IPS-1 plays an essential role in dsRNA-induced stress granule formation by interacting with PKR and promoting its activation.

The formation of cytoplasmic stress granules and the innate immune response are two distinct cellular stress responses. Our study investigated the involvement of four innate immune proteins - retinoic-acid-inducible gene I (RIG-I, also known as DDX58), melanoma differentiation-associated gene 5 (MDA5, also known as IFIH1), IFN-ß promoter stimulator (IPS-1, also known as MAVS) and protein kinase regulated by dsRNA (PKR, also known as EIF2AK2) in the formation of stress granules. Knockdown of IPS-1 or PKR significantly decreased the formation of stress granules induced by double-stranded (ds)RNA. IPS-1 depletion markedly attenuated the phosphorylation of PKR and eIF2α that was triggered by dsRNA, and IPS-1 facilitated the in vitro autophosphorylation of PKR. In IPS-1-depleted cells, the dsRNA-mediated dimerization of PKR through its dsRNA-binding domains was significantly abrogated, suggesting that IPS-1 might be involved in PKR dimerization. By co-immunoprecipitation and pulldown assays, our data demonstrate that IPS-1 directly binds to PKR through the IPS-1 caspase activation and recruitment domain (CARD), suggesting that the effect of IPS-1 on the formation of stress granules might be exerted through interacting with PKR and mediating its activation. PKR was recruited into stress granules upon activation, whereas the majority of IPS-1 protein formed clusters on mitochondrial membranes. Our work provides the first evidence that the innate signaling molecule IPS-1 plays an essential role in stress granule formation.

Cytotoxic and PPARs transcriptional activities of sterols from the Vietnamese soft coral Lobophytum laevigatum.

A new unusual sterol, named lobophytosterol (1), and five known metabolites (2-6) were isolated from the methanol extract of the soft coral Lobophytum laevigatum. Their chemical structures were elucidated by extensive spectroscopic analysis and comparison with those reported in the literature. The absolute stereochemistry of 1 was determined using a modified Mosher's method. Compounds 1-3 showed cytotoxic activity against HCT-116 cells with IC(50) values of 3.2, 6.9 and 18.1 µM, respectively. Compound 1 additionally displayed cytotoxic effects on A549 and HL-60 cells with IC(50) values of 4.5 and 5.6 µM, respectively. Treatment of these cells with compound 1 resulted in an induction of apoptosis evident by chromatin condensation in treated cells. Besides, compounds 2, 4, and 6 significantly upregulated PPARs transcriptional activity dose-dependently in Hep-G2 cells. Taken together, these data suggest that compound 1 might inhibit the growth of the cancer cells by the induction of apoptosis, and compounds 2, 4, and 6 might act as specific agonists for PPARα, PPARδ, and PPARγ and may therefore regulate cellular glucose, lipid, and cholesterol metabolism.

Screening Reveals Sterol Derivatives with Pro-Differentiation, Pro-Survival, or Potent Cytotoxic Effects on Oligodendrocyte Progenitor Cells.

Inducing the formation of new oligodendrocytes from oligodendrocyte progenitor cells (OPCs) represents a potential approach to repairing the loss of myelin observed in multiple sclerosis and other diseases. Recently, we demonstrated that accumulation of specific cholesterol precursors, 8,9-unsaturated sterols, is a dominant mechanism by which dozens of small molecules enhance oligodendrocyte formation. Here, we evaluated a library of 56 sterols and steroids to evaluate whether other classes of bioactive sterol derivatives may also influence mouse oligodendrocyte precursor cell (OPC) differentiation or survival. From this library, we identified U-73343 as a potent enhancer of oligodendrocyte formation that induces 8,9-unsaturated sterol accumulation by inhibition of the cholesterol biosynthesis enzyme sterol 14-reductase. In contrast, we found that mouse OPCs are remarkably vulnerable to treatment with the glycosterol OSW-1, an oxysterol-binding protein (OSBP) modulator that induces Golgi stress and OPC death in the low picomolar range. A subsequent small-molecule suppressor screen identified mTOR signaling as a key effector pathway mediating OSW-1's cytotoxic effects in mouse OPCs. Finally, evaluation of a panel of ER and Golgi stress-inducing small molecules revealed that mouse OPCs are highly sensitive to these perturbations, more so than closely related neural progenitor cells. Together, these studies highlight the wide-ranging influence of sterols and steroids on OPC cell fate, with 8,9-unsaturated sterols positively enhancing differentiation to oligodendrocytes and OSW-1 able to induce lethal Golgi stress with remarkable potency.

Detection of estrogenic activity from kraft mill effluents by the yeast estrogen screen.

Estrogenic activity of kraft pulp mill effluents (P. radiata, E. globulus and mixed -50% E. globulus and 50% P. radiata) was evaluated by the yeast estrogen screen assay. The estrogenic activity values were relatively low, ranking between 1.475 and 0.383 ng/L of EE2 eq. (Estrogenic equivalent of 17 alpha-ethynylestradiol), where the highest value corresponds to the E. globulus effluent and the lowest value to the P. radiata effluent. Analysis by solid phase extraction (SPE) and gas chromatography-mass spectrometry (GC-MS) of chemical compounds present in all three effluents detected at least five major groups of organic compounds, corresponding to fatty acids, hydrocarbons, phenols, sterols and triterpenes. Comparison of analytical and biological data suggests that sterols could be the cause of the estrogenic activity in the evaluated effluent.

Synthesis and cytotoxicities of icogenin analogues with disaccharide residues.

For further structure-activity relationships (SAR) research of furostan saponin, two icogenin analogues: (25R)-22-O-methyl-furost-5-en-3beta,26-diol-3-O-alpha-l-rhamnopyranosyl-(1-->2)-beta-d-glucopyranoside 1 and (25R)-22-O-methyl-furost-5-en-3beta,26-diol-3-O-alpha-l-rhamnopyranosyl-(1-->2)-alpha-d-glucopyranoside 2, with valuable disaccharide moieties, were synthesized from diosgenin through eight steps. Both of the analogues behaved the similar cytotoxic activities with icogenin, towards nine types of human tumor cells herein.

Candida albicans gains azole resistance by altering sphingolipid composition.

Fungal infections by drug-resistant Candida albicans pose a global public health threat. However, the pathogen's diploid genome greatly hinders genome-wide investigations of resistance mechanisms. Here, we develop an efficient piggyBac transposon-mediated mutagenesis system using stable haploid C. albicans to conduct genome-wide genetic screens. We find that null mutants in either gene FEN1 or FEN12 (encoding enzymes for the synthesis of very-long-chain fatty acids as precursors of sphingolipids) exhibit resistance to fluconazole, a first-line antifungal drug. Mass-spectrometry analyses demonstrate changes in cellular sphingolipid composition in both mutants, including substantially increased levels of several mannosylinositolphosphoceramides with shorter fatty-acid chains. Treatment with fluconazole induces similar changes in wild-type cells, suggesting a natural response mechanism. Furthermore, the resistance relies on a robust upregulation of sphingolipid biosynthesis genes. Our results shed light into the mechanisms underlying azole resistance, and the new transposon-mediated mutagenesis system should facilitate future genome-wide studies of C. albicans.

New steroidal alkaloid and furostanol glycosides isolated from Solanum lyratum with cytotoxicity.

Two previously undescribed steroidal compounds, 16, 23-epoxy-22, 26-epimino-cholest-22(N), 23, 25(26)-trien-3ß-ol-3-O-ß-D-glucopyranosyl-(1→2)-ß-D-glucopyranosyl-(1→4)-ß-D-galactopyranoside (1) and 26-O-ß-D-glucopyranosyl-(25R)-5α-furost-20(22)-en-3ß, 26-diol (2), together with 7 known ones including 26-O-ß-D-glucopyranosyl-(25R)-5, 20(22)-dien-furost-3ß, 26-diol (3), (25R)-5-en-spirost-3ß-ol-O-ß-D-glucopyranosyl-(1→4)-[α-L-rhmanopyranosyl-(1→2)]-ß-D-galactopyranoside (4), funkioside D (5), aspidistrin (6), tigogenin-3-O-ß-D-lucotrioside (7), desglucolanatigonin II (8), and degalactotigonin (9), were isolated from Solanum lyratum Thunb. Their cytotoxic activities were tested in two cancer cell lines by MTT method. One of the steroidal glycosides (6) showed significant cytotoxic activity against gastric cancer SGC7901 and liver cancer BEL-7402 cells.

Preclinical Safety Assessment of Furostanol Glycoside-Based Standardized Fenugreek Seed Extract in Laboratory Rats.

The present work is aimed at studying acute oral toxicity (AOT), subchronic oral toxicity, mutagenicity, and genotoxicity of furostanol glycosides-based standardized fenugreek seed extract (Fenu-FG) using the Organization for Economic Co-operation and Development (OECD) guidelines. The AOT and subchronic (90-day repeated dose) toxicity studies were performed on Wistar rats as per OECD 423 and OECD 408 guidelines, respectively. The mutagenicity (reverse mutation assay, Ames test) and genotoxicity (mammalian chromosome aberration test) were assessed in vitro using OECD 471 and OECD 473 guidelines, respectively. At an acute oral limit dose of 2,000 mg/kg, Fenu-FG did not show any mortality or treatment-related adverse signs. Ninety days of subchronic oral administration of Fenu-FG (250, 500, or 1,000 mg/kg) in rats did not induce any treatment-related significant changes with respect to body weight, hematology, blood biochemistry, urinalysis, gross pathology, or histopathology. The no-observed-adverse-effect-level of Fenu-FG was 1,000 mg/kg/day. Furthermore, Fenu-FG did not demonstrate mutagenic potential up to a concentration of 5,000 µg/plate (Ames test) and did not induce structural chromosome aberrations up to 2,000 µg/ml (in human lymphocyte cells in vitro). In conclusion, Fenu-FG was found safe during preclinical safety assessments.

Autoinhibitory sterol sulfates mediate programmed cell death in a bloom-forming marine diatom.

Cell mortality is a key mechanism that shapes phytoplankton blooms and species dynamics in aquatic environments. Here we show that sterol sulfates (StS) are regulatory molecules of a cell death program in Skeletonema marinoi, a marine diatom-blooming species in temperate coastal waters. The molecules trigger an oxidative burst and production of nitric oxide in a dose-dependent manner. The intracellular level of StS increases with cell ageing and ultimately leads to a mechanism of apoptosis-like death. Disrupting StS biosynthesis by inhibition of the sulfonation step significantly delays the onset of this fatal process and maintains steady growth in algal cells for several days. The autoinhibitory activity of StS demonstrates the functional significance of small metabolites in diatoms. The StS pathway provides another view on cell regulation during bloom dynamics in marine habitats and opens new opportunities for the biochemical control of mass-cultivation of microalgae.

Dissolution of resin acids, retene and wood sterols from contaminated lake sediments.

The dissolution potency of hydrophobic resin acids (RAs), retene and wood sterols from sediments was studied. These wood extractives and their metabolites are sorbed from pulp and paper mill effluents to downstream sediments. With harmful components like these, sediments can pose a hazard to the aquatic environment. Therefore, sediment elutriates with water were produced under variable conditions (agitation rate and efficiency, time), and concentrations of the dissoluted compounds were analyzed. Both naturally contaminated field sediments and artificially spiked sediments were studied. By vigorous agitation RAs can be released fast from the sediment matrix and equilibrium reached within 3 days. Compared to RAs, desorption of retene from lake sediment was slower and did not completely reach equilibrium in 23 days. Sterols spiked to pristine sediment with a 33-day contact time desorbed faster than those associated authentically with industrial sediment of from a contaminated lake. Simulating the water turbulence adjacent to a sediment surface by low and high rate of agitation in the laboratory, an increase in the mixing rate after 43-day elutriation suddenly released a high amount of wood sterols. The results indicate wide variation between hazardous chemicals in their tendency to dissolution from sediment solids. Erosion and hydrology adjacent to the sediment surface, as well as risks from dredging activities of sediments, may expose lake biota to bioactive chemicals.

Asterosaponins from the starfish Culcita novaeguineae and their bioactivities.

Bioassay-guided fractionation of the n-BuOH extract of the starfish Culcita novaeguineae resulted in the isolation of one new sulfated steroidal glycoside (asterosaponin) (1), along with three known asterosaponins, thornasteroside A (2), marthasteroside A(1) (3) and regularoside A (4), as active compounds causing morphological abnormality of Pyricularia oryzae mycelia. Their structures were elucidated by extensive spectral studies and chemical evidences. All the saponins showed moderate cytotoxicity against cancer cell lines K-562 and BEL-7402.

Cytotoxic sterols from marine-derived fungus Pennicillium sp.

A new sterol, ergosta-8(14), 22-diene-3,5,6,7-tetraol(3beta, 5alpha, 6beta, 7alpha, 22E) (1), together with four known sterols ergosta-8(9), 22-diene-3,5,6,7-tetraol (3beta, 5alpha, 6beta, 7alpha, 22E) (2), 5alpha,8alpha-epidioxy-24(S)-methylcholesta-6,22-diene-3beta-ol (3), 5alpha,8alpha-epidioxy-24(S)-methylcholesta-6,9(11), 22-triene-3beta-ol (4), 3beta,5alpha,9alpha-trihydroxyergosta-7,22-diene-6-one (5) was isolated from marine fungus Pennicillium sp. Their structures were determined based on chemical analysis and spectral methods (IR, 1D and 2D NMR, HR-FAB-MS). Compounds 1-5 were evaluated for cytotoxicity against human liver cancer cell (Hep G), and most of them exhibited potent activity. Compound 1 display the highest potency with IC50 values 10.4 microg mL-1.

Halichondria sulfonic acid, a new HIV-1 inhibitory guanidino-sulfonic acid, and halistanol sulfate isolated from the marine sponge Halichondria rugosa Ridley & Dendy.

A new sulfur-containing guanidino derivative, halichondria sulfonic acid (1) showing anti-HIV-1 activity, and halistanol trisulfate (2) with anti-tumor activity have been isolated from the marine sponge Halichondria rugosa Ridley & Dendy collected in the Chinese Southern Sea. The structure of 1 was elucidated by analysis of spectroscopic and crystal data.

Ultrahigh-Performance Liquid Chromatography-High-Resolution Quadrupole Time-of-Flight Mass Spectrometry Based Metabolomics Reveals Key Differences between Brachiaria decumbens and B. brizantha, Two Similar Pastures with Different Toxicities.

Several species of Brachiaria (Poaceae) currently cover extensive grazing areas in Brazil, providing valuable source of feed for a large cattle population. However, numerous cases of toxicity outbreaks in livestock have raised concerns on safety of using these plants, especially B. decumbens. In this study, chemometric analysis of ultrahigh-performance liquid chromatography-high-resolution quadrupole time-of-flight mass spectrometry (UHPLC-HR-QTOF-MS) data has for the first time uncovered qualitative and quantitative differences between metabolomes of toxic B. decumbens and nontoxic B. brizantha. The steroidal saponin protoneodioscin was established as the main biomarker for B. decumbens when compared to B. brizantha, and therefore the key explanation for their phytochemical differentiation. Quantification of protodioscin in both plants showed no significant differences; consequently, the idea that this compound is solely responsible for toxicity outbreaks must be discarded. Instead, we propose that the added occurrence of its stereoisomer, protoneodioscin, in B. decumbens, can be considered as the probable cause of these events. Interestingly, the greatest concentrations of saponins for both species were reached during winter (B. decumbens = 53.6 ± 5.1 mg·g(-1) dry weight (D.W.); B. brizantha = 25.0 ± 1.9 mg·g(-1) D.W.) and spring (B. decumbens = 49.4 ± 5.0 mg·g(-1) D.W.; B. brizantha = 27.9 ± 1.4 mg·g(-1) D.W.), although in the case of B. decumbens these values do not vary significantly among seasons.

A retrospective evaluation of species-specific sensitivity for neurological signs in toxicological studies: Is the dog more sensitive than the non-human primate?

Selection of the appropriate non-rodent species in preclinical programs is crucial for good translatability and human safety. There is no data available in the literature which provides exact comparison of dog and non-human primate (NHP) sensitivity regarding neurological signs in toxicological studies. We performed a retrospective analysis of 174 toxicity studies with 15 neuroscience substances. Neurological signs in dogs and NHPs were evaluated in correlation to exposure data. Overall incidence of substance induced convulsions was similar in both species and no gender differences were observed. The reported liability of beagles to spontaneous convulsions was not confirmed in our studies. The symptom tremor showed the best inter-species translatability. The current toxicological study design does not include exposure assessment at the time-point of neurological signs, therefore, we propose to include additional toxicokinetic samples. Our analysis revealed factors including housing, handling, and behavior, which prevents direct species comparison. In addition only one non-rodent species is routinely tested in development programs, therefore data for both species is rare. We however, had sufficient data which enabled comparison for one compound. In the spirit of 3Rs further examples should be evaluated.

Cytotoxic 5α,8α-epidioxy sterols from the marine sponge Monanchora sp.

Three new sterols, 5α,8α-epidioxy-24-norcholesta-6,9(11),22-trien-3ß-ol (1), 5α,8α-epidioxy-cholesta-6,9(11),24-trien-3ß-ol (2), and 5α,8α-epidioxy-cholesta-6,23-dien-3ß,25-diol (3), with four known sterols (4-7) were isolated from a marine sponge Monanchora sp. Their chemical structures were elucidated by extensive spectroscopic analysis. Compounds 1 and 3-7 showed moderate cytotoxicity against several human carcinoma cell lines including renal (A-498), pancreatic (PANC-1 and MIA PaCa-2), and colorectal (HCT 116) cancer cell lines.

An excess concentration of oxysterols in the plasma is cytotoxic to cultured endothelial cells.

To test if there is an excess concentration of oxysterols in the plasma of the patients with cardiovascular disease, we analyzed the oxysterol content in the plasma from 105 cardiac catheterized patients with angina and 80+/-8% stenosis in their coronary arteries. The result showed that the plasma contained a significantly higher concentration of oxysterols than did plasma from 105 age- and sex-matched, non-catheterized and angina-free controls (P<0.05). We used endothelial cells (ECs) cultured in medium containing either [3H]thymidine, [3H]mevalonolactone or 45Ca(2+) to determine how the plasma from the patients influences cell growth and function. We found that less [3H]thymidine (P<0.05), less [3H]mevalonolactone (P<0.05) and more 45Ca(2+) (P<0.001) was incorporated into ECs cultured in the plasma from 36 patients with 83+/-4% stenosis than from the 36 controls. When synthetic 7beta-hydroxycholesterol, cholesterol 5beta,6beta-epoxide, cholesterol 5alpha,6alpha-epoxide and 7-ketocholesterol were added to the plasma from the controls, the influx of 45Ca(2+) into ECs then equaled that in the plasma of patients. The enhanced incorporation of 45Ca(2+) into the ECs cultured in the plasma both from the patients and from controls with added synthetic oxysterols substantiates in vitro the hypothesis that oxysterols increase the influx of calcium into cells. These data indicated that an excess of oxysterols in the plasma of the patients was cytotoxic to the cultured cells.

Structure and cytotoxicity of new polyhydroxylated sterols from the Caribbean gorgonian Plexaurella grisea.

The gorgonian Plexaurella grisea contains the new steroids 9-hydroxygorgosterol (1), 9,11 alpha,14-trihydroxygorgosterol (2), 5 beta,6 beta-epoxyergost-24(28)-ene-3 beta,7 beta-diol (3), ergost-24(28)-ene-3 beta,5 alpha,6 beta,7 beta-tetrol (4), an unseparable 1:1 mixture of the epimers (25R) and (25S)-26-acetoxy-3 beta,5 alpha-dihydroxyergost-24(28)-en-6-one (5/6), and seven related, known compounds (7-13). The structures of these new compounds were defined by spectroscopic analysis. All the compounds (1-13) isolated from P. grisea were tested against P 388, A 549, and HT 29 tumor cell lines. Compounds 3, 5/6, and 12 exhibited selective activity against the HT 29 cell line (ED(50) = 0.1 microg/ml).

Transformation of hamster embryo cells by neutral sterols and bile acids.

Toxicity and cell transformation of Syrian hamster embryo cells in culture by certain neutral sterols and bile acids show interesting trends related to their structures: cholesterol-alpha-epoxide and cholestan-3 beta, 5 alpha, 6 beta-triol were more toxic and induced transformation to these cells, whereas their metabolic precursor, cholesterol, was inactive. The secondary bile acids, lithocholic and deoxycholic acids, were more toxic than their primary bile acid precursors, cholic and chenodeoxycholic acids and transformed the cells. These data suggest that mammalian cell transformation is a useful short-term assay to measure the potential toxicity and carcinogenicity of steroid derivatives.

Biological effects of oxysterols: current status.

A review of relevant literature on biological activities of oxysterols (OS) and cholesterol is presented. The data clearly demonstrate manifold biological activities, often detrimental, for OS compared with little or no such activity of a deleterious nature for cholesterol itself. Cholesterol is perhaps the single most important compound in animal tissue and, as such, it is difficult to imagine it as a toxin or hazard. In contrast, OS exhibit cytotoxicity to a wide variety of cells leading to angiotoxic and atherogenic effects; alter vascular permeability to albumin; alter prostaglandin synthesis and stimulate platelet aggregation, an important process facilitating atherosclerosis and thrombosis; alter the functionality of low density lipoprotein (LDL) receptors, possibly stimulating hypercholesterolaemia; modify cholesteryl ester accumulation in various cells, inducing foam cell formation; and enrich the LDL particle in cholesteryl esters, possibly increasing its atherogenicity. Furthermore, OS are mutagenic and carcinogenic, although some have been studied as antitumour agents based on their cytotoxic properties. Moreover, numerous studies have implicated OS in membrane and enzyme alterations that are interrelated with many of the foregoing effects. The authors find that OS deserve much more attention than cholesterol itself in terms of research activity but that unfortunately the reverse is true with regard to funding.