIC2a. In cultured mouse cortical neurons, it inhibits homomeric ASIC1a and heteromeric ASIC1a-containing channels. The inhibition is use-dependent but voltage- and pH-independent. Structure-activity relationship analysis suggests that hydroxyls at the 5 and 6 positions of the A/B ring are critical functional groups. Triol alleviates acidosis-mediated injury of cultured mouse cortical neurons and protects against middle cerebral artery occlusion-induced brain injury in an ASIC1a-dependent manner. CONCLUSIONS: Our study identifies Triol as a novel ASIC inhibitor, which may serve as a new pharmacological tool for studying ASICs and may also be developed as a potential drug for treating stroke.

Effects of brassinosteroids on cancer cells: A review.

Brassinolide is a new type of steroidal hormone with strong activities, which is well known as an efficient and low-toxicity plant growth regulator for a long time. Because steroidal hormones have a wide application prospect, brassinosteroids have been gradually explored in pharmacology and animal cells in recent decades. Brassinolide could effectively reverse the resistance of human T lymphoblastoid cell line CCRF-VCR 1000 by inhibiting the effusion of drug transported by P-glycoprotein. Brassinosteroids could also accelerate wound healing by positively eliminating inflammation and stimulating reepithelialization of the reparation stage. The occurrence of cancer is a multistep process mediated by a variety of factors. Until now, cancer has always been one group of the major diseases that threaten human health. Many studies have found that brassinosteroids have attracted a great deal of potential as an anticancer agent in the treatment of cancer cells, and most of them exert anticancer activity by inducing apoptosis in cancer cells. There are few articles on the relationship between brassinosteroids and cancer so far. Accordingly, in this article, we summarized current research about the brassinosteroids and cancers. Through the review, researchers could know more about brassinosteroids which might become a new tool for the treatment of cancer in the future, and not only a plant hormone.

Marine Bile Natural Products as Agonists of the TGR5 Receptor.

Agonism of the G protein-coupled bile acid receptor "Takeda G-protein receptor 5" (TGR5) aids in attenuating cholesterol accumulation due to atherosclerotic progression. Although mammalian bile compounds can activate TGR5, they are generally weak agonists, and more effective compounds need to be identified. In this study, two marine bile compounds (5ß-scymnol and its sulfate) were compared with mammalian bile compounds deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA) using an in vitro model of TGR5 agonism. The response profiles of human embryonic kidney 293 cells (HEK293) transfected to overexpress TGR5 (HEK293-TGR5) and incubated with subcytotoxic concentrations of test compounds were compared to nontransfected HEK293 control cells using the specific calcium-binding fluorophore Fura-2AM to measure intracellular calcium [Ca2+]i release. Scymnol and scymnol sulfate caused a sustained increase in [Ca2+]i within TGR5 cells only, which was abolished by a specific inhibitor for Gαq protein (UBO-QIC). Sustained increases in [Ca2+]i were seen in both cell types with DCA exposure; this was unaffected by UBO-QIC, indicating that TGR5 activation was not involved. Exposure to UDCA did not alter [Ca2+]i, suggesting a lack of TGR5 bioactivity. These findings demonstrated that both scymnol and scymnol sulfate are novel agonists of TGR5 receptors, showing therapeutic potential for treating atherosclerosis.

Agonism for the bile acid receptor GPBAR1 reverses liver and vascular damage in a mouse model of steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is associated with an increased risk of developing cardiovascular complications and mortality, suggesting that treatment of NASH might benefit from combined approaches that target the liver and the cardiovascular components of NASH. Using genetic and pharmacologic approaches, we show that G protein-coupled bile acid-activated receptor 1 (GPBAR1) agonism reverses liver and vascular damage in mouse models of NASH. NASH is associated with accelerated vascular inflammation representing an independent risk factor for development of cardiovascular diseases and cardiovascular-related mortality. GPBAR1, also known as TGR5, is a G protein-coupled receptor for secondary bile acids that reduces inflammation and promotes energy expenditure. Using genetic and pharmacologic approaches, we investigated whether GPBAR1 agonism by 6ß-ethyl-3α,7ß-dihydroxy-5ß-cholan-24-ol (BAR501) reverses liver and vascular damage induced by exposure to a diet enriched in fat and fructose (HFD-F). Treating HFD-F mice with BAR501 reversed liver injury and promoted the browning of white adipose tissue in a Gpbar1-dependent manner. Feeding HFD-F resulted in vascular damage, as shown by the increased aorta intima-media thickness and increased expression of inflammatory genes (IL-6,TNF-α, iNOS, and F4/80) and adhesion molecules (VCAM, intercellular adhesion molecule-1, and endothelial selectin) in the aorta, while reducing the expression of genes involved in NO and hydrogen sulfide generation, severely altering vasomotor activities of aortic rings in an ex vivo assay. BAR501 reversed this pattern in a Gpbar1-dependent manner, highlighting a potential role for GPBAR1 agonism in treating the liver and vascular component of NASH.-Carino, A., Marchianò, S., Biagioli, M., Bucci, M., Vellecco, V., Brancaleone, V., Fiorucci, C., Zampella, A., Monti, M. C., Distrutti, E., Fiorucci, S. Agonism for the bile acid receptor GPBAR1 reverses liver and vascular damage in a mouse model of steatohepatitis.

A natural product inhibits the initiation of α-synuclein aggregation and suppresses its toxicity.

The self-assembly of α-synuclein is closely associated with Parkinson's disease and related syndromes. We show that squalamine, a natural product with known anticancer and antiviral activity, dramatically affects α-synuclein aggregation in vitro and in vivo. We elucidate the mechanism of action of squalamine by investigating its interaction with lipid vesicles, which are known to stimulate nucleation, and find that this compound displaces α-synuclein from the surfaces of such vesicles, thereby blocking the first steps in its aggregation process. We also show that squalamine almost completely suppresses the toxicity of α-synuclein oligomers in human neuroblastoma cells by inhibiting their interactions with lipid membranes. We further examine the effects of squalamine in a Caenorhabditis elegans strain overexpressing α-synuclein, observing a dramatic reduction of α-synuclein aggregation and an almost complete elimination of muscle paralysis. These findings suggest that squalamine could be a means of therapeutic intervention in Parkinson's disease and related conditions.

Endosomal signaling of the receptor for calcitonin gene-related peptide mediates pain transmission.

G protein-coupled receptors (GPCRs) are considered to function primarily at the plasma membrane, where they interact with extracellular ligands and couple to G proteins that transmit intracellular signals. Consequently, therapeutic drugs are designed to target GPCRs at the plasma membrane. Activated GPCRs undergo clathrin-dependent endocytosis. Whether GPCRs in endosomes control pathophysiological processes in vivo and are therapeutic targets remains uncertain. We investigated the contribution of endosomal signaling of the calcitonin receptor-like receptor (CLR) to pain transmission. Calcitonin gene-related peptide (CGRP) stimulated CLR endocytosis and activated protein kinase C (PKC) in the cytosol and extracellular signal regulated kinase (ERK) in the cytosol and nucleus. Inhibitors of clathrin and dynamin prevented CLR endocytosis and activation of cytosolic PKC and nuclear ERK, which derive from endosomal CLR. A cholestanol-conjugated antagonist, CGRP8-37, accumulated in CLR-containing endosomes and selectively inhibited CLR signaling in endosomes. CGRP caused sustained excitation of neurons in slices of rat spinal cord. Inhibitors of dynamin, ERK, and PKC suppressed persistent neuronal excitation. CGRP8-37-cholestanol, but not unconjugated CGRP8-37, prevented sustained neuronal excitation. When injected intrathecally to mice, CGRP8-37-cholestanol inhibited nociceptive responses to intraplantar injection of capsaicin, formalin, or complete Freund's adjuvant more effectively than unconjugated CGRP8-37 Our results show that CLR signals from endosomes to control pain transmission and identify CLR in endosomes as a therapeutic target for pain. Thus, GPCRs function not only at the plasma membrane but also in endosomes to control complex processes in vivo. Endosomal GPCRs are a drug target that deserve further attention.

7-Keto-Cholesterol and Cholestan-3beta, 5alpha, 6beta-Triol Induce Eryptosis through Distinct Pathways Leading to NADPH Oxidase and Nitric Oxide Synthase Activation.

BACKGROUND/AIMS: We showed that patho-physiological concentrations of either 7-keto-cholesterol (7-KC), or cholestane-3beta, 5alpha, 6beta-triol (TRIOL) caused the eryptotic death of human red blood cells (RBC), strictly dependent on the early production of reactive oxygen species (ROS). The goal of the current study was to assess the contribution of the erythrocyte ROS-generating enzymes, NADPH oxidase (RBC-NOX), nitric oxide synthase (RBC-NOS) and xanthine oxido-reductase (XOR) to the oxysterol-dependent eryptosis and pertinent activation pathways. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, reactive oxygen/nitrogen species (RONS) and nitric oxide formation from 2',7'-dichloro-dihydrofluorescein (DCF-DA) and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) -dependent fluorescence, respectively; Akt1, phospho-NOS3 Ser1177, and PKCζ from Western blot analysis. The activity of individual 7-KC (7 µM) and TRIOL (2, µM) on ROS-generating enzymes and relevant activation pathways was assayed in the presence of Diphenylene iodonium chloride (DPI), N-nitro-L-arginine methyl ester (L-NAME), allopurinol, NSC23766 and LY294002, inhibitors in this order of RBC-NOX, RBC-NOS, XOR and upstream regulatory proteins Rac GTPase and phosphoinositide3 Kinase (PI3K); hemoglobin oxidation from spectrophotometric analysis. RESULTS: RBC-NOX was the target of 7-KC, through a signaling including Rac GTPase and PKCζ, whereas TRIOL caused activation of RBC-NOS according to the pathway PI3K/Akt, with the concurrent activity of a Rac-GTPase. In concomitance with the TRIOL-induced .NO production, formation of methemoglobin with global loss of heme were observed, ascribable to nitrosative stress. XOR, activated after modification of the redox environment by either RBC-NOX or RBC-NOS activity, concurred to the overall oxidative/nitrosative stress by either oxysterols. When 7-KC and TRIOL were combined, they acted independently and their effect on ROS/RONS production and PS exposure appeared the result of the effects of the oxysterols on RBC-NOX and RBC-NOS. CONCLUSION: Eryptosis of human RBCs may be caused by either 7-KC or TRIOL by oxidative/nitrosative stress through distinct signaling cascades activating RBC-NOX and RBC-NOS, respectively, with the complementary activity of XOR; when combined, the oxysterols act independently and both concur to the final eryptotic effect.

The Bile Acid Receptor GPBAR1 Regulates the M1/M2 Phenotype of Intestinal Macrophages and Activation of GPBAR1 Rescues Mice from Murine Colitis.

GPBAR1 (TGR5 or M-BAR) is a G protein-coupled receptor for secondary bile acids that is highly expressed in monocytes/macrophages. In this study, we aimed to determine the role of GPBAR1 in mediating leukocyte trafficking in chemically induced models of colitis and investigate the therapeutic potential of BAR501, a small molecule agonist for GPBAR1. These studies demonstrated that GPBAR1 gene ablation enhanced the recruitment of classically activated macrophages in the colonic lamina propria and worsened the severity of inflammation. In contrast, GPBAR1 activation by BAR501 reversed intestinal inflammation in the trinitrobenzenesulfonic acid and oxazolone models by reducing the trafficking of Ly6C+ monocytes from blood to intestinal mucosa. Exposure to BAR501 shifted intestinal macrophages from a classically activated (CD11b+, CCR7+, F4/80-) to an alternatively activated (CD11b+, CCR7-, F4/80+) phenotype, reduced the expression of inflammatory genes (TNF-α, IFN-γ, IL-1ß, IL-6, and CCL2 mRNAs), and attenuated the wasting syndrome and severity of colitis (≈70% reduction in the Colitis Disease Activity Index). The protective effect was lost in Gpbar1-/- mice. Exposure to BAR501 increased the colonic expression of IL-10 and TGF-ß mRNAs and the percentage of CD4+/Foxp3+ cells. The beneficial effects of BAR501 were lost in Il-10-/- mice. In a macrophage cell line, regulation of IL-10 by BAR501 was GPBAR1 dependent and was mediated by the recruitment of CREB to its responsive element in the IL-10 promoter. In conclusion, GPBAR1 is expressed in circulating monocytes and colonic macrophages, and its activation promotes a IL-10-dependent shift toward an alternatively activated phenotype. The targeting of GPBAR1 may offer therapeutic options in inflammatory bowel diseases.

Castasterone attenuates insecticide induced phytotoxicity in mustard.

In the current investigation, we studied role of castasterone (CS), (a bioactive brassinosteroid) in Brassica juncea grown under imidacloprid (IMI) stress. We observed that CS-seed treatment resulted in the recovery of seedling growth under IMI toxicity. Seed treatment with CS, significantly enhanced the contents of pigments like chlorophylls, carotenoids, anthocyanins and xanthophylls under stress. Oxidative stress generated by the production of reactive oxygen species (ROS) like hydrogen peroxide and superoxide anion, was reduced after CS treatment under IMI toxicity. Antioxidative defense system got activated after CS-seed treatment, resulting in the increased activities of enzymes. Moreover, CS-seed treatment under IMI stress also stimulated the biosynthesis of organic acids of Krebs cycle (citrate, succinate, fumarate and malate) and phenolics. We also noticed that CS is also involved in the regulation of the gene expression of some key enzymes involved in pigment metabolism (CHLASE, PSY, CHS), carbon fixation (RUBISCO), Krebs cycle (CS, SUCLG1, SDH, FH), ROS generation (RBO), antioxidative enzymes (SOD, CAT, POD, DHAR, GR, GST), phenolic biosynthesis (PAL) and pesticide detoxification system (CXE, P450, NADH). This modulated gene expression after CS-treatment activated the insecticide detoxification, leading to the reduction of IMI residues. Data analysis using multivariate statistical technique i.e. multiple linear regression, also supported the fact that CS can efficiently reduce IMI induced phytotoxicity in B. juncea.

Potent Antitrypanosomal Activities of 3-Aminosteroids against African Trypanosomes: Investigation of Cellular Effects and of Cross-Resistance with Existing Drugs.

Treatment of animal African trypanosomiasis (AAT) requires urgent need for safe, potent and affordable drugs and this has necessitated this study. We investigated the trypanocidal activities and mode of action of selected 3-aminosteroids against Trypanosoma brucei brucei. The in vitro activity of selected compounds of this series against T. congolense (Savannah-type, IL3000), T. b. brucei (bloodstream trypomastigote, Lister strain 427 wild-type (427WT)) and various multi-drug resistant cell lines was assessed using a resazurin-based cell viability assay. Studies on mode of antitrypanosomal activity of some selected 3-aminosteroids against Tbb 427WT were also carried out. The tested compounds mostly showed moderate-to-low in vitro activities and low selectivity to mammalian cells. Interestingly, a certain aminosteroid, holarrhetine (10, IC50 = 0.045 ± 0.03 µM), was 2 times more potent against T. congolense than the standard veterinary drug, diminazene aceturate, and 10 times more potent than the control trypanocide, pentamidine, and displayed an excellent in vitro selectivity index of 2130 over L6 myoblasts. All multi-drug resistant strains of T. b. brucei tested were not significantly cross-resistant with the purified compounds. The growth pattern of Tbb 427WT on long and limited exposure time revealed gradual but irrecoverable growth arrest at ≥ IC50 concentrations of 3-aminosteroids. Trypanocidal action was not associated with membrane permeabilization of trypanosome cells but instead with mitochondrial membrane depolarization, reduced adenosine triphosphate (ATP) levels and G2/M cell cycle arrest which appear to be the result of mitochondrial accumulation of the aminosteroids. These findings provided insights for further development of this new and promising class of trypanocide against African trypanosomes.

Castasterone confers copper stress tolerance by regulating antioxidant enzyme responses, antioxidants, and amino acid balance in B. juncea seedlings.

The aim of the present study was to explore the effect of exogenous application of castasterone (CS) on physiologic and biochemical responses in Brassica juncea seedlings under copper (Cu) stress. Seeds were pre-soaked in different concentrations of CS and grown for 7 days under various levels of Cu. The exposure of B. juncea to higher levels of Cu led to decrease of morphologic parameters, with partial recovery of length and fresh weight in the CS pre-treated seedlings. Metal content was high in both roots and shoots under Cu exposure while the CS pre-treatment reduced the metal uptake. Accumulation of hydrogen peroxide (H2O2) and superoxide anion radical (O2-) were chosen as stress biomarker and higher levels of H2O2 (88.89%) and O2- (62.11%) showed the oxidative stress in metal treated B. juncea seedlings, however, CS pre-treatment reduced ROS accumulation in Cu-exposed seedlings. The Cu exposures lead to enhance the plant's enzymatic and non-enzymatic antioxidant system. It was observed that enzymatic activities of ascorbate peroxidase (APOX), dehydroascorbate reductase (DHAR), and glutathione reductase (GR), glutathione perxoidase (GPOX) and gultrathione-s-transferase increased while activity of monodehydroascorbate reductase (MDHAR) decreased under Cu stress. The pre-treatment with CS positively affected the activities of enzymes. RT-PCR analysis showed that mRNA transcript levels were correlated with total enzymatic activity of DHAR, GR, GST and GSH. Increase in the gene expression of DHAR (1.85 folds), GR (3.24 folds), GST-1 (2.00 folds) and GSH-S (3.18 folds) was noticed with CS pre-treatment. Overall, the present study shows that Cu exposure induced severe oxidative stress in B. juncea plants and exogenous application of CS improved antioxidative defense system by modulating the ascorbate-glutathione cycle and amino acid metabolism.

Brassinolide-like activity of castasterone analogs with varied side chains against rice lamina inclination.

Brassinolide (BL) and castasterone (CS) are the representative members of brassinosteroid class of plant steroid hormone having plant growth promoting activity. In this study, eleven CS analogs bearing a variety of side chains were synthesized to determine the effect of the side chain structures on the BL-like activity. The plant hormonal activity was evaluated in a dwarf rice lamina inclination assay, and the potency was determined as the reciprocal logarithm of the 50% effective dose (ED50) from each dose-response curve. The reciprocal logarithm of ED50 (pED50) was decreased dramatically upon deletion of the C-28 methyl group of CS. The introduction of oxygen-containing groups such as hydroxy, methoxy, and ethoxycarbonyl was also unfavorable to the activity. The pED50 was influenced by the geometry of carbon-carbon double bond between C-24 and C-25 (cis and trans), but the introduction of a fluorine atom at the C-25 position of the double bond did not significantly change the activity. The binding free energy (ΔG) was calculated for all ligand-receptor binding interactions using molecular dynamics, resulting that ΔG is linearly correlated with the pED50.

Castasterone and citric acid treatment restores photosynthetic attributes in Brassica juncea L. under Cd(II) toxicity.

Cadmium(II) toxicity is a serious environmental issue warranting effective measures for its mitigation. In the present study, ameliorative effects of a bioactive brassinosteroid, castasterone (CS) and low molecular weight organic acid, citric acid (CA) against the Cd(II) toxicity to Brassica juncea L. were evaluated. Seeds of B. juncea treated with CS (0, 0.01, 1 and 100nM) were sown in cadmium spiked soils (0 and 0.6mmolkg-1 soil). CA (0.6mmolkg-1soil) was added to soil one week after sowing seeds. Plants were harvested 30 days after sowing. Phytotoxicity induced by Cd(II) was evident from stunted growth of the plants, malondialdehyde accumulation, reduction in chlorophyll and carotenoid contents, and leaf gas exchange parameters. Cd(II) toxicity was effectively alleviated by seed soaking with CS (100nM) and/ or soil amendment with CA (0.6mMkg-1 soil). Relative gene expression of genes encoding for some of the key enzymes of pigment metabolism were also analysed. Expression of chlorophyllase (CHLASE) was reduced, while that of phytoene synthase (PSY), and chalcone synthase (CHS) genes were enhanced with CS and/or CA treatments with respect to plants treated with Cd(II) only. Cd also affected the activities of antioxidative enzymes. Plants responded to Cd(II) by accumulation of total sugars. CS (100nM) and CA treatments further enhanced the activities of these parameters and induced the contents of secondary plant pigments (flavonoids and anthocyanins) and proline. The results imply that seed treatment with CS and soil application with CA can effectively alleviate Cd(II) induced toxicity in B. juncea by strengthening its antioxidative defence system and enhancing compatible solute accumulation.

When cholesterol meets histamine, it gives rise to dendrogenin A: a tumour suppressor metabolite.

Dendrogenin A (DDA) is the first steroidal alkaloid (SA) to be identified in human tissues to date and arises from the stereoselective enzymatic conjugation of 5,6α-epoxycholesterol (5,6α-EC) with histamine (HA). DDA induces the re-differentiation of cancer cellsin vitroandin vivoand prevents breast cancer (BC) and melanoma development in mice, evidencing its protective role against oncogenesis. In addition, DDA production is lower in BCs compared with normal tissues, suggesting a deregulation of its biosynthesis during carcinogenesis. The discovery of DDA reveals the existence of a new metabolic pathway in mammals which lies at the crossroads of cholesterol and HA metabolism and which leads to the production of this metabolic tumour suppressor.

Elucidation of the hepatoprotective moiety of 5ß-scymnol that suppresses paracetamol toxicity in mice.

The shark bile alcohol, 5ß-scymnol, protects mice from the hepatotoxic effects of paracetamol (APAP) overdose. To elucidate the hepatoprotective structural moiety of scymnol, we compared its effect with that of its analogue and natural bile salt, sodium scymnol sulfate, in a clinically relevant model of APAP-induced toxicity. Exposure of healthy male Swiss mice to a toxic overdose of APAP (350 mg/kg, ip) significantly increased serum hepatocellular enzyme activities, decreased hepatocellular glutathione (GSH) levels, and induced severe centrilobular hepatocellular necrosis. Repeated low-dose scymnol (5 mg/kg/day for 7 days, ip) significantly reduced the extent of APAP-induced hepatotoxicity without preventing GSH depletion. Sodium scymnol sulfate, which lacks the tri-hydroxyl-substituted aliphatic side chain of scymnol, failed to reduce the APAP hepatotoxicity or prevent GSH depletion when tested under the same experimental conditions. We conclude that the tri-hydroxyl-substituted aliphatic side chain is the hepatoprotective structural moiety of 5ß-scymnol that suppresses APAP-induced cytotoxicity in mice.

A new bioactive steroidal ketone from the South China Sea sponge Xestospongia testudinaria.

A new steroidal ketone (1), with an ergosta-22,25-diene side chain, was obtained from the South China Sea marine sponge Xestospongia testudinaria. The structure of 1 was determined on the basis of detailed spectroscopic analysis and by comparison with literature. Compound 1 exhibited significant inhibitory activity against protein tyrosine phosphatase 1B (PTP1B), a key target for the treatment of type II diabetes and obesity, with an IC50 value of 4.27 ± 0.55 µM, which is comparable with the positive control oleanolic acid (IC50 = 2.63 ± 0.22 µM).

Side-chain oxysterols modulate cholesterol accessibility through membrane remodeling.

Side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), are key regulators of cholesterol homeostasis. New evidence suggests that the alteration of membrane structure by 25-HC contributes to its regulatory effects. We have examined the role of oxysterol membrane effects on cholesterol accessibility within the membrane using perfringolysin O (PFO), a cholesterol-dependent cytolysin that selectively binds accessible cholesterol, as a sensor of membrane cholesterol accessibility. We show that 25-HC increases cholesterol accessibility in a manner dependent on the membrane lipid composition. Structural analysis of molecular dynamics simulations reveals that increased cholesterol accessibility is associated with membrane thinning, and that the effects of 25-HC on cholesterol accessibility are driven by these changes in membrane thickness. Further, we find that the 25-HC antagonist LY295427 (agisterol) abrogates the membrane effects of 25-HC in a nonenantioselective manner, suggesting that agisterol antagonizes the cholesterol-homeostatic effects of 25-HC indirectly through its membrane interactions. These studies demonstrate that oxysterols regulate cholesterol accessibility, and thus the availability of cholesterol to be sensed and transported throughout the cell, by modulating the membrane environment. This work provides new insights into how alterations in membrane structure can be used to relay cholesterol regulatory signals.

The novel steroidal alkaloids dendrogenin A and B promote proliferation of adult neural stem cells.

Dendrogenin A (DDA) and dendrogenin B (DDB) are new aminoalkyl oxysterols which display re-differentiation of tumor cells of neuronal origin at nanomolar concentrations. We analyzed the influence of dendrogenins on adult mice neural stem cell proliferation, sphere formation and differentiation. DDA and DDB were found to have potent proliferative effects in neural stem cells. Additionally, they induce neuronal outgrowth from neurospheres during in vitro cultivation. Taken together, our results demonstrate a novel role for dendrogenins A and B in neural stem cell proliferation and differentiation which further increases their likely importance to compensate for neuronal cell loss in the brain.

Squalamine as a broad-spectrum systemic antiviral agent with therapeutic potential.

Antiviral compounds that increase the resistance of host tissues represent an attractive class of therapeutic. Here, we show that squalamine, a compound previously isolated from the tissues of the dogfish shark (Squalus acanthias) and the sea lamprey (Petromyzon marinus), exhibits broad-spectrum antiviral activity against human pathogens, which were studied in vitro as well as in vivo. Both RNA- and DNA-enveloped viruses are shown to be susceptible. The proposed mechanism involves the capacity of squalamine, a cationic amphipathic sterol, to neutralize the negative electrostatic surface charge of intracellular membranes in a way that renders the cell less effective in supporting viral replication. Because squalamine can be readily synthesized and has a known safety profile in man, we believe its potential as a broad-spectrum human antiviral agent should be explored.

[The role of reactive oxygen species and calcium ions in the implementation of the stress-protective effect of brassinosteroids on plant cells].

The effect of the brassinosteroids (BS) 24-epibrassiniolide and 24-epicastasterone on the thermoresistance of wheat coleoptile (Triticum aestivum L.) and their generation of the superoxide anion radical and antioxidant enzymes activity were investigated. The treatment of coleoptiles with 10 nM solutions of BS caused a transient increase in O2·- generation and a subsequent increase in the activity of superoxide dismutase and catalase and an improvement in heat resistance. Pretreatment of coleoptiles with the NADPH oxidase inhibitor imidazole leveled the increase in production of the superoxide anion radical and prevented an increase in the activity of antioxidant enzymes and the development of cell thermostability. The investigated effects of BS were also depressed by the pretreatment of coleoptile segments with extracellular calcium chelator EGTA and inhibitor of ADP-ribosyl cyclase nicotinamide. A conclusion was made about the participation of calcium ions and reactive oxygen species generated by the action of NADPH oxidase in the implementation of the stress-protective effect of the BS in the cells of wheat coleoptile.