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Steroid hormones are essential for the biological processes of eukaryotic organisms. The steroid endocrine system of C. elegans, which includes dafachronic acids (DA) and the nuclear receptor ceDAF-12, provides a simple model for exploring the role of steroid hormone signaling pathways in animals. In this study, we show for the first time the feasibility of designing synthetic steroids that can modulate different physiological processes, such as development, reproduction and ageing, in relation to ceDAF-12. Our results not only confirm the conclusions derived from genetic studies linking these processes but also provide new chemical tools to selectively manipulate them, as we found that different compounds produce different phenotypic results. The structures of these compounds are much more diverse than those of endogenous hormones and analogues previously described by other researchers, allowing further development of the chemical modulation of the steroid endocrine system in C. elegans and related nematodes., (© 2024 Wiley-VCH GmbH.)

Extracts from Taiwan's traditional medicinal mushroom, Antrodia cinnamomea , exhibit anti-inflammatory activities in cellular and preclinical studies. However, this paper is the first to report that Antcin K, a triterpenoid isolated from A. cinnamomea , inhibits proinflammatory cytokine production in human rheumatoid synovial fibroblasts (RASFs), which are major players in rheumatoid arthritis (RA) disease. In our analysis of the mechanism of action, Antcin K inhibited the expression of three cytokines (tumor necrosis factor alpha [TNF-α], interleukin 1 beta [IL-1β] and IL-8) in human RASFs; cytokines that are crucial to RA synovial inflammation. Notably, incubation of RASFs with Antcin K reduced the phosphorylation of the focal adhesion kinase (FAK), phosphoinositide 3-kinase (PI3K), protein kinase B (AKT) and nuclear factor-κB (NF-κB) signaling cascades, all of which promote cytokine production in RA. Intraperitoneal injections of Antcin K (10 mg/kg or 30 mg/kg) attenuated paw swelling, cartilage degradation and bone erosion, and decreased serum levels of TNF-α, IL-1β, IL-8 in collagen-induced arthritis (CIA) mice; in further experiments, IL-6 levels were similarly reduced. The inhibitory effects of Antcin K upon TNF-α, IL-1β and IL-8 expression in human RASFs was achieved through the downregulation of the FAK, PI3K, AKT and NF-κB signaling cascades. Our data support clinical investigations using Antcin K in RA disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Achudhan, Liu, Lin, Huang, Tsai, Ko, Chiang, Kuo and Tang.)

Prevention therapy against Dirofilaria immitis in companion animals is currently threatened by the emergence of isolates resistant to macrocyclic lactone anthelmintics. Understanding the control over developmental processes in D. immitis is important for elucidating new approaches to heartworm control. The nuclear receptor DAF-12 plays a role in the entry and exit of dauer stage in Caenorhabditis elegans and in the development of free-living infective third-stage larvae (iL3) of some Clade IV and V parasitic nematodes. We identified a DAF-12 ortholog in the clade III nematode D. immitis and found that it exhibited a much higher affinity for dafachronic acids than described with other nematode DAF-12 investigated so far. We also modelled the DimDAF-12 structure and characterized the residues involved with DA binding. Moreover, we showed that cholesterol derivatives impacted the molting process from the iL3 to the fourth-stage larvae. Since D. immitis is unable to synthesize cholesterol and only completes its development upon host infection, we hypothesize that host environment contributes to its further molting inside the host vertebrate. Our discovery contributes to a better understanding of the developmental checkpoints of D. immitis and offers new perspectives for the development of novel therapies against filarial infections.

Follicular fluid meiosis-activating sterol (FF-MAS) exerts beneficial effects on the meiotic resumption of mammalian oocytes and their subsequent early embryonic development, but the signaling pathway underlying these effects has not been elucidated. Therefore, the objective of the present study was to investigate whether the mitogen-activated protein kinase (MAPK) pathway is involved in the FF-MAS-induced in vitro resumption of meiosis in porcine oocytes. Porcine cumulus oocyte complexes (COCs) were allocated in several groups cultured in TCM-199 medium with different concentration of AY 9944-A-7 (20, 30, 40 μmol/L) or ketoconazole (20 μmol/L) to increase or decrease endogenous accumulation of FF-MAS. Each experimental condition was repeated at least six times. After maturation for 44 h, the resumption of meiosis was assessed by the frequency of germinal vesicle breakdown (GVBD) and the first polar body (PBI) extrusion, The relative expressions of related genes in MAPK pathway [c-mos, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase 1/2 (ERK1/2)] at both transcriptional and translational levels were detected to investigate the kinetic trend of expression throughout oocyte maturation in vitro in response to the addition of AY 9944-A-7 or ketoconazole to the maturation medium. Results indicated that AY 9944-A-7 promoted, while ketoconazole inhibited, the in vitro maturation (IVM) of porcine oocytes. Relative expression of meiosis related genes was upregulated by AY 9944-A-7 and downregulated by ketoconazole. With extended culturing time, c-mos mRNA expression levels reached their peak at 12 h of maturation and decreased gradually thereafter, while MEK, ERK1 and ERK2 expression increased after an initial decrease peaking at 44 h of culture in the AY 9944-A-7-group. And the trend of the protein expression of c-mos, MEK, ERK1/2 was basically consistent with the mRNA expression of these genes. These results imply that the endogenous accumulation of FF-MAS is beneficial to resumption of meiosis in porcine oocytes and that MAPK signaling is involved in FF-MAS-induced meiotic resumption., Competing Interests: Declaration of competing interest All the authors involved in this work declare that there are no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there are no financial or other contractual agreements that might cause a conflict of interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Liver X Receptors (LXRs) are ligand dependent transcription factors activated by oxidized cholesterol metabolites (oxysterols) that play fundamental roles in the transcriptional control of lipid metabolism, cholesterol transport and modulation of inflammatory responses. In the last decade, LXRs have become attractive pharmacological targets for intervention in human metabolic diseases and thus, several efforts have concentrated on the development of synthetic analogues able to modulate LXR transcriptional response. In this sense, we have previously found that cholestenoic acid analogues with a modified side chain behave as LXR inverse agonists. To further investigate the structure-activity relationships and to explore how cholestenoic acid derivatives interact with the LXRs, we evaluated the LXR biological activity of new analogues containing a C24-C25 double bond. Furthermore, a microarray assay was performed to evaluate the recruitment of coregulators to recombinant LXR LBD upon ligand binding. Also, conventional and accelerated molecular dynamics simulations were applied to gain insight on the molecular determinants involved in the inverse agonism. As LXR inverse agonists emerge as very promising candidates to control LXR activity, the cholestenoic acid analogues here depicted constitute a new relevant steroidal scaffold to inhibit LXR action., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Ethnopharmacological Relevance: Drug induced liver damage remains a prevalent concern in healthcare and may reduce the effectiveness of therapy by compromising therapeutic regimens. Many Commiphora species are known for their medicinal properties, and some of them are used traditionally for hepatoprotective effect. In the course of our drugs discovery from natural sources, phytosterols (lophenol (Lop) and lathosterol (Lat)), isolated from Commiphora kua were studied to evaluate their hepatoprotective effects in acetaminophen (APAP) induced hepatotoxicity in mice., Aims and Objective: To evaluate the hepatoprotective effects of phytosterols isolated from C. kua using in vivo experimental model., Materials and Methods: Mice of either sex were divided into 7 groups: Vehicle, silymarin (SLY), acetaminophen (APAP), Lop 25, Lop 50, Lat 25, Lat 50 (n = 5). Vehicle group received only vehicle (0.1% DMSO solution) for 7 days, APAP group received single dose of acetaminophen on day 7 and SLY group received silymarin for 7 days. Lop 25 and Lop 50 received low and high doses of Lop (25 μg/kg BW and 50 μg/kg BW), respectively, for 7 days, while Lat 25 and Lat 50 received low and high doses of Lat (25 μg/kg BW and 50 μg/kg BW) for 7 days. On day 7, all animals except Vehicle group kept fasted for 18 h and received APAP i. p. 400 mg/kg BW. After 20 h of APAP administration, the animals anesthetized with light chloroform and scarified by cervical decapitation. The blood serum and liver tissue samples were collected for biochemical and histopathological analysis. Liver function tests (LFTs) including lactate deydrogenase (LDH), alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST) and direct bilirubin) were used as biochemical parameters. While catalase (CAT), superoxide dismutase (SOD) and reduced glutathione (GSH) were taken as anti-oxidant enzymes., Results: Significant increase in levels of ALT, AST, ALP, LDH and direct bilirubin, and significant decrease in concentration of anti-oxidant enzymes (SOD, CAT and GSH) was observed in APAP-treated group. Similarly, histological slides showed obvious signs of damage to liver cells, reflecting acetaminophen induced hepatotoxicity. Treatment of test animals with phytosterols resulted in significant recovery of LFTs profile and concentration of anti-oxidant enzymes. Similarly, significant improvement of liver tissues was noted in histological analysis., Conclusions: Both phytosterols possessed hepatoprotective potential and should be further evaluated for acute toxicity studies and pharmacokinetics/pharmacodynamics profile., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: While immune responses to the murine hookworm Nippostrongylus brasiliensis have been investigated, signaling pathways regulating development of infectious larvae (iL3) are not well understood. We hypothesized that N. brasiliensis would use pathways similar to those controlling dauer development in the free-living nematode Caenorhabditis elegans, which is formally known as the "dauer hypothesis.", Methods: To investigate whether dafachronic acid activates the N. brasiliensis DAF-12 homolog, we utilized an in vitro reporter assay. We then utilized RNA-Seq and subsequent bioinformatic analyses to identify N. brasiliensis dauer pathway homologs and examine regulation of these genes during iL3 activation., Results: In this study, we demonstrated that dafachronic acid activates the N. brasiliensis DAF-12 homolog. We then identified N. brasiliensis homologs for members in each of the four canonical dauer pathways and examined their regulation during iL3 activation by either temperature or dafachronic acid. Similar to C. elegans, we found that transcripts encoding antagonistic insulin-like peptides were significantly downregulated during iL3 activation, and that a transcript encoding a phylogenetic homolog of DAF-9 increased during iL3 activation, suggesting that both increased insulin-like and DAF-12 nuclear hormone receptor signaling accompanies iL3 activation. In contrast to C. elegans, we observed a significant decrease in transcripts encoding the dauer transforming growth factor beta ligand DAF-7 during iL3 activation, suggesting a different role for this pathway in parasitic nematode development., Conclusions: Our data suggest that canonical dauer pathways indeed regulate iL3 activation in the hookworm N. brasiliensis and that DAF-12 may be a therapeutic target in hookworm infections.

Parasitic nematodes transition between dramatically different free-living and parasitic stages, with correctly timed development and migration crucial to successful completion of their lifecycle. However little is known of the mechanisms controlling these transitions. microRNAs (miRNAs) negatively regulate gene expression post-transcriptionally and regulate development of diverse organisms. Here we used microarrays to determine the expression profile of miRNAs through development and in gut tissue of the pathogenic nematode Haemonchus contortus. Two miRNAs, mir-228 and mir-235, were enriched in infective L3 larvae, an arrested stage analogous to Caenorhabditis elegans dauer larvae. We hypothesized that these miRNAs may suppress development and maintain arrest. Consistent with this, inhibitors of these miRNAs promoted H. contortus development from L3 to L4 stage, while genetic deletion of C. elegans homologous miRNAs reduced dauer arrest. Epistasis studies with C. elegans daf-2 mutants showed that mir-228 and mir-235 synergise with FOXO transcription factor DAF-16 in the insulin signaling pathway. Target prediction suggests that these miRNAs suppress metabolic and transcription factor activity required for development. Our results provide novel insight into the expression and functions of specific miRNAs in regulating nematode development and identify miRNAs and their target genes as potential therapeutic targets to limit parasite survival within the host.

Antrodia camphorata, a well-known and highly valued edible medicinal mushroom with intriguing activities like liver protection, has been traditionally used for the treatment of alcoholic liver disease. A. camphorata shows highly medicinal and commercial values with the demand far exceeds the available supply. Thus, the petri-dish cultured A. camphorata (PDCA) is expected to develope as a substitute. In this paper, nineteen triterpenes were isolated from PDCA, and thirteen of them were the unique anthroic acids in A. camphorata, including the main content antcin K, which suggested that PDCA produced a large array of the same anthroic acids as the wild one. Furthermore, no obvious acute toxicity was found suggesting the edible safety of PDCA. In mice alcohol-induced liver injury model, triglyceride (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and malondialdehyde (MDA) had been reduced by the PDCA powder as well as the main content antcin K, which indicated that the PDCA could protect alcoholic liver injury in mice model and antcin K could be the effective component responsible for the hepatoprotective activities of PDCA against alcoholic liver diseases., (Copyright © 2019 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Background: Our previous study showed that (+)-cholesten-3-one (CN) has the potential to induce the osteoblastic differentiation of mesenchymal stem cells (MSCs). However, the roles of CN in targeting miRNA-mRNA-lncRNA interactions to regulate osteoblast differentiation remain poorly understood., Results: A total of 77 miRNAs (36 upregulated and 41 downregulated) and 295 lncRNAs (281 upregulated and 14 downregulated) were significantly differentially expressed during CN-induced MSC osteogenic differentiation. Bioinformatic analysis identified that several pathways may play vital roles in MSC osteogenic differentiation, such as the vitamin D receptor signalling, TNF signalling, PI3K-Akt signalling, calcium signalling, and mineral absorption pathways. Further bioinformatic analysis revealed 16 core genes, including 6 mRNAs (Vdr, Mgp, Fabp3, Fst, Cd38, and Col1a1), 5 miRNAs (miR-483, miR-298, miR-361, miR-92b and miR-155) and 5 lncRNAs (NR_046246.1, NR_046239.1, XR_086062.1, XR_145872.1 and XR_146737.1), that may play important roles in regulating the CN-induced osteogenic differentiation of MSCs. Verified by the luciferase reporter, AR-S, qRT-PCR and western blot assays, we identified one miRNA (miR-298) that may enhance the osteogenic differentiation potential of MSCs via the vitamin D receptor signalling pathway., Conclusions: This study revealed the global expression profile of miRNAs and lncRNAs involved in the Chinese medicine active ingredient CN-induced osteoblast differentiation of MSCs for the first time and provided a foundation for future investigations of miRNA-mRNA-lncRNA interaction networks to completely illuminate the regulatory role of CN in MSC osteoblast differentiation.

Strongyloides stercoralis hyperinfection causes high mortality rates in humans, and, while hyperinfection can be induced by immunosuppressive glucocorticoids, the pathogenesis remains unknown. Since immunocompetent mice are resistant to infection with S. stercoralis , we hypothesized that NSG mice, which have a reduced innate immune response and lack adaptive immunity, would be susceptible to the infection and develop hyperinfection. Interestingly, despite the presence of large numbers of adult and first-stage larvae in S. stercoralis -infected NSG mice, no hyperinfection was observed even when the mice were treated with a monoclonal antibody to eliminate residual granulocyte activity. NSG mice were then infected with third-stage larvae and treated for 6 wk with methylprednisolone acetate (MPA), a synthetic glucocorticoid. MPA treatment of infected mice resulted in 50% mortality and caused a significant >10-fold increase in the number of parasitic female worms compared with infected untreated mice. In addition, autoinfective third-stage larvae, which initiate hyperinfection, were found in high numbers in MPA-treated, but not untreated, mice. Remarkably, treatment with Δ7-dafachronic acid, an agonist of the parasite nuclear receptor Ss -DAF-12, significantly reduced the worm burden in MPA-treated mice undergoing hyperinfection with S. stercoralis Overall, this study provides a useful mouse model for S. stercoralis autoinfection and suggests a therapeutic strategy for treating lethal hyperinfection., Competing Interests: The authors declare no conflict of interest.

Aim: Antrodia Camphorate (AC) is a mushroom that is widely used in Asian countries to prevent and treat various diseases, including liver diseases. However, the active ingredients that contribute to the biological functions remain elusive. The purpose of the present study is to test the hepatoprotective effect of Antcin H, a major triterpenoid chemical isolated from AC, in murine models of acute liver injury., Results: We found that Antcin H pretreatment protected against liver injury in both acetaminophen (APAP) and galactosamine/tumor necrosis factor (TNF)α models. More importantly, Antcin H also offered a significant protection against acetaminophen-induced liver injury when it was given 1 h after acetaminophen. The protection was verified in primary mouse hepatocytes. Antcin H prevented sustained c-Jun-N-terminal kinase (JNK) activation in both models. We excluded an effect of Antcin H on acetaminophen metabolism and TNF receptor signaling and excluded a direct effect as a free radical scavenger or JNK inhibitor. Since the sustained JNK activation through its interaction with mitochondrial Sab, leading to increased mitochondrial reactive oxygen species (ROS), is pivotal in both models, we examined the effect of Antcin H on p-JNK binding to mitochondria and impairment of mitochondrial respiration. Antcin H inhibited the direct effect of p-JNK on isolated mitochondrial function and binding to isolated mitochondria. Innovation and Conclusion: Our study has identified Antcin H as a novel active ingredient that contributes to the hepatoprotective effect of AC, and Antcin H protects against liver injury through disruption of the binding of p-JNK to Sab, which interferes with the ROS-dependent self-sustaining activation of MAPK cascade. Antioxid. Redox Signal. 26, 207-220.

Cellular cholesterol homeostasis relies on precise control of the sterol content of organelle membranes. Obtaining insight into cholesterol trafficking pathways and kinetics by live-cell imaging relies on two conditions. First, one needs to develop suitable analogs that resemble cholesterol as closely as possible with respect to their biophysical and biochemical properties. Second, the cholesterol analogs should have good fluorescence properties. This interferes, however, often with the first requirement, such that the imaging instrumentation must be optimized to collect photons from suboptimal fluorophores, but good cholesterol mimics, such as the intrinsically fluorescent sterols, cholestatrienol (CTL) or dehydroergosterol (DHE). CTL differs from cholesterol only in having two additional double bonds in the ring system, which is why it is slightly fluorescent in the ultraviolet (UV). In the first part of this protocol, we describe how to synthesize and image CTL in living cells relative to caveolin, a structural component of caveolae. In the second part, we explain in detail how to perform time-lapse experiments of commercially available BODIPY-tagged cholesterol (TopFluor-cholesterol ® ; TF-Chol) in comparison to DHE. Finally, using two-photon time-lapse imaging data of TF-Chol, we demonstrate how to use our imaging toolbox SpatTrack for tracking sterol rich vesicles in living cells over time.

Antrodia cinnamomea (A. cinnamomea), a popular medicinal mushroom in Taiwan, is widely used to prevent or treat liver diseases. Systematic studies on the anti-inflammatory effect of A. cinnamomea and its molecular mechanisms have not yet been fully investigated. HPLC fingerprint analysis identified seven ergostane-type triterpenoids from A. cinnamomea water extract (ACW), including high amounts of Antcin K (AC), Antcin C, Antcin H, Dehydrosulphurenic acid, Antcin B, Antcin A and Dehydroeburicoic acid. Here, we explored the effects and mechanisms of ACW and the highest content AC on N-nitrosodiethylamine (DEN) induced liver inflammation, fibrosis and carcinogenesis in rats. In the in vitro study, we measured how ACW and AC dose-dependently scavenged O[Formula: see text], H 2 O 2 and HOCl by a chemiluminescence analyzer. In the in vivo experiment, oral intake ACW and AC significantly inhibited DEN-enhanced hepatocellular inflammation, fibrosis and carcinoma by pathologic observation, the elevated bile and liver reactive oxygen species (ROS) amounts, plasma [Formula: see text]-glutamyl transpeptidase, and oxidative stress including 3-nitrotyrosine, 4-hydroxynonenal and Kuppfer cell infiltration (ED-1 stains) in the inflammatory livers. DEN enhanced nuclear factor-[Formula: see text]B (NF-[Formula: see text]B) translocation, whereas ACW and AC suppressed DEN-enhanced NF-[Formula: see text]B translocation through the inhibition of its upstream signaling of p85/phosphoinositide-3-kinase, mitogen activated protein kinase and CYP2E1 expression. In conclusion, DEN can induce hepatocellular inflammation, fibrosis and carcinoma by increasing NF-[Formula: see text]B translocation to the nucleus, and oxidative injury. ACW and its active component, Antcin K, counteract DEN-induced hepatic injury and inflammation by the protective and therapeutic mechanisms of a direct scavenging ROS activity and an upregulation of anti-oxidant defense mechanisms.

Background: Liver X receptors (LXRs) are transcription factors activated by cholesterol metabolites containing an oxidized side chain. Due to their ability to regulate lipid metabolism and cholesterol transport, they have become attractive pharmacological targets. LXRs are closely related to DAF-12, a nuclear receptor involved in nematode lifespan and regulated by the binding of C-27 steroidal acids. Based on our recent finding that the lack of the C-25 methyl group does not abolish their DAF-12 activity, we evaluated the effect of removing it from the (25R)-cholestenoic acid, a LXR agonist., Methods: The binding mode and the molecular basis of action of 27-nor-5-cholestenoic acid were evaluated using molecular dynamics simulations. The biological activity was investigated using reporter gene expression assays and determining the expression levels of endogenous target genes. The in vitro MARCoNI assay was used to analyze the interaction with cofactors., Results: 27-Nor-5-cholestenoic acid behaves as an inverse agonist. This correlates with the capacity of the complex to better bind corepressors rather than coactivators. The C-25 methyl moiety would be necessary for the maintenance of a torsioned conformation of the steroid side chain that stabilizes an active LXRβ state., Conclusion: We found that a 27-nor analog is able to act as a LXR ligand. Interestingly, this minimal structural change on the steroid triggered a drastic change in the LXR response., General Significance: Results contribute to improve our understanding on the molecular basis of LXRβ mechanisms of action and provide a new scaffold in the quest for selective LXR modulators., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Dengue, transmitted primarily by the bites of infected Aedes aegypti L., is transmitted to millions of individuals each year in tropical and subtropical areas. Dengue control strategies are primarily based on controlling the vector, using insecticides, but the appearance of resistance poses new challenges. Recently, highly selective protease inhibitors by phage display were obtained for digestive enzymes of the 4th instar larvae (L4) midgut. These mutants were not confirmed as a larvicide due to the low yield of the expression of these inhibitors. In the present study, chimera molecules were constructed based on the mutations at positions P1-P4' selected previously. The T6, T23 and T149 mutants were mixed with another Kunitz inhibitor, domain 1 of the inhibitor boophilin (D1)., Methods: The chimeras T6/D1, T149/D1 and T23/D1 were expressed at high levels in P. pastoris yeast, purified by ionic exchange chromatography and their homogeneity was analyzed by SDS-PAGE. The chimera inhibitors were assayed against larval trypsin, chymotrypsin and elastase using specific chromogenic substrates. The inhibitors were assayed for their larvicide potential against L4., Results: The chimeras exhibited strong inhibitory activities against the larval digestive enzymes in a dose-dependent manner. T6/D1, T149/D1 and T23/D1 exhibited strong larvicidal activity against L4 of Ae. aegypti with inhibitor concentrations in the μM range. A synergistic increase in mortality was observed when a mixture of the three chimeric inhibitors was tested., Conclusions: The strategy for constructing the chimeric inhibitors was successful. The chimeras showed strong larvicidal activity against Ae. aegypti. In the future, our findings can be used to design synthetic inhibitors for larvae digestive enzymes as an alternative method to control the dengue vector.

Previous research demonstrated that the ethyl acetate extract from Antrodia cinnamomea suppresses the invasive potential of human breast and hepatoma cells, but the effective compounds are not identified. The main bioactive compounds of A. cinnamomea are ergostane-type triterpenoids, and the content of antcin K is the highest. The objective of this study was to evaluate the antimetastatic activity and mechanisms of antcin K purified from the fruiting body of basswood-cultivated A. cinnamomea on human liver cancer Hep 3B cells. The results showed that adhesion, migration, and invasion of Hep 3B cells were effectively inhibited by antcin K within 24 h of treatment. Antcin K not only reduced the protein expression and activity of MMP-2 and MMP-9 but also down-regulated vimentin and up-regulated E-cadherin in Hep 3B cells. In depth investigation for the molecular mechanism revealed that antcin K could reduce the protein expression of integrin β1, β3, α5, and αv and suppress phosphorylation of FAK, Src, PI3K, AKT, MEK, ERK, and JNK. These results suggested that antcin K was able to inhibit the metastasis of human hepatoma cells through suppression of integrin-mediated adhesion, migration, and invasion. Coupled with these findings, antcin K has a good potential to reduce the risk of liver cancer metastasis.

Dafachronic acids (DAs) are 3-keto cholestenoic acids bearing a carboxylic acid moiety at the end of the steroid side chain. These compounds interact with the DAF-12 receptor, a ligand-dependent transcription factor that acts as a molecular switch mediating the choice between arrest at diapause or progression to reproductive development and adult lifespan in different nematodes. Recently, we reported that the 27-nor-Δ4-DA was able to directly activate DAF-12 in a transactivation cell-based luciferase assay and rescued the Mig phenotype of daf-9(rh50) Caenorhabditis elegans mutants. In the present paper, to investigate further the relationship between the structure of the steroid side chain and DAF-12 activity, we evaluated the in vitro and in vivo activity of Δ4-DA analogues with modified side chains using transactivation cell-based assays and daf-9(dh6) C. elegans mutants. Our results revealed that introduction of a 24,25-double bond on the cholestenoic acid side chain did not affect DAF-12 activity, whereas shortening the side chain lowered the activity. Most interestingly, the C24 alcohol 24-hydroxy-4-cholen-3-one (6) was an antagonist of the DAF-12 receptor both in vitro and in vivo.

Three new sterols, 5α,8α-epidioxy-24-norcholesta-6,9(11),22-trien-3β-ol (1), 5α,8α-epidioxy-cholesta-6,9(11),24-trien-3β-ol (2), and 5α,8α-epidioxy-cholesta-6,23-dien-3β,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.

A new steroidal alkaloid, (20S,22R,24R)-24-ethyl-3-oxocholest-4-en-22-amino, named as nandsterine (1), together with 10 known alkaloids, palmatine (2), O-methylbulbocapnine (3), nantenine (4), dehydronantenine (5), glaucine (6), didehydroglaucine (7), dehydrocorydaline (8), jatrorrhizine (9), magnoflorine (10) and berberine (11), was isolated from the fruit of Nandina domestica Thunb. Their structures were elucidated by using spectroscopic methods as well as by comparing with the published data. Compound 1 was a new class of steroidal alkaloid isolated from the family Berberidaceae, meanwhile compounds 2, 3, 6-8 and 10 were obtained from N. domestica for the first time. Compound 1 exhibited cytotoxicity against HL-60 cells (human leukaemia) with IC50 values of 52.1 μM.

The pinewood nematode Bursaphelenchus xylophilus has caused severe damage to pine forests in large parts of the world [1-4]. Dispersal of this plant-parasitic nematode occurs when the nematode develops into the dispersal fourth larval stage (LIV) upon encountering its insect vector, the Monochamus pine sawyer beetle, inside an infected pine tree [5-9]. Here, we show that LIV formation in B. xylophilus is induced by C16 and C18 fatty acid ethyl esters (FAEEs), which are produced abundantly on the body surface of the vector beetle specifically during the late development pupal, emerging adult, and newly eclosed adult stages. The LIV can then enter the tracheal system of the adult beetle for dispersal to a new pine tree. Treatment of B. xylophilus with long-chain FAEEs, or the PI3 kinase inhibitor LY294002, promotes LIV formation, while Δ7-dafachronic acid blocks the effects of these chemicals, suggesting a conserved role for the insulin/IGF-1 and DAF-12 pathways in LIV formation. Our work provides a mechanism by which LIV formation in B. xylophilus is specifically coordinated with the life cycle of its vector beetle. Knowledge of the chemical signals that control the LIV developmental decision could be used to interfere with the dispersal of this plant-parasitic nematode., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The cattle tick Rhipicephalus (Boophilus) microplus lays eggs in the soil near the roots of grass, or in similar highly moist environments that are prone to biofilm formation. Tick eggs have a protective wax coating that may be a source of nutrients for microorganisms. However, as the eggs remain viable and show no visible signs of microbial colonization, we hypothesized that the coating might have anti-biofilm properties. We show here that the coating inhibits biofilm formation by both Gram-negative and Gram-positive bacteria, though by different mechanisms. We have identified the anti-biofilm molecule as N-(3-sulfooxy-25-cholest-5-en-26-oyl)-L-isoleucine (boophiline), and we show that it inhibits the expression of fliC (flagellin) and cdrA (biofilm scaffold), whose products are necessary for biofilm formation in Pseudomonas aeruginosa. Boophiline is a novel biofilm inhibitor being also effective against Staphylococcus epidermidis biofilm. In our study we show evidences of the boophiline mode of action in the protection of arthropod eggs against biofilm colonization., (© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.)

27-Nor-Δ(4)-dafachronic acid was prepared in nine steps and 14% overall yield by two sequential 2-carbon homologations from 20β-carboxyaldehyde-4-pregnen-3-one. Its activity was evaluated in vivo, where it rescued the Mig phenotype of daf-9(rh50) Caenorhabditis elegans mutants and restored their normal resistance to oxidative stress. 27-Nor-Δ(4)-dafachronic acid was also able to directly bind and activate DAF-12 in a transactivation cell-based luciferase reporter assay, although it was less active than the corresponding 25R-and 25S dafachronic acids. The binding mode of the 27-Nor steroid was studied by molecular dynamics using a homology model of the CeDAF-12 receptor., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The experiment in vivo in growing male Wistar rats was carried out. The animals of the experimental 2-4 groups were daily intragastrically injected water solutions of the dried extract from the leaves of Seratulla coronata L. in volume of 1.0 ml, containing 2, 20 and 50 mg of phytoecdysteroids per kg of animal weight, accordingly. Animals of control group were daily injected 1.0 ml of water. The content of phytoecdysteroids in the dry extract was analyzed by high performance liquid chromatography (HPLC). The concentration of the sum of phytoecdysteroids in dry extract was 6.15%, 66% of which was 20-hydroxyecdysone and 23% was 25S-inokosteron. On the 15th day animals were taken out of the experiment by the decapitation. The content of corticosterone, prostaglandin E2 and beta-endorphin in rat blood plasma were determined by ELISA test. The pathological--anatomical analysis and weighing of the liver did not reveal any adverse changes of this organ in the animals of all groups. The average concentration of blood plasma corticosterone reduced with increasing of the dose of the extract injected to the animals, reaching significant differences relative to the control group (60.9 +/- 9.4 ng/ml) for 3 and 4 groups (22.7 +/- 6.6 and 17.6 +/- 7.3 ng/ml, accordingly). Beta-endorphin and prostaglandin E2 levels did not differ. The ratio of the mediator of stress (corticosterone) and inhibitors of stress (beta-endorphin and prostaglandin E2) has been calculated. A monotonic decrease of corticosterone/beta-endorphin and corticosterone/prostaglandin E2 ratio has been found with extract dose increasing. Taken together the results of determination of biochemical parameters of the general adaptation syndrome the dose-dependent stress-protective effect of Seratulla coronata L extract has been demonstrated.

A stereoselective synthesis of (25S)-Δ(1)-, (25S)-Δ(1,4)-, (25S)-Δ(1,7)-, (25S)-Δ(8(14))-, (25S)-Δ(4,6,8(14))-dafachronic acid, methyl (25S)-Δ(1,4)-dafachronate and (25S)-5α-hydroxy-3,6-dioxocholest-7-en-26-oic acid is described. (25S)-Δ(1,4)-Dafachronic acid and its methyl ester are natural products isolated from corals and have been obtained by synthesis for the first time. (25S)-5α-Hydroxy-3,6-dioxocholest-7-en-26-oic acid represents a promising synthetic precursor for cytotoxic marine steroids.

In Caenorhabditis elegans and Drosophila melanogaster, removing the germline precursor cells increases lifespan. In worms, and possibly also in flies, this lifespan extension requires the presence of somatic reproductive tissues. How the somatic gonad signals other tissues to increase lifespan is not known. The lifespan increase triggered by loss of the germ cells is known to require sterol hormone signaling, as reducing the activity of the nuclear hormone receptor DAF-12, or genes required for synthesis of the DAF-12 ligand dafachronic acid, prevents germline loss from extending lifespan. In addition to sterol signaling, the FOXO transcription factor DAF-16 is required to extend lifespan in animals that lack germ cells. DAF-12/NHR is known to assist with the nuclear accumulation of DAF-16/FOXO in these animals, yet we find that loss of DAF-12/NHR has little or no effect on the expression of at least some DAF-16/FOXO target genes. In this study, we show that the DAF-12-sterol signaling pathway has a second function to activate a distinct set of genes and extend lifespan in response to the somatic reproductive tissues. When germline-deficient animals lacking somatic reproductive tissues are given dafachronic acid, their expression of DAF-12/NHR-dependent target genes is restored and their lifespan is increased. Together, our findings indicate that in C. elegans lacking germ cells, the somatic reproductive tissues promote longevity via steroid hormone signaling to DAF-12., Competing Interests: The authors have declared that no competing interests exist.

Morphological novelties are lineage-specific traits that serve new functions. Developmental polyphenisms have been proposed to be facilitators of phenotypic evolution, but little is known about the interplay between the associated genetic and environmental factors. Here, we study two alternative morphologies in the mouth of the nematode Pristionchus pacificus and the formation of teeth-like structures that are associated with bacteriovorous feeding and predatory behaviour on fungi and other worms. These teeth-like denticles represent an evolutionary novelty, which is restricted to some members of the nematode family Diplogastridae but is absent from Caenorhabditis elegans and related nematodes. We show that the mouth dimorphism is a polyphenism that is controlled by starvation and the co-option of an endocrine switch mechanism. Mutations in the nuclear hormone receptor DAF-12 and application of its ligand, the sterol hormone dafachronic acid, strongly influence this switch mechanism. The dafachronic acid-DAF-12 module has been shown to control the formation of arrested dauer larvae in both C. elegans and P. pacificus, as well as related life-history decisions in distantly related nematodes. The comparison of dauer formation and mouth morphology switch reveals that different thresholds of dafachronic acid signalling provide specificity. This study shows how hormonal signalling acts by coupling environmental change and genetic regulation and identifies dafachronic acid as a key hormone in nematode evolution.

(25S)-3-Oxocholesta-1,4-dien-26-oic acid (1) and a new (25S)-18-acetoxy-3-oxocholesta-1,4-dien-26-oic acid (2) were isolated from a soft coral Minabea sp. (cf. aldersladei) collected in North Sulawesi, Indonesia, together with two known cholic-acid-type compounds, 3-oxochol-1,4-dien-24-oic acid (3) and 3-oxochol-4-en-24-oic acid (4). The structures of these compounds were determined on the basis of their spectroscopic data. The absolute stereochemistry at C-25 of 2 was determined by comparative (1)H NMR study using chiral anisotropic reagents [(S)- and (R)-phenylglycine methyl esters]. This is the first to report compound 1 as a natural product.

MicroRNAs (miRNAs) posttranscriptionally regulate gene expression, but the factors that direct transcription of miRNAs are not well characterized. Activation versus repression of key developmental miRNAs in Caenorhabditis elegans is directly mediated by ligand occupancy of a nuclear hormone receptor that acts to couple nutrient availability to developmental programs.

We describe the stereoselective transformation of diosgenin (4a) to (25R)-Delta(4)-dafachronic acid (1a),(25R)-Delta(7)-dafachronic acid (2a), and (25R)-cholestenoic acid (3a), which represent potential ligands forthe hormonal receptor DAF-12 in Caenorhabditis elegans. Key-steps of our synthetic approach are amodified Clemmensen reduction of diosgenin (4a) and a double bond shift from the 5,6- to the 7,8-position. In the 25R-series, the Delta(7)-dafachronic acid 2a exhibits the highest hormonal activity.

Under harsh environmental conditions, Caenorhabditis elegans larvae undergo arrest and form dauer larvae that can attach to other animals to facilitate dispersal. It has been argued that this phenomenon, called phoresy, represents an intermediate step toward parasitism. Indeed, parasitic nematodes invade their hosts as infective larvae, a stage that shows striking morphological similarities to dauer larvae. Although the molecular regulation of dauer entry in C. elegans involves insulin and TGF-beta signaling, studies of TGF-beta orthologs in parasitic nematodes didn't provide evidence for a common origin of dauer and infective larvae. To identify conserved regulators between Caenorhabditis and parasitic nematodes, we used an evolutionary approach involving Pristionchus pacificus as an intermediate. We show by mutational and pharmacological analysis that Pristionchus and Caenorhabditis share the dafachronic acid-DAF-12 system as the core endocrine module for dauer formation. One dafachronic acid, Delta7-DA, has a conserved role in the mammalian parasite Strongyloides papillosus by controlling entry into the infective stage. Application of Delta7-DA blocks formation of infective larvae and results in free-living animals. Conservation of this small molecule ligand represents a fundamental link between dauer and infective larvae and might provide a general strategy for nematode parasitism.

Steriodal heterocyclic systems namely cholest-5-en-3-oxazolo and thiazoloquinoxaline have been synthesized via the reaction of cholest-5-en-3-one semicarbazone/thiosemicarbazone with 2,3-dichloroquinoxaline at 80 degrees C in high yield. Cholest-5-en-3-one semicarbazone is obtained by the condensation of cholest-5-en-3-one with semicarbazide in the presence of AcONa in ethanol and cholest-5-en-one thiosemicarbazone is obtained by the condensation of cholest-5-en-3-one with thiosemicarbazide in ethanol in the presence of a few drops of HCl. The structures of these compounds were evident by elemental analysis, IR, 1H NMR and FAB mass spectral analysis. These synthesized compounds were investigated for antibacterial activity first by the disk-diffusion assay against two Gram-positive and two Gram-negative bacteria and then the minimum inhibitory concentration (MIC) of these compounds were determined and the results were compared with the standard drug Amoxicillin. The results showed that these compounds oxazolo/thiazoloquinoxaline are better antibacterial agents as compared to the standard drug Amoxicillin.

In Vitro maturation (IVM) of human oocytes is an emerging infertility treatment with great promise. To be successful this future assisted reproductive technology must entail both nuclear and cytoplasmic maturation of the oocytes and give rise to human embryos that have the same developmental potential as embryos resulting from the golden standard of human IVF. The aspiration of immature oocytes from small to medium size antral follicles followed by their maturation In Vitro present an attractive alternative to the hormonal stimulation of patients in In Vitro fertilization (IVF) treatment, since administration of exogenous hormones is a costly treatment and may cause severe health problems. Of the long list of side effect and health concern ovarian hyper-stimulation syndrome (OHSS) is by far the most severe although long term effect on cancer prevalence is another concern. Another potential group of patients that could benefit from future IVM treatments are the young women undergoing anticancer therapy (radiation- or chemotherapy). Thus, ovarian and oocyte cryopreservation techniques are emerging, however such treatments can only be fully realized when IVM becomes an efficient means of obtaining healthy birth. At present, the In Vitro maturation techniques are highly successful in mice, variable successful in domestic species and still regarded experimental in the human clinic due to suboptimal fertilization rates and embryo quality. This review discusses comparative studies of the processes of oocyte maturation In Vivo and In Vitro, in various mammalian species including human. Among the substances that have been reported to influence oocyte maturation there is an interesting endogenous signaling molecule: FF-MAS (4,4-dimethyl-5 alpha-cholest-8,14,24-trien-3 beta -ol), an intermediate in the cholesterol biosynthetic pathway present in all cells. This review gives special focus to FF-MAS, the effect seen in animal and human studies so far and its potential use in treatment of human infertility is being discussed, including both the safety and efficacy issues that need to be addressed. It is being reviewed how FF-MAS and related MAS analogues by our group and other scientific groups have been observed to mediate a dose-dependant response on both the nuclear maturation and especially the cytoplasmic maturation during oocyte maturation In Vitro thus giving rise to pre-embryos of higher developmental potential. Studies are reviewed regarding the family of meiosis activating sterols, its In Vivo regulation by gonadotropins (especially LH) and suggestions to the signaling pathways as the putative MAS receptor eliciting the important cytoplasmic maturation signaling cascade that involves mos/MAP kinase. The pharmacological effect of synthetic FF-MAS has been observed in various models and species, including murine, porcine and humane oocytes. Finally, the chromosome status of IVM human oocytes has been the focus of a large prospective clinical trial, documenting that FF-MAS acting on human oocytes during In Vitro maturation presents a safe procedure evaluated on numerical chromosome aberration rates in metaphase-II oocytes. In conclusion the In Vitro maturation of human oocytes is already now a valuable clinical treatment alternative for a subset of infertile patients, especially the Polycystic Ovarian Syndrome (PCOS) patients. IVM has the promise of being tomorrow's gold standard in treatment of human infertility if most of the important components of oocyte maturation are understood and can be adequately addressed In Vitro. Considering the present low frequency of successful fertilization and pre-implantation development following In Vitro maturation of human oocytes, the addition of FF-MAS or MAS analogues to the maturation medium to improve the cytoplasmic maturation and to yield higher quality pre-embryos may prove highly beneficial.

Background: Oocyte competence for maturation and embryogenesis is associated with diameter in many mammals. We aimed to test whether this relationship exists in humans and to quantify its impact upon in vitro maturation (IVM)., Methods: We used computer-assisted image analysis daily to measure average diameter, zona thickness and other parameters in oocytes. Immature oocytes originated from unstimulated patients with polycystic ovaries, and from stimulated patients undergoing intracytoplasmic sperm injection (ICSI). Some were cultured with meiosis activating sterol (FF-MAS). Matured oocytes were inseminated using ICSI and embryo development was monitored. In vivo matured oocytes were also measured., Results: Immature oocytes were smaller at collection than in vivo matured oocytes. Maturation was related to oocyte diameter and many oocytes grew in culture. FF-MAS stimulated growth in oocytes derived from ICSI patients, but only stimulated growth in PCO derived oocytes if they matured in vitro. Degenerating oocytes showed cytoplasmic shrinkage. Neither zona thickness, perivitelline space, nor the total diameter of the oocyte plus zona were informative regarding maturation capacity., Conclusions: Immature oocytes grow during maturation culture. FF-MAS promotes oocyte growth in vitro. Oocytes from different sources have different growth profiles in vitro. Measuring oocytes in clinical IVM may provide additional non-invasive information that could potentially avoid the use of growing oocytes.

The petroleum ether, chloroform, ethyl acetate and methanol extracts of the whole plant of Oldenlandia herbacea were screened for in vitro antioxidant activity by using ABTS, DPPH, nitric oxide and hydrogen peroxide assay methods. The chloroform, ethyl acetate and methanol extracts showed potent antioxidant activity against ABTS free radicals. In the hydrogen peroxide method, the methanol and ethyl acetate extracts showed potent activity. Chemical investigation of the petroleum ether extract yielded a long chain hydrocarbon, 9,9-dimethyl hexacosane (1), and a steroid, 23-ethyl-cholest-23-en-3beta-ol (2).

Objective: To search for chemical constituents with structural diversity from Laurencia tristicha to supply for biological assay., Method: Compounds were isolated by means of column chromatography over normal phase silica gel and Sephadex LH-20, recrystallization and HPLC. Structures were identified by spectroscopic methods including 1D NMR, IR and MS. Cytotoxicities of the purified compounds were evaluated by MTT method., Result: Seven compounds were isolated from L. tristicha. Their structures were elucidated as cholesterol (1), cholesta- 5-en-3beta, 7alpha-diol (2), beta-stigmasterol (3), phytol (4), zeaxanthin (5), 4 -hydroxybenzaldehyde (6), indolyl-3-carbaldehyde (7). In the cytotoxic assay compound 2 was active against human cancer cell lines HCT-8, Bel-7402, BGc-823, A549 and HELA with IC50 values of 1.90, 2.02, 1.99, 6.52 and 1.20 microg x mL(-1), respectively. Compound 4 showed cytotoxicity against HCT-8 and HELA with IC50 value of 3.51 and 2.04 microg x mL(-1), and other compounds were inactive ( IC50 > 10 microg x mL(-1))., Conclusion: Compounds 1-7 were isolated from L. tristicha for the first time. In additon, compounds 2 and 4 were cytotoxic against several human cancer cell lines.

Meiosis of vertebrate oocytes is a protracted process initiated within differentiated oocytes before the first meiotic arrest of the first meiotic division. Meiosis normally resumes in response to the stimulation of ovulation, proceeding to metaphase of the second meiotic division. In fish and amphibian oocytes, this resumption is triggered by follicular steroids. By contrast, the role of steroids in the resumption of mammalian oocyte maturation is less clear. Specifically, mammalian meiotic maturation proceeds undisturbed even when steroid production is severely suppressed. This puzzling mammalian divergence has been reexamined recently. Here, we review the published data and conclude that steroids are not necessary for the resumption of mammalian meiosis. Nevertheless, steroids are probably involved in follicular growth, somatic-cell differentiation and the acquisition of developmental competence of mature ova.

The antiradiation properties of the ecdysteroid-containing preparations ("serpisten" and inokosterone) are studied under their application before or after the 22.6 cGy chronic low intensity gamma-irradiation of mice. It is shown that the antiradiation of these compounds depend on the dose of preparations and time of the application before or after irradiation of mice. "Serpisten" prevented the decrease of the growth of the body mass of irradiated mice. The normalization of the phospholipid composition of the mice liver and blood erythrocytes for the most investigated parameters revealed under the application of this compound at the dose of 50 mg/kg after the irradiation of animals. The capacity of "serpisten" to decompose of peroxides is shown in vitro. Inokosterone had the certain anabolic properties, caused the normalization of the total peroxidase activity of blood and intensity of the lipid peroxidation (LPO) in brain and in liver, and also the repair of the interrelation between the LPO intensity and catalase activity in the irradiated mice liver. The obtained results allow to conclude that the antiradiation properties of the ecdysteroid-containing preparations under the chronic low intensity irradiation of animals at the low dose due to their capacity to depend on the LPO regulatory system parameters.

Background: Follicular fluid meiosis-activating sterol (FF-MAS) protects young oocytes from precocious chromatid separation (predivision). Reduced expression of cohesion and checkpoint proteins and predivision has been hypothesized to occur in age-related aneuploidy in oocytes., Methods: To know whether FF-MAS also protects aged oocytes from predivision and from age-related non-disjunction, we analysed chromosome constitution in mouse oocytes matured spontaneously with or without 10 microM FF-MAS and in hypoxanthine (HX)-arrested young and aged oocytes induced to resume maturation by FF-MAS. Messenger RNA for checkpoint protein MAD2 and cohesion protein SMC1beta was compared between oocytes matured with or without FF-MAS., Results: Aged oocytes possessed many bivalents with single distal chiasma at meiosis I. Predivision was especially high in aged oocytes cultured sub-optimally to metaphase II in alpha-minimum essential medium (alpha-MEM). FF-MAS reduced predivision significantly (P < 0.001) but neither reduced non-disjunction nor induced aneuploidy in aged oocytes. Polyploidy was high in FF-MAS-stimulated maturation, in particular in the aged oocytes (P > 0.001). Relative levels of Smc1beta mRNA appeared increased by maturation in FF-MAS, and mitochondrial clustering was restored., Conclusions: Sister chromatids of aged oocytes appear to be highly susceptible to precocious chromatid separation, especially when maturation is under sub-optimal conditions, e.g. in the absence of cumulus and FF-MAS. This may relate to some loss of chromatid cohesion during ageing. FF-MAS protects aged oocytes from predivision during maturation, possibly by supporting Smc1beta expression, thus reducing risks of meiotic errors, but it cannot prevent age-related non-disjunction. Aged oocytes appear prone to loss of co-ordination between nuclear maturation and cytokinesis suggesting age-related relaxed cell cycle control.

Synthesis of five novel Delta8(14)-15-ketosterols comprising modified side chains starting from ergosterol is described. Ergosteryl acetate was converted into (22E)-3beta-acetoxy-5alpha-ergosta-8(14),22-dien-15-one through three stages in 32% overall yield; further transformations of the product obtained led to (22E)-3beta-hydroxy-5alpha-ergosta-8(14),22-dien-15-one, (22S,23S)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one, (22R,23R)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one, (22R,23R)-5alpha-ergost-8(14)-en-15-on-3beta,22,23-triol and (22R,23R)-3beta-hydroxy-22,23-isopropylidenedioxy-5alpha-ergost-8(14)-en-15-one. New Delta8(14)-15-ketosterols were evaluated for their cytotoxicity and effects on sterol biosynthesis in human hepatoma Hep G2 cells in comparison with known 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one. Among the compounds tested, (22R,23R)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one was found to be the most potent inhibitor of sterol biosynthesis (IC(50)=0.6+/-0.2microM), whereas (22R,23R)-5alpha-ergost-8(14)-en-15-on-3beta,22,23-triol exhibited the highest cytotoxicity (TC(50)=12+/-3microM at a 24h incubation).

Purpose: Studies in mice and humans have shown that Follicular Fluid - Meiosis Activating Sterol (FF-MAS) induces meiotic maturation of immature oocytes in vitro. A multicenter, prospectively randomised study evaluated chromosomal status of embryos from FSH/hCG primed human immature oocytes, cultured with or without FF-MAS., Methods: Denuded immature oocytes (n=365) were randomly allocated into inert control, FF-MAS 5 microM or 20 microM. Seventy +/-2 hours after ICSI on matured oocytes, all cleaved embryos were fixed for fluorescence in situ hybridisation analysis., Results: Only 15% of oocytes resulted in cleaved embryos. GV oocytes matured at significantly lower rates (14% and 7%) in the two FF-MAS groups compared to the inert control group (47%). High rates of chromosomal abnormalities were found in all groups., Conclusion: Immature oocytes showed poor development with high rates of embryo chromosomal abnormalities. Exposure to FF-MAS in the concentrations, duration and/or formulation used in this study did not improve the results.

The potential to promote neovascularization in ischemic tissues using exogenous agents has become an exciting area of therapeutics. In an attempt to identify novel small molecules with angiogenesis promoting activity, we screened a library of natural products and identified a sulfated steroid, sokotrasterol sulfate, that induces angiogenesis in vitro and in vivo. We show that sokotrasterol sulfate promotes endothelial sprouting in vitro, new blood vessel formation on the chick chorioallantoic membrane, and accelerates angiogenesis and reperfusion in a mouse hindlimb ischemia model. We demonstrate that sulfation of the steroid is critical for promoting angiogenesis, as the desulfated steroid exhibited no endothelial sprouting activity. We thus developed a chemically synthesized sokotrasterol sulfate analog, 2beta,3alpha,6alpha-cholestanetrisulfate, that demonstrated equivalent activity in the hindlimb ischemia model and resulted in the generation of stable vessels that persisted following cessation of therapy. The function of sokotrasterol sulfate was dependent on cyclooxygenase-2 activity and vascular endothelial growth factor induction, as inhibition of either cyclooxygenase-2 or vascular endothelial growth factor blocked angiogenesis. Surface expression of alpha(v)beta(3) integrin was also necessary for function, as neutralization of alpha(v)beta(3) integrin, but not beta(1) integrin, binding abrogated endothelial sprouting and antiapoptotic activity in response to sokotrasterol sulfate. Our findings indicate that sokotrasterol sulfate and its analogs can promote angiogenesis in vitro and in vivo and could potentially be used for promoting neovascularization to relieve the sequelae of vasoocclusive diseases.

Population density, temperature and food availability all regulate the formation of the Caenorhabditis elegans dauer larva by modulating endocrine signaling pathways. The orphan nuclear receptor DAF-12 is pivotal for the decision to form a dauer or to undergo normal reproductive development. The DAF-12 ligand has been predicted to be a sterol that is metabolized by DAF-9, a cytochrome P450. Here we chemically characterize purified lipophilic nematode extracts and show that the ligand for DAF-12 contains a carboxyl moiety and is likely to be derived from a sterol. Using a candidate ligand approach we find that the C27 bile acid cholestenoic acid (5-cholesten-3beta-ol-(25S)-carboxylic acid) promotes reproductive growth in dauer-constitutive mutants in a daf-9- and daf-12-dependent manner. Furthermore, we find that cholestenoic acid can act as a DAF-12 ligand by activating DAF-12 in a cell-based transcription assay. Analysis of dauer-rescuing lipophilic extracts from nematodes by gas chromatography-mass spectrometry indicates the presence of several regioisomers of cholestenoic acid that are distinct from Delta(5)-cholestenoic acid and are not present in extracts from daf-9 mutants. These data suggest that carboxylated sterols may be key determinants of life history.

Follicular fluid meiosis-activating sterol (FF-MAS) has been isolated from the follicular fluid (FF) of several species including man. FF-MAS increases the quality of in vitro oocyte maturation, and thus the developmental potential of oocytes exposed to FF-MAS during in vitro maturation is improved. The aim of the present study was to investigate the effects of FF-MAS on porcine oocyte maturation and pronucleus formation in vitro. Porcine cumulus-oocyte complexes (COCs) were isolated from abattoir ovaries and in vitro matured for 48 h in NCSU 37 medium supplemented with 1 mg/l cysteine, 10 ng/ml epidermal growth factor and 50 microM 2-mercaptoethanol with or without 10% porcine follicular fluid (pFF). For the first 22 h, 1 mM db-cAMP and 10 I.E PMSG/hCG was added. The medium was supplemented with 1 microM, 3 microM, 10 microM, 30 microM or 100 microM FF-MAS dissolved in ethanol. After maturation the COCs were denuded mechanically using a fine glass pipette under constant pH and in vitro fertilized with fresh semen (5 x 10(5) spermatozoa/ml). The presumptive zygotes were evaluated 18 h after fertilization. The addition of pFF increased the monospermic as well as the polyspermic penetration of oocytes. In the absence of pFF, the addition of FF-MAS decreased the polyspermic penetration rate, whereas FF-MAS in combination with pFF decreased monospermic and increased polyspermic penetration. The degeneration rate of ova decreased in the presence of FF-MAS irrespective of the presence or absence of pFF. In the absence of pFF, FF-MAS at 3-10 microM increased the number of zygotes with advanced maternal pronuclear stages. In supraphysiological doses, i.e. 30-100 microM, FF-MAS dose-dependently and reversibly inhibited nuclear maturation in the absence of pFF.

Objective: To evaluate the effect of adding follicular-fluid meiosis-activating sterol (FF-MAS) in a novel 0.2% recombinant human albumin-based formulation to cumulus-enclosed oocytes on chromosomal status and development of pre-embryos., Design: Multicenter, prospective, randomized, open (double-blind for vehicle and FF-MAS groups), four parallel groups, controlled trial., Setting: Four public IVF clinics in Denmark., Patient(s): Two hundred eighteen women undergoing IVF donated 483 oocytes., Intervention(s): Follicle-stimulating hormone/hCG-primed cumulus-enclosed oocytes randomized to 4 hours of exposure to medium with 1 or 10 micromol/L of FF-MAS dissolved in 0.2% recombinant human albumin, medium with 0.2% recombinant human albumin (vehicle control), or medium alone (control) before insemination., Main Outcome Measure(s): Primary endpoint: incidence of human pre-embryos with chromosomal abnormalities. Secondary endpoint: fertilization rate, cleavage rate, and pre-embryo quality assessed after 68 hours of culture., Result(s): At pre-embryo level, the overall abnormality rates in the control, vehicle control, and 1- and 10-micromol/L FF-MAS groups were 53%, 39%, 42%, 53%, respectively, and at blastomere level 49%, 44%, 44%, and 48%, respectively. After 20 and 26 hours, the fertilization rates were between 67% and 71% in all groups. No differences in the cleavage rates were observed., Conclusion(s): The concentrations of FF-MAS in a novel 0.2% recombinant human albumin-based formulation of FF-MAS did not increase the risk of chromosomal abnormalities in pre-embryos or blastomeres. No statistically significant differences in fertilization rate, cleavage rate, or number of good quality pre-embryos were found among the four groups.

The endothelium forms a continuous monolayer at the interface between blood and tissue and contributes significantly to the sensing and transducing of signals between blood and tissue. New blood vessel formation, or angiogenesis, is initiated by the activation of endothelial cells and is an important process required for various pathological and physiological situations. This study used cleavable isotope-coded affinity tag reagents combined with mass spectrometry to investigate the molecular basis of a recently discovered angiogenesis-promoting steroid, sokotrasterol sulfate. Changes in the relative abundances of over 1000 proteins within human endothelial cells treated with sokotrasterol sulfate and vehicle-treated cells were identified and quantitated using this technique. A method that examines the entire ensemble of quantitative measurements was developed to identify proteins that showed a statistically significant change in relative abundance resulting from treatment with sokotrasterol sulfate. A total of 93 proteins was significantly up-regulated, and 37 were down-regulated in response to sokotrasterol sulfate stimulation of endothelial cells. Among the up-regulated proteins, several were identified that are novel to endothelial cells and are likely involved in cell communication and morphogenesis. These findings are consistent with a role for sokotrasterol sulfate in endothelial sprouting.

Meiosis-activating sterol (MAS) was shown to overcome the inhibitory effect of hypoxanthine on spontaneous maturation of mouse oocytes and was suggested to mediate the stimulation of meiosis by gonadotropins. Follicular fluid (FF)-MAS is synthesized by cytochrome P450 lanosterol 14alpha-demethylase (LDM). Follicular LDM was preferentially localized in oocytes by immunohistochemistry. Using [3H]acetate or R-[5-3H]mevalonate as precursors as well as high-performance liquid chromatographic and thin-layer chromatographic separation, we have measured the concentrations of de novo-synthesized lanosterol, FF-MAS, and cholesterol in rat graafian follicles, cumulus-oocyte complexes (COCs), and denuded oocytes (DOs) treated with LH, AY-9944 (an inhibitor of Delta14-reductase, which was anticipated to increase FF-MAS levels by inhibiting its metabolism), or both after 8 h of culture. In follicles, both LH and AY-9944 increased the accumulation of FF-MAS as compared to controls. In COCs, AY-9944 caused a marked increase in FF-MAS, but we were unable to detect accumulation of FF-MAS in DOs. Neither the endogenous increases in FF-MAS accumulation nor the addition of FF-MAS to the culture medium could overcome the inhibition on resumption of meiosis by phosphodiesterase inhibitors. Compared to LH-induced resumption of meiosis in follicles, that induced by AY-9944 was much delayed. These results call into question any role of FF-MAS as an obligatory mediator of LH activity on germinal vesicle breakdown. The discrepancy between the positive staining for LDM in oocytes and our inability to detect de novo synthesized FF-MAS in DOs may relate to the sensitivity of the methodology employed and either the number of oocytes used or a deficiency in LDM synthetic activity in such oocytes. Further studies are required to confirm any of these alternatives.

Background: In this study, we compared the relative ability of FSH (100 mIU/ml), epidermal growth factor (EGF) (10 ng/ml), and follicular-fluid meiosis-activating sterol (FF-MAS, 10 micromol/l) to induce meiotic resumption and polar body I (PBI) extrusion in mouse oocytes., Methods: Cumulus-enclosed oocytes (CEO) were co-incubated with meiosis-arresting agents, including 4 mmol/l hypoxanthine (Hx), 0.3 mmol/l dibutyryl cAMP (dbcAMP), and 8.5 micromol/l cilostamide, a selective inhibitor of the oocyte-specific phosphodiesterase 3 (PDE 3)., Results: In Hx-treated oocytes, FSH, EGF and FF-MAS induced meiosis resumption at very high rates, but only FSH and EGF also promoted PBI extrusion with high frequency. In experiments conducted in the presence of dbcAMP, FF-MAS was unable to promote an increase in germinal vesicle breakdown (GVBD) rate, whereas FSH and EGF generated a response similar to the Hx groups. Neither FSH, EGF nor FF-MAS caused any change in the meiotic status of CEO when meiotic arrest at the germinal vesicle (GV) stage was maintained by cilostamide. In the presence of Hx, naked oocytes (NkO) co-cultured with their cumulus cells were able to respond to the GVBD-inducing effect of FSH and EGF by resuming meiosis at high rate., Conclusions: Collectively, these results indicate that: (i) a signal triggered in cumulus cells by either FSH or EGF, but not necessarily coincident with FF-MAS, may contribute to meiotic maturation, supporting GVBD and extrusion of PBI; (ii) the transmission of this signal can occur in a paracrine fashion, at least with reference to the breakdown of the GV. It also appears that concomitant regulation of intra-oocyte cAMP degradation is a prerequisite for meiosis resumption.

Background: Follicular fluid-meiosis-activating sterol (FF-MAS) is a factor present in the pre-ovulatory follicle during the time of oocyte maturation. In mouse oocytes maturing in vitro, FF-MAS promotes the completion of meiotic maturation to metaphase II (MII) and improves competence to complete the 2-cell stage to blastocyst transition. We produced analogues of FF-MAS and selected three on the basis of potency to promote the resumption of meiosis by mouse oocytes maintained in meiotic arrest by hypoxanthine. The objective of this study was to determine whether these FF-MAS analogues also affect the quality of oocytes maturing in vitro with respect to the completion of meiotic maturation and augmenting the frequency of development to the blastocyst stage after fertilization in vitro., Methods: Cumulus cell-enclosed oocytes were isolated from the small antral follicles of 18 or 20 day post-natal mice. These oocytes normally have a reduced competence to complete meiotic maturation and preimplantation embryo development. Oocytes were isolated at the germinal vesicle stage and matured in vitro using media supplemented with 0.1% ethanol, 1 micromol/l FF-MAS, or 0.1-10 micromol/l FF-MAS analogues ZK255884 (884), ZK255933 (933) and ZK255991 (991). Oocytes that progressed to MII were fertilized in vitro and the percentage developing to the 2-cell and blastocyst stages was determined., Results: At 1 micromol/l, 991 and 933 increased the portion of oocytes progressing to MII, whereas the lowest dose of 991 and 884 was ineffective. Treatment of maturing oocytes with either 0.1 or 1 micromol/l 933 dramatically increased oocyte competence to complete preimplantation development., Conclusions: The synthetic analogue of FF-MAS, ZK255933, is a potent agonist that improves the quality of mouse oocytes matured in vitro. This compound may therefore have therapeutic value for treatment of oocytes from women undergoing therapy for infertility owing to poor oocyte quality.

The effect on cholesterol metabolism in Hep G2 hepatoma cells was studied for new analogues of 15-ketosterol [3beta-hydroxy-5alpha-cholest-8(14)-en-15-one] (I): (24S)-3beta-hydroxy-24-methyl-5alpha-cholesta-8(14),22-diene-15-one (II), (24S)-3alpha-hydroxy-24-methyl-5-alpha-cholesta-8(14),22-diene-15-one (III), and (24S)-24-methyl-5alpha-cholesta-8(14),22-diene-3,15-dione (IV). Analogues (I) and (II) were found to be equally effective inhibitors of cholesterol biosynthesis after a 3-h incubation with Hep G2 cells; however, (II) produced a stronger inhibitory effect after a 24-h incubation or after an incubation of cells preliminarily treated with the inhibitor in a medium containing no ketosterol. The ability of ketosterols to inhibit cholesterol biosynthesis decreased in the order (II) > (IV) > (III). Ketosterol (II) inhibited, whereas ketosterol (III) stimulated the biosynthesis of cholesteryl esters. (IV) stimulated the biosynthesis of cholesteryl esters at a concentration of 1-10 microM and exerted no marked effect at a concentration of 30 microM. These results indicate that delta8(14)-15-ketosterols containing a modified side chain are of interest as regulators of cholesterol metabolism in liver cells. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.