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Cholesterol oxidase is a highly demanded enzyme used in medicine, pharmacy, agriculture, chemistry, and biotechnology. It catalyzes oxidation of 3β-hydroxy-5-ene- to 3-keto-4-ene- steroids with the formation of hydrogen peroxide. Here, we expressed 6xHis-tagged mature form of the extracellular cholesterol oxidase (ChO) from the actinobacterium Nocardioides simplex VKM Ac-2033D (55.6 kDa) in Escherichia coli cells. The recombinant enzyme (ChO

Auto-oxidized oxysterols are implicated in the pathogenesis of various chronic diseases. Their concentrations are indicators of oxidative stress in vivo and associated with atherosclerosis. Subclinical hypothyroidism is related with cardiac diseases and oxidative stress, but the exact mechanisms underlying these associations are not clear yet.To investigate the auto-oxidized oxysterols, 7-ketocholesterol (7-KC) and cholestane-3β,5α,6β-triol (chol-triol), in patients with subclinical hypothyroidism, as well as to evaluate the impact of restoring euthyroidism on oxysterol concentrations.In this prospective observational study, 64 patients with newly diagnosed autoimmune thyroiditis (41 with subclinical hypothyroidism and 23 euthyroidism), and 45 healthy controls were enrolled. Age, gender, and body mass index were matched among patient groups and healthy controls. Anthropometric measurements were obtained and fasting plasma 7-ketocholesterol and cholestane-3β,5α,6β-triol concentrations were measured by using liquid chromatography coupled with tandem mass spectrometry. Levothyroxine was then administered to all patients with subclinical-hypothyroidism. After three months, measurements of the oxysterols and serum cholesterols from the patients who have become euthyroid were repeated.Concentrations of 7-ketocholesterol and cholestane-3β,5α,6β-triol were significantly higher in patients with subclinical-hypothyroidism when compared to both euthyroid patients and healthy controls (p 0.001 for both oxysterols). After restoration of euthyroidism, concentrations of 7-ketocholesterol and cholestane-3β,5α,6β-triol decreased significantly and reached similar concentrations observed in healthy controls (p 0.001 for both oxysterols).Auto-oxidized oxysterol species are higher in patients with mild thyroid dysfunction, and supported the rationale for treating subclinical-hypothyroidism.

Previous investigations of the

Previous research complemented with results on BIA1 enzymatic activities shows that the enzyme regulates brassinosteroid homeostasis via mono- and diacetylation of castasterone

ObjectiveTo characterize subclinical abnormalities in asymptomatic heterozygote NPC1 mutation carriers as markers of neurodegeneration.MethodsMotor function, cognition, mood, sleep, and smell function were assessed in 20 first-degree heterozygous relatives of patients with Niemann-Pick disease type C (NPC) (13 male, age 52.7 ± 9.9 years). Video-oculography and abdominal ultrasound with volumetry were performed to assess oculomotor function and size of liver and spleen. NPC biomarkers in blood were analyzed. 18F-fluorodesoxyglucose PET was performed (n = 16) to detect patterns of brain hypometabolism.ResultsNPC heterozygotes recapitulated characteristic features of symptomatic NPC disease and demonstrated the oculomotor abnormalities typical of NPC. Hepatosplenomegaly (71%) and increased cholestantriol (33%) and plasma chitotriosidase (17%) levels were present. The patients also showed signs seen in other neurodegenerative diseases, including hyposmia (20%) or pathologic screening for REM sleep behavior disorder (24%). Cognitive function was frequently impaired, especially affecting visuoconstructive function, verbal fluency, and executive function. PET imaging revealed significantly decreased glucose metabolic rates in 50% of participants, affecting cerebellar, anterior cingulate, parieto-occipital, and temporal regions, including 1 with bilateral abnormalities.ConclusionNPC heterozygosity, which has a carrier frequency of 1:200 in the general population, is associated with abnormal brain metabolism and functional consequences. Clinically silent heterozygous gene variations in NPC1 may be a risk factor for late-onset neurodegeneration, similar to the concept of heterozygous GBA mutations underlying Parkinson disease.

International audience; Tumour cells are characterized by having lost their differentiation state. They constitutively secrete small extracellular vesicles (sEV) called exosomes when they come from late endosomes. Dendrogenin A (DDA) is an endogenous tumour suppressor cholesterol-derived metabolite. It is a new class of ligand of the nuclear Liver X receptors (LXR) which regulate cholesterol homeostasis and immunity. We hypothesized that DDA, which induces tumour cell differentiation, inhibition of tumour growth and immune cell infiltration into tumours, could functionally modify sEV secreted by tumour cells. Here, we have shown that DDA differentiates tumour cells by acting on the LXRβ. This results in an increased production of sEV (DDA-sEV) which includes exosomes. The DDA-sEV secreted from DDA-treated cells were characterized for their content and activity in comparison to sEV secreted from control cells (C-sEV). DDA-sEV were enriched, relatively to C-sEV, in several proteins and lipids such as differentiation antigens, "eat-me" signals, lipidated LC3 and the endosomal phospholipid bis(monoacylglycero)phosphate, which stimulates dendritic cell maturation and a Th1 T lymphocyte polarization. Moreover, DDA-sEV inhibited the growth of tumours implanted into immunocompetent mice compared to control conditions. This study reveals a pharmacological control through a nuclear receptor of exosome-enriched tumour sEV secretion, composition and immune function. Targeting the LXR may be a novel way to reprogram tumour cells and sEV to stimulate immunity against cancer.

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

This review comprehensively describes the recent advances in the synthesis and pharmacological evaluation of steroid polyamines squalamine, trodusquemine, ceragenins, claramine, and their diverse analogs and derivatives, with a special focus on their complete synthesis from cholic acids, as well as an antibacterial and antiviral, neuroprotective, antiangiogenic, antitumor, antiobesity and weight-loss activity, antiatherogenic, regenerative, and anxiolytic properties. Trodusquemine is the most-studied small-molecule allosteric PTP1B inhibitor. The discovery of squalamine as the first representative of a previously unknown class of natural antibiotics of animal origin stimulated extensive research of terpenoids (especially triterpenoids) comprising polyamine fragments. During the last decade, this new class of biologically active semisynthetic natural product derivatives demonstrated the possibility to form supramolecular networks, which opens up many possibilities for the use of such structures for drug delivery systems in serum or other body fluids.

Covering: 1993 to 2021 (mainly 2017–2021)Alzheimer's and Parkinson's diseases are neurodegenerative conditions affecting over 50 million people worldwide. Since these disorders are still largely intractable pharmacologically, discovering effective treatments is of great urgency and importance. These conditions are characteristically associated with the aberrant deposition of proteinaceous aggregates in the brain, and with the formation of metastable intermediates known as protein misfolded oligomers that play a central role in their aetiology. In this Highlight article, we review the evidence at the physicochemical, cellular, animal model and clinical levels on how the natural products squalamine and trodusquemine offer promising opportunities for chronic treatments for these progressive conditions by preventing both the formation of neurotoxic oligomers and their interaction with cell membranes.

Covering: 1993 to 2021 (mainly 2017-2021)Alzheimer's and Parkinson's diseases are neurodegenerative conditions affecting over 50 million people worldwide. Since these disorders are still largely intractable pharmacologically, discovering effective treatments is of great urgency and importance. These conditions are characteristically associated with the aberrant deposition of proteinaceous aggregates in the brain, and with the formation of metastable intermediates known as protein misfolded oligomers that play a central role in their aetiology. In this Highlight article, we review the evidence at the physicochemical, cellular, animal model and clinical levels on how the natural products squalamine and trodusquemine offer promising opportunities for chronic treatments for these progressive conditions by preventing both the formation of neurotoxic oligomers and their interaction with cell membranes.

Brassinosteroids (BRs) are steroid phytohormones that are known to regulate plant growth and nutrient uptake and distribution. However, how BRs regulate nutrient uptake and balance in legume species is not fully understood. Here, we show that optimal BR levels are required for soybean (Glycine max L.) seedling growth, as treatments with both 24-epicastasterone (24-epiCS) and the BR biosynthesis inhibitor propiconazole (PPZ) inhibit root growth, including primary root elongation and lateral root formation and elongation. Specifically, 24-epiCS and PPZ reduced the total phosphorus and potassium levels in the shoot and affected several minor nutrients, such as magnesium, iron, manganese, and molybdenum. A genome-wide transcriptome analysis identified 3774 and 4273 differentially expressed genes in the root tip after brassinolide and PPZ treatments, respectively. The gene ontology (GO) analysis suggested that genes related to “DNA-replication”, “microtubule-based movement”, and “plant-type cell wall organization” were highly responsive to the brassinolide and PPZ treatments. Furthermore, consistent with the effects on the nutrient concentrations, corresponding mineral transporters were found to be regulated by BR levels, including the GmPHT1s, GmKTs, GmVIT2, GmZIPs, and GmMOT1 genes. Our study demonstrates that optimal BR levels are important for growth and mineral nutrient homeostasis in soybean seedlings.

Brassinosteroids (BRs) are essential phytohormones, which bind to the plant receptor, BRI1, to regulate various physiological processes. The molecular mechanism of the perception of BRs by the ectodomain of BRI1 remains not fully understood. It also remains elusive why a substantial difference in biological activity exists between the BRs. In this work, we study the binding mechanisms of the two most bioactive BRs, brassinolide (BLD) and castasterone (CAT), using molecular dynamics simulations. We report free-energy landscapes of the binding processes of both ligands, as well as detailed ligand binding pathways. Our results suggest that CAT has a lower binding affinity compared to BLD due to its inability to form hydrogen-bonding interactions with a tyrosine residue in the island domain of BRI1. We uncover a conserved nonproductive binding state for both BLD and CAT, which is more stable for CAT and may further contribute to the bioactivity difference. Finally, we validate past observations about the conformational restructuring and ordering of the island domain upon BLD binding. Overall, this study provides new insights into the fundamental mechanism of the perception of the two most bioactive BRs, which may create new avenues for genetic and agrochemical control of their signaling cascade.

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

Cholestane-3β,5α,6β-triol (3β,5α,6β-triol) is formed from cholestan-5,6-epoxide (5,6-EC) in a reaction catalysed by cholesterol epoxide hydrolase, following formation of 5,6-EC through free radical oxidation of cholesterol. 7-Oxocholesterol (7-OC) and 7β-hydroxycholesterol (7β-HC) can also be formed by free radical oxidation of cholesterol. Here we investigate how 3β,5α,6β-triol, 7-OC and 7β-HC are metabolised to bile acids. We show, by monitoring oxysterol metabolites in plasma samples rich in 3β,5α,6β-triol, 7-OC and 7β-HC, that these three oxysterols fall into novel branches of the acidic pathway of bile acid biosynthesis becoming (25R)26-hydroxylated then carboxylated, 24-hydroxylated and side-chain shortened to give the final products 3β,5α,6β-trihydroxycholanoic, 3β-hydroxy-7-oxochol-5-enoic and 3β,7β-dihydroxychol-5-enoic acids, respectively. The intermediates in these pathways may be causative of some phenotypical features of, and/or have diagnostic value for, the lysosomal storage diseases, Niemann Pick types C and B and lysosomal acid lipase deficiency. Free radical derived oxysterols are metabolised in human to unusual bile acids via novel branches of the acidic pathway, intermediates in these pathways are observed in plasma., Graphical abstract Image 1, Highlights • Free radical derived oxysterols metabolised to unusual bile acids. • Metabolism proceeds via novel branches of the acidic pathway. • Bile acids with:- 3β,5α,6β-triol; 3β,7β-diol-5-ene or 3β-ol-5-en-7-one in A/B-ring. • Unusual bile acids found in plasma from patients with NPC, NPB and LALD. • Intermediates in the biosynthesis of unusual bile acids found in plasma.

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

The bile alcohol 5β-scymnol ([24R]-(+)-5β-cholestan-3α,7α,12α,24,26,27-hexol) is a therapeutic nutraceutical derived from marine sources, however very little is known about its potential for biotransformation as a xenobiotic in higher vertebrates. In this study, biotransformation products of scymnol catalysed by liver microsomes isolated from normal and streptozotocin (STZ)-treated male Wistar rats were characterised by liquid chromatography-tandem mass spectroscopy (LC-MSMS). In order of increasing polarity relative to the reversed phase sorbent, structural assignments were made for four biotransformation products, namely 3-oxoscymnol (5β-cholestan-3-one-7α,12α,24,26,27-pentol); 7-oxoscymnol (5β-cholestan-7-one-3α,12α,24,26,27-pentol); 3β-scymnol (5β-cholestan-3β,7α,12α,24,26,27-hexol) and 6β-hydroxyscymnol (5β-cholestan-3α,6β,7α,12α,24,26,27-heptol). In addition, a total of eight biotransformation products were characterised from microsomal incubations of crude oxoscymnol compounds, namely 7β-scymnol; 3,12-dioxoscymnol; 3,7-dioxoscymnol; 7,12-dioxoscymnol; 12-oxo-3β-scymnol; 7-oxo-3β-scymnol; 6β-hydroxy-12-oxoscymnol and 6β-hydroxy-7-oxoscymnol. Collectively, the results indicate hepatic enzyme-catalysed hydroxylation, dehydrogenation and epimerisation reactions on the steroid nucleus of scymnol, and provide an insight into biotransformation pathways for scymnol use as a therapeutic nutraceutical in higher vertebrates.

Evidence from clinical, genetic, and medical studies has shown the neuronal developmental disorder aspect of schizophrenia (SZ). Whereas oxysterols are vital factors in neurodevelopment, it is still unknown whether they are involved in the pathophysiology of SZ. The current study aims to explore the profile of oxysterols in plasma, ratio to total cholesterol (Tchol) and the association with clinical factors in patients with SZ. Forty men diagnosed with SZ and forty healthy controls matched for age and sex were included in the study. The ratios of cholestane-3β,5α,6β-triol, 27-hydroxycholesterol (27-OHC) and Cholestanol to Tchol increased in the schizophrenic group compared to controls. However, levels of 24S-hydroxycholesterol (24-OHC) were not significantly different between patients and controls. For the SZ patients, the plasma 24-OHC levels were positively correlated with the positive and negative syndrome total scores (PANSS) but negatively correlated with the Montreal Cognitive Assessment scores (MOCA). Moreover, the ratio Cholestanol to Tchol was negatively correlated with MOCA scores and positively correlated with PANSS general. The binary logistic regression analysis revealed that the ratio Cholestane-3β,5α,6β-triol/TChol could be considered as an independent risk factor for SZ. On the other hand, the receiver's operating characteristics analysis corresponding to potential biomarkers on SZ showed Areas Under the Curve (AUCs) of 82.1%; 69.7% and 77.6% for the ratio of Cholestane-3β,5α,6β-triol/TChol, 27-OHC/TChol and Cholestanol/TChol respectively. The relevance of Cholestane-3β,5α,6β-triol, 27-OHC and Cholestanol assays as biomarkers of this disease deserves further investigation.

Background: Parkinson’s disease (PD) is a progressive neurodegenerative disorder thought to be caused by accumulation of α-synuclein (α-syn) within the brain, autonomic nerves, and the enteric nervous system (ENS). Involvement of the ENS in PD often precedes the onset of the classic motor signs of PD by many years at a time when severe constipation represents a major morbidity. Studies conducted in vitro and in vivo, have shown that squalamine, a zwitterionic amphipathic aminosterol, originally isolated from the liver of the dogfish shark, effectively displaces membrane-bound α-syn. Objective: Here we explore the electrophysiological effect of squalamine on the gastrointestinal (GI) tract of mouse models of PD engineered to express the highly aggregating A53T human α-syn mutant. Methods: GI motility and in vivo response to oral squalamine in PD model mice and controls were assessed using an in vitro tissue motility protocol and via fecal pellet output. Vagal afferent response to squalamine was measured using extracellular mesenteric nerve recordings from the jejunum. Whole cell patch clamp was performed to measure response to squalamine in the myenteric plexus. Results: Squalamine effectively restores disordered colonic motility in vivo and within minutes of local application to the bowel. We show that topical squalamine exposure to intrinsic primary afferent neurons (IPANs) of the ENS rapidly restores excitability. Conclusion: These observations may help to explain how squalamine may promote gut propulsive activity through local effects on IPANs in the ENS, and further support its possible utility in the treatment of constipation in patients with PD.

Background & Aims GPBAR1, also known as TGR5, is a G protein–coupled receptor activated by bile acids. Hepatic innate immune cells are involved in the immunopathogenesis of human liver diseases and in several murine hepatitis models. Here, by using genetic and pharmacological approaches, we provide evidence that GPBAR1 ligation attenuates the inflammation in rodent models of hepatitis. Material and methods Hepatitis was induced by concanavalin A (Con A) or α-galactosyl-ceramide (α-GalCer). 6b-Ethyl-3a,7b-dihydroxy-5b-cholan-24-ol (BAR501), a selective agonist of GPBAR1, was administrated by o.s. Results In the mouse models of hepatitis, the genetic ablation of Gpabar1 worsened the severity of liver injury and resulted in a type I NKT cells phenotype that was biased toward a NKT1, a proinflammatory, IFN-γ producing, NKT cells subtype. Further on, NKT cells from GPBAR1–/– mice were sufficient to cause a severe hepatitis when transferred to naïve mice. In contrast, GPBAR1 agonism rescued wild-type mice from acute liver damage and redirects the NKT cells polarization toward a NKT10, a regulatory, IL-10 secreting, type I NKT cell subset. In addition, GPBAR1 agonism significantly expanded the subset of IL-10 secreting type II NKT cells. RNAseq analysis of both NKT cells type confirmed that IL-10 is a major target for GPABR1. Accordingly, IL-10 gene ablation abrogated protection afforded by GPBAR1 agonism in the Con A model. Conclusion Present results illustrate a role for GPBAR1 in regulating liver NKT ecology. Because NKT cells are an essential component of liver immune system, our data provide a compelling evidence for a GPBAR1-IL-10 axis in regulating of liver immunity., Graphical abstract

Sewage pollution is a principal factor of decreasing water quality, although it has not been considered a real impact in Amazonia that is still considered a pristine environment around the world. Thus, this study aimed to assess the levels of sewage contamination in sediments from three streams crossing Manaus − a Brazilian city of 2,403,796 inhabitants in the heart of the Amazon rain forest. Cholesterol, cholestanol, brassicasterol, ergosterol, stigmasterol, β-sitosterol, campesterol, stigmastanol, coprostanol, and epicoprostanol levels were determined by liquid chromatography tandem mass spectrometry (LC−MS/MS). The fecal indicator, coprostanol, was found in high concentrations (509−12 830 ng g−1) and high relative proportions (21–54%) in all samples collected in the Mindu stream that crosses many heavily populated districts of the city, and in the Quarenta stream that crosses the Industrial District of Manaus. The sediments of the Tarumã-Açu stream also presented coprostanol; however, concentrations (

Gas chromatography-mass spectrometry (GC-MS) analysis revealed that castasterone and its biosynthetic precursors are found in

When a spermatozoon shows chemotactic behavior, transient [Ca2+]i increases in the spermatozoon are induced by an attractant gradient. The [Ca2+]i increase triggers a series of stereotypic responses of flagellar waveforms that comprise turning and straight-swimming. However, the molecular mechanism of [Ca2+]i modulation controlled by the attractants is not well defined. Here, we examined receptive mechanisms for the sperm attractant, SAAF, in the ascidian, Ciona intestinalis, and identified a plasma membrane Ca2+-ATPase (PMCA) as a SAAF-binding protein. PMCA is localized in sperm flagella membranes and seems to interact with SAAF through basic amino acids located in the second and third extracellular loops. ATPase activity of PMCA was enhanced by SAAF, and PMCA inhibitors, 5(6)-Carboxyeosin diacetate and Caloxin 2A1, inhibited chemotactic behavior of the sperm. Furthermore, Caloxin 2A1 seemed to inhibit efflux of [Ca2+]i in the sperm, and SAAF seemed to competitively reduce the effect of Caloxin 2A1. On the other hand, chemotactic behavior of the sperm was disordered not only at low-Ca2+, but also at high-Ca2+ conditions. Thus, PMCA is a potent candidate for the SAAF receptor, and direct control of Ca2+ efflux via PMCA is a fundamental mechanism to mediate chemotactic behavior in the ascidian spermatozoa.

Purpose of review To update researchers of recently discovered metabolites of cholesterol and of its precursors and to suggest relevant metabolic pathways. Recent findings Patients suffering from inborn errors of sterol biosynthesis, transport and metabolism display unusual metabolic pathways, which may be major routes in the diseased state but minor in the healthy individual. Although quantitatively minor, these pathways may still be important in healthy individuals. Four inborn errors of metabolism, Smith-Lemli-Opitz syndrome, cerebrotendinous xanthomatosis and Niemann Pick disease types B (NPB) and C (NPC) result from mutations in different genes but can generate elevated levels of the same sterol metabolite, 7-oxocholesterol, in plasma. How this molecule is metabolized further is of great interest as its metabolites may have an important role in embryonic development. A second metabolite, abundant in NPC and NPB diseases, cholestane-3β,5α,6β-triol (3β,5α,6β-triol), has recently been shown to be metabolized to the corresponding bile acid, 3β,5α,6β-trihydroxycholanoic acid, providing a diagnostic marker in plasma. The origin of cholestane-3β,5α,6β-triol is likely to be 3β-hydroxycholestan-5,6-epoxide, which can alternatively be metabolized to the tumour suppressor dendrogenin A (DDA). In breast tumours, DDA levels are found to be decreased compared with normal tissues linking sterol metabolism to cancer. Summary Unusual sterol metabolites and pathways may not only provide markers of disease, but also clues towards cause and treatment.

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

5α-Cyprinol 27-sulfate is the major biliary bile salt present in cypriniform fish including the zebrafish (Danio rerio). The current study was designed to identify the zebrafish cytosolic sulfotransferase (Sult) enzyme(s) capable of sulfating 5α-cyprinol and to characterize the zebrafish 5α-cyprinol-sulfating Sults in comparison with human SULT2A1. Enzymatic assays using zebrafish homogenates showed 5α-cyprinol-sulfating activity. A systematic analysis, using a panel of recombinant zebrafish Sults, revealed two Sult2 subfamily members, Sult2st2 and Sult2st3, as major 5α-cyprinol-sulfating Sults. Both enzymes showed higher activities using 5α-cyprinol as the substrate, compared to their activity with DHEA, a representative substrate for mammalian SULT2 family members, particularly SULT2A1. pH-Dependence and kinetics experiments indicated that the catalytic properties of zebrafish Sult2 family members in mediating the sulfation of 5α-cyprinol were different from those of either zebrafish Sult3st4 or human SULT2A1. Collectively, these results imply that both Sult2st2 and Sult2st3 have evolved to sulfate specifically C27-bile alcohol, 5α-cyprinol, in Cypriniform fish, whereas the enzymatic characteristics of zebrafish Sult3 members, particularly Sult3st4, correlated with those of human SULT2A1.

Plant hormones (PH) adjust plant growth to environmental conditions such as nutrient availability. Although responses of individual PHs to growth-determining nutrient supplies have been reported, little is known about simultaneous dynamics in the metabolism of different PH species. Brassica napus seedlings were grown under increasing supply of B, and LC-MS/MS was used to characterize bioactive forms of different PH species together with several of their precursors, storage and inactivated forms. Increasing shoot B concentrations in response to B supply were accompanied by decreasing concentrations of abscisic acid (ABA) and indole-3-acetic acid (IAA), which appeared to be synthesized under B deficiency mainly via indole-3-acetonitrile (IAN). By contrast, shoot B concentrations correlated closely with cytokinins, and the B-dependent growth response appeared to be triggered primarily by de-novo synthesis of cytokinins and by re-routing less active towards highly active forms of cytokinin. Also gibberellin biosynthesis strongly increased with B supply, in particular gibberellin species from the non-13-hydroxylation pathway. The brassinosteroid castasterone appeared to support shoot growth primarily at suboptimal B nutrition. These results indicate that a variable B nutritional status causes coordinated changes in PH metabolism as prerequisite for an adjusted growth response.

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

Niemann-Pick type C (NPC) is a neurological disease caused by an intracellular cholesterol accumulation. Cholesterol oxidation product cholestane-3β,5α,6β-triol (C-triol) serves as diagnostic biomarker for NPC, but its measurement in the routine laboratory remains difficult. We developed an isotope dilution gas chromatography-mass spectrometry (GC-MS) method permitting screening for NPC in plasma. 1440 plasma samples obtained from clinically suspicious patients were subjected to alkaline saponification. C-triol was extracted with carbon tetrachloride, transformed into the trimethylsilylethers, separated on a fused silica capillary column with a nonpolar silicone stationary phase, and analyzed by GC-MS. NPC diagnosis was confirmed by DNA sequencing. The method was linear over a concentration range of 0.03-200ng/mL with a mean recovery rate of 98.6%. The intra- and inter-day variation coefficients assessed at two concentrations were below 15%. Limits of quantification (LOQ) and detection (LOD) were 0.03ng/mL and 0.01ng/mL, respectively. Receiver operating characteristic (ROC) analysis estimated that the area under curve was 0.997 implying a significant discriminatory power to identify subjects with NPC. Nevertheless, 13 NPC patients and 29 control subjects confirmed by sequencing showed false negative or positive results, respectively. Two patients with cerebrotendinous xanthomatosis showed a 5-10-fold increase in C-triol levels. We developed a quick and sensitive GC-MS method for determination of C-triol, which may serve as a simple and inexpensive diagnostic tool aiding NPC diagnosis in a routine hospital laboratory. As C-triol elevation is not limited to NPC, the NPC diagnosis has to be confirmed by DNA sequencing.

In recent years the oxysterol species cholestane-3β, 5α, 6β-triol (C-triol) has found application as a diagnostic biomarker for Niemann-Pick disease type C. Other studies have described increased C-triol in patients with Niemann-Pick disease type A/B and milder increases in lysosomal acid lipase deficiency (LALD), whereas they note normal C-triol levels in Smith-Lemli-Opitz syndrome (SLOS) and familial hypercholesterolaemia (FH) patients. Herein, we review data collected in our laboratory during method evaluation along with 5 years of routine analysis and present findings which differ from those reported by other groups with respect to LALD, SLOS and FH in particular, whilst providing further evidence regarding the clinical sensitivity and specificity of this biomarker, which are difficult to accurately assess. All of our Wolman disease (severe LALD) patients have demonstrated gross elevations of C-triol at diagnosis, with reduction to normal levels after induction of enzyme replacement therapy. In diagnostic specimens from SLOS patients we observed very low or undetectable C-triol levels whereas in post-therapeutic SLOS patients demonstrated normalised levels; we also describe a homozygous FH patient in which C-triol is significantly elevated. Upon investigation, we found that C-triol was formed artefactually from cholesterol during our sample preparation, i.e. this is a false positive of analytical origin; at present it is unclear whether similar effects occur during sample preparation in other laboratories. Our data demonstrates clinical sensitivity of 100% during routine application to diagnostic specimens; this is in keeping with other estimates, yet in a small proportion of patients diagnosed prior to C-triol measurement, either by Filipin staining of fibroblasts or molecular genetics, we have observed normal C-triol concentrations. Clinical specificity of C-triol alone is 93.4% and 95.3% when performed in conjunction with lysosomal enzymology. These performance statistics are very similar to those achieved with Filipin staining of cultured fibroblasts in the 5 years preceding introduction of C-triol to routine use in our laboratory. It is increasingly apparent to us that although this analyte is a very useful addition to the diagnostic tools available for NPC, with considerable advantages over more invasive and time-consuming methods, the interpretation of results is complex and should be undertaken only in light of clinical details and results of other analyses including enzymology for lysosomal acid lipase and acid sphingomyelinase.

International audience; Dendrogenin A (DDA) is a newly-discovered steroidal alkaloid, which remains to date the first ever found in mammals. DDA is a cholesterol metabolites that induces cancer cell differentiation and death in vitro and in vivo, and thus behave like a tumor suppressor metabolite. Preliminary studies performed on 10 patients with estrogen receptor positive breast cancers (ER(+)BC) showed a strong decrease in DDA levels between normal matched tissue and tumors. This suggests that a deregulation on DDA metabolism is associated with breast carcinogenesis. To further investigate DDA metabolism on large cohorts of patients we have developed an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS) procedure for the quantification of DDA in liquid and in solid tissues. This method enabled the identification of DDA analogues such as its geometric isomer C17 and dendrogenin B (C26) in human samples showing that other 5,6α-epoxycholesterol conjugation products with biogenic amines exist as endogenous metabolites . We report here the first complete method of quantification of DDA in liquid and solid tissues using hydrophilic interaction liquid chromatography (HILIC). Two different methods of extraction using either a Bligh and Dyer organic extraction or protein precipitation were successfully applied to quantify DDA in solid and liquid tissues. The protein precipitation method was the fastest. The fact that this method is automatable opens up possibilities to study DDA metabolism in large cohorts of patients.

International audience; Dendrogenin A (DDA) is a tumor suppressor mammalian cholesterol-derived metabolite and a new class of ligand of the Liver X receptor (LXR), which displays tumor cell differentiation. In human MCF7 breast adenocarcinoma cells, DDA-induced cell differentiation was associated with an increased accumulation of neutral lipids and proteins found in milk indicating that DDA re-activates some functions of lactating cells. Active iodide transport occurs in the normal lactating mammary cells through the sodium/iodide symporter (NIS) and iodide (I) is secreted into milk to be used by the nursing newborn for thyroid hormones biosynthesis. In the present study, we assessed whether DDA may induce other characteristic of lactating cells such as NIS expression and iodine uptake in MCF7 breast cancer cells and extended this study to the papillary B-CPAP and undifferentiated anaplastic 8505c thyroid cancer cells. Moreover, we evaluated DDA impact on the expression of thyroid specific proteins involved in thyroid hormone biogenesis. We report here that DDA induces NIS expression in MCF7 cells and significantly increases the uptake of 131-I by acting through the LXR. In addition, DDA induces phenotypic, molecular and functional characteristics of redifferentiation in the two human thyroid carcinoma cell lines and the uptake of 131-I in the undifferentiated 8505c cells was associated with a strong expression of all the specific proteins involved in thyroid hormone biosynthesis, TSH receptor, thyroperoxidase and thyroglobulin. 131-I incorporation in the 8505c cells was stimulated by DDA as well as by the synthetic LXR ligand, GW3965. Together these data show that the re-differentiation of breast and thyroid cancer cells by DDA, is associated with the recovery of functional NIS expression and involves an LXR-dependent mechanism. These results open new avenues of research for the diagnosis of thyroid cancers as well as the development of new therapeutic approaches for radioiodine refractory thyroid cancers.

Sickle cell disease (SCD) causes anemia, oxidative stress, chronic inflammation, and lipid abnormalities. Oxysterols are oxidized derivatives of cholesterol and affect cholesterol metabolism and eryptosis. Our aim was to determine whether the plasma concentrations of 7-ketocholesterol (7-KC) and cholestane-3β,5α,6β-triol (C-triol) were associated with hemolysis and lipid profile in patients with SCD. A total of 32 steady-state pediatric patients with SCD (22 HbSS and 10 HbSs+) and 25 healthy controls were included in the study. Hemolysis parameters, ferritin, serum iron, lipids, 7-KC and C-triol concentrations of all subjects were measured. Oxysterols were quantified with N,N-dimethylglycine derivatization via LC-MS/MS. 7-KC and C-triol concentrations were found to be increased in SCD patients, while there was no difference between the HbSS and HbSs+ subgroups. 7-KC concentrations s were correlated negatively with hemoglobin and positively with lactate dehydrogenase concentrations, while C-triol concentrations were negatively correlated with HDL cholesterol. Furthermore, while 7-KC and C-triol concentrations were highly correlated among controls, there was no correlation in patients. The findings of our study suggest that 7-KC and C-triol may have a role in SCD pathophysiology. The lack of correlation in patients' 7-KC and C-triol concentrations suggest alterations in oxysterol production in patients with SCD.

Prey are under selection to minimize predation losses. In aquatic environments, many prey use chemical cues released by predators, which initiate predator avoidance. A prominent example of behavioral predator-avoidance constitutes diel vertical migration (DVM) in the freshwater microcrustacean

Angiogenesis is critical for breast cancer progression. Overexpression of HER-2/neu receptors occur in 25-30% of breast cancers, and treatment with trastuzumab inhibits HER-2-overexpressing tumor growth. Notably, HER-2-mediated signaling enhances vascular endothelial growth factor (VEGF) secretion to increase tumor-associated angiogenesis. Squalamine (aminosterol compound) suppresses VEGF-induced activation of kinases in vascular endothelial cells and inhibits tumor-associated angiogenesis. We assessed antitumor effects of squalamine either alone or with trastuzumab in nude mice bearing breast tumor xenografts without (MCF-7) or with HER2-overexpression (MCF-7/HER-2). Squalamine alone inhibited progression of MCF-7 tumors lacking HER2 overexpression, and squalamine combined with trastuzumab elicited marked inhibition of MCF-7/HER2 growth exceeding that of trastuzumab alone. MCF-7/HER-2 cells secrete higher levels of VEGF than MCF-7 cells, but squalamine elicited no growth inhibition of either MCF-7/HER-2 or MCF-7 cells in vitro. However, squalamine did stop growth of human umbilical vein endothelial cells (HUVECs) and reduced VEGF-induced endothelial tube-like formations in vitro. These effects correlated with blockade of focal adhesion kinase phosphorylation and stress fiber assembly in HUVECs. Thus, squalamine effectively inhibits growth of breast cancers with or without HER-2-overexpression, an effect due in part to blockade of tumor-associated angiogenesis.

International audience; Squalamine is a natural polycationic aminosterol extracted from the shark Squalus acanthias. Squalamine displays remarkable efficacy against antimicrobial-resistant Gram-negative and Gram-positive bacteria. Its membranolytic activity and low cytotoxicity make squalamine one of the most promising agents to fight nosocomial pathogens such as Acinetobacter baumannii. In the context of chronic diseases and therapeutic failures associated with this pathogen, the presence of dormant cells, i.e. persisters and viable but non-culturable cells (VBNCs), highly tolerant to antimicrobial compounds is problematic. The aim of this study was to investigate the antibacterial activity of squalamine against this bacterial population of A. baumannii. Bacterial dormancy was induced by cold shock and nutrient starvation in the presence of high doses of either colistin, ciprofloxacin or squalamine. Persisters and VBNCs induced by these treatments were then challenged with 100 mg/L squalamine. The efficacy of each treatment was determined by evaluating culturability on agar medium, membrane integrity (LIVE/DEAD®BacLightTM staining) and respiratory activity (BacLightTM RedoxSensorTM CTC staining) of bacteria. A. baumannii ATCC 17978 generated persisters as well as VBNCs in the presence of high doses of ciprofloxacin but not colistin or squalamine. Squalamine at 100 mg/L (below its haemolytic concentration) was able to kill dormant cells. Squalamine did not induce persister cell or VBNC formation in A. baumannii ATCC 17978. Interestingly, squalamine was significantly active against this type of dormant population generated by ciprofloxacin, making it a very promising anti-persister agent.

Tamoxifen (Tam) was developed as a ligand and modulator of estrogen receptor α (ERα) and is one of the main drugs used globally for the hormonotherapy of breast cancers. Besides ERα, Tam also binds with high affinity to the microsomal antiestrogen binding site (AEBS). The AEBS is a hetero-oligomeric proteinaceous complex with cholesterol-5,6-epoxide hydrolase (ChEH) activity that is associated with an intracellular histamine (HA) binding site. The enzymatic activities of the ChEH subunits control developmental programs in mammals and transform cholesterol-5,6-epoxides (5,6-EC) into cholestane-3β,5α,6β-triol. Inhibition of the ChEH activity by pharmacological agents such as Tam induce cancer cell re-differentiation through the accumulation of 5,6-EC. A few years ago, the putative chemical reactivity of the 5,6-EC epoxide group towards nucleophiles led our group to hypothesize that 5,6-EC could react with HA that was co-localized at the AEBS to give a new molecule involved in cell differentiation. This hypothesis was chemically tested and the conjugation of 5,6α-EC: with HA was found possible but only under catalytic conditions. It gave a stereo-selective single product of transformation which was named dendrogenin A (DDA). DDA was found to display potent cancer cell differentiation and anticancer properties in vitro and in vivo, suggesting that it was a tumor suppressor metabolite. The presence of DDA was then established in several mammalian tissues, providing the first evidence of a steroidal alkaloid metabolite in mammals. The discovery of DDA highlights a new metabolic pathway in mammals which lies at the crossroads of cholesterol and histamine metabolism and produces this tumor suppressor metabolite.

During the process of icogenin analog research, we obtained two cytotoxic steroids: compound 4 and compound 6 casually. Their in vitro antitumor activities were tested by the standard MTT assay. The results disclosed that compound 4 (IC50 = 3.65–6.90 μM) showed potential antitumor activities against HELA, KB cell lines and compound 6 (IC50 = 2.40–9.05 μM) showed potential antitumor activities against HELA, BGC-823, KB, A549, HCT-8 cell lines.

The control of the secretory activity of serotonergic neurons has been pointed out to reduce motor and non-motor side effects of the antiparkinsonian drug L-DOPA. This strategy deserves further investigation because it is presently unclear whether L-DOPA promotes a non-vesicular release of dopamine and serotonin from serotonergic neurons. To get a full neurochemical picture compatible with the existence of such a mechanism, we combined multisite intracerebral microdialysis, post mortem tissue measurement and single unit extracellular recordings in the dorsal raphe nucleus from hemiparkinsonian rats. L-DOPA (3-100mg/kg, ip.) non-homogeneously decreased extracellular serotonin levels in the striatum, substantia nigra pars reticulata, hippocampus and prefrontal cortex and homogenously serotonin tissue content in the striatum, cortex and cerebellum. L-DOPA (12mg/kg) did not modify the firing rate or pattern of serotonergic-like neurons recorded in the dorsal raphe nucleus. When focusing on serotonin release in the prefrontal cortex and the hippocampus, we found that L-DOPA (12 or 100mg/kg) enhanced serotonin extracellular levels in both regions upon Ca(2+) removal. Concomitantly, L-DOPA-stimulated dopamine release partly persisted in the absence of Ca(2+) in a region-dependent manner. Local application of the serotonin reuptake inhibitor citalopram (1µM) blunted the responses to L-DOPA (3-12mg/kg), measured as extracellular dopamine levels, most prominently in the hippocampus. These data stress that L-DOPA, already at low to moderate doses, promotes non-vesicular releases of serotonin and dopamine in a region-dependent manner.