Non-Skeletal Activities of Vitamin D: From Physiology to Brain Pathology.

Vitamin D is a secosteroid hormone regulating the expression of almost 900 genes, and it is involved in the regulation of calcium and phosphate metabolism, immune response, and brain development. Low blood vitamin D levels have been reported in patients affected by various diseases. Despite a large amount of literature data, there is uncertainty surrounding the role of vitamin D as a serum biomarker in Alzheimer's disease (AD) and Parkinson's disease (PD). Indeed, the lack of internationally recognized 25(OH)D3 reference measurement procedures and standard materials in the past led to unstandardized serum total 25(OH)D3 results among research and clinical care laboratories. Thus, most of the literature studies reported unstandardized data, which are of little use and make it difficult to draw conclusions of the role of vitamin D in AD and PD. This review summarizes the extra-skeletal actions of vitamin D, focusing its role in immunomodulation and brain function, and reports the issue of lacking standardized literature data concerning the usefulness of vitamin D as a biomarker in AD and PD.

Synthesis of Strophasterol†A Guided by a Proposed Biosynthesis and Innate Reactivity.

The synthesis of strophasterol A, a moderator of endoplasmatic reticulum (ER) stress in Alzheimer's disease, and the first member of a structurally unprecedented class of secosterols, was achieved through the implementation of a key step of its proposed biosynthesis and two C-H oxidations. Analysis of the innate reactivity of the intermediates enabled the identification of a novel way to prepare an α-chloro-γ-hydroxy-δ-keto enone, as well as its vinylogous α-ketol rearrangement to a δ-keto carboxylic acid.

Chemical alterations and regulations of biomolecules in lifestyle-related diseases.

We know experientially that not only nutrient factors but also non-nutritive functional food factors are playing important roles in maintenance of homeostasis, health promotion, and disease prevention. Although some of these effective behaviors are supported by accumulating scientific evidences, it is in general difficult to determine properly in human. Therefore, the discovering of novel biomarker and developments of the analytical method are one of the prudent strategies to understand disease etiology and evaluate efficacies of functional food factors via monitoring the pathophysiological alteration in live body, tissue, and cells. This review describes recent our findings on (1) formation mechanism, bioactivities, quantitative determination of cholesterol ozonolysis product, secosterol as possible biomarker for lifestyle-related disease, and (2) chemical biology approach for the investigating molecular mechanisms of most promising cancer chemopreventive food factors, isothiocyanate-inducing bioactivities.

Detection of novel CYP11A1-derived secosteroids in the human epidermis and serum and pig adrenal gland.

To investigate whether novel pathways of vitamin D3 (D3) and 7-dehydrocholesterol (7DHC) metabolism initiated by CYP11A1 and previously characterized in vitro, occur in vivo, we analyzed samples of human serum and epidermis, and pig adrenals for the presence of intermediates and products of these pathways. We extracted human epidermis from 13 individuals and sera from 13 individuals and analyzed them by LC/qTOF-MS alongside the corresponding standards. Pig adrenal glands were also analyzed for these steroids and secosteroids. Epidermal, serum and adrenal samples showed the presence of D3 hydroxy-derivatives corresponding to 20(OH)D3, 22(OH)D3, 25(OH)D3, 1,25(OH)2D3, 20,22(OH)2D3, 20,23(OH)2D3, 20,24(OH)2D3, 20,25(OH)2D3, 20,26(OH)2D3, 1,20,23(OH)3D3 and 17,20,23(OH)3D3, plus 1,20(OH)2D3 which was detectable only in the epidermis. Serum concentrations of 20(OH)D3 and 22(OH)D3 were only 30- and 15-fold lower than 25(OH)D3, respectively, and at levels above those required for biological activity as measured in vitro. We also detected 1,20,24(OH)3D3, 1,20,25(OH)3D3 and 1,20,26(OH)3D3 in the adrenals. Products of CYP11A1 action on 7DHC, namely 22(OH)7DHC, 20,22(OH)27DHC and 7-dehydropregnenolone were also detected in serum, epidermis and the adrenal. Thus, we have detected novel CYP11A1-derived secosteroids in the skin, serum and adrenal gland and based on their concentrations and biological activity suggest that they act as hormones in vivo.

9,11-Secosteroids and polyhydroxylated steroids from two South China Sea soft corals Sarcophyton trocheliophorum and Sinularia flexibilis.

A new 9,11-secosteroid, 25(26)-dehydrosarcomilasterol (1), two new polyhydroxylated steroids, 7α-hydroxy-crassarosterol A (2) and 11-acetoxy-7α-hydroxy-crassarosterol A (3), together with three known related ones (4-6), were isolated from the South China Sea soft corals Sarcophyton trocheliophorum and Sinularia flexibilis, respectively. The structures of the new steroids were elucidated on the basis of extensive spectroscopic analyses, comparison with the literature data and chemical correlation. Compound 2 exhibited a moderate protein tyrosine phosphatase 1B (PTP1B) inhibitory activity with an IC50 value of 33.05µM. Compounds 1-3 showed weak in vitro cytotoxicities against the tumor cell lines K562 and HL-60.

Implications of cholesterol autoxidation products in the pathogenesis of inflammatory diseases.

There is rising interest in non-enzymatic cholesterol oxidation because the resulting oxysterols have biological activity and can be used as non-invasive markers of oxidative stress in vivo. The preferential site of oxidation of cholesterol by highly reactive species is at C7 having a relatively weak carbon-hydrogen bond. Cholesterol autoxidation is known to proceed via two distinct pathways, a free radical pathway driven by a chain reaction mechanism (type I autoxidation) and a non-free radical pathway (type II autoxidation). Oxysterols arising from type II autoxidation of cholesterol have no enzymatic correlates, and singlet oxygen ((1)ΔgO2) and ozone (O3) are the non-radical molecules involved in the mechanism. Four primary derivatives are possible in the reaction of cholesterol with singlet oxygen via ene addition and the formation of 5α-, 5ß-, 6α- and 6ß-hydroxycholesterol preceded by their respective hydroperoxyde intermediates. The reaction of ozone with cholesterol is very fast and gives rise to a complex array of oxysterols. The site of the initial ozone reaction is at the Δ5,6 -double bond and yields 1,2,3-trioxolane, a compound that rapidly decomposes into a series of unstable intermediates and end products. The downstream product 3ß-hydroxy-5-oxo-5,6-secocholestan-6-al (sec-A, also called 5,6-secosterol), resulting from cleavage of the B ring, and its aldolization product (sec-B) have been proposed as a specific marker of ozone-associated tissue damage and ozone production in vivo. The relevance of specific ozone-modified cholesterol products is, however, hampered by the fact sec-A and sec-B can also arise from singlet oxygen via Hock cleavage of 5α-hydroperoxycholesterol or via a dioxietane intermediate. Whatever the mechanism may be, sec-A and sec-B have no enzymatic route of production in vivo and are reportedly bioactive, rendering them attractive biomarkers to elucidate oxidative stress-associated pathophysiological pathways and to develop pharmacological agents.

New vitamin D analogs as potential therapeutics in melanoma.

Extensive evidence shows that the active form of vitamin D3--1α,25-dihydroxyvitamin D3--plays an important role in cancer prevention, has tumorostatic activity and may potentially be used in therapy for melanoma. Vitamin D3 and its analogs (secosteroids) exert multiple effects on cancer cells, including inhibition of cell growth and induction of differentiation. Activity of secosteroids depends on multiple cellular factors, including expression of the vitamin D receptor. Despite its endogenous origin, the key drawback for the use of pharmacologically effective doses of 1α,25-dihydroxyvitamin D3 is its hypercalcemic effect leading to profound toxicity. The solution may lie in properties of vitamin D3 analogs with modified side chains, which demonstrate low calcemic activity but conserve the anti-tumor properties. Noncalcemic vitamin D compounds were found to be potent in multiple studies that mandate further clinical testing. Finally, recent studies revealed alternative metabolic pathways for secosteroids and new targets in the cells, which opens up new therapeutic possibilities.

Quantitative and transformation product analysis of major active physalins from Physalis alkekengi var. franchetii (Chinese lantern) using ultraperformance liquid chromatography with electrospray ionisation tandem mass spectrometry and time-of-flight mass spectrometry.

INTRODUCTION: Chinese lantern is the calyx or calyx-with-fruit of the plant Physalis alkekengi .var. franchetii (Solanaceae), and is potential material for the food and pharmaceutical industries. Physalins are the most active and representative secondary metabolites of Chinese lantern. A separation and quantification method based on UPLC-ESI-MS/MS was developed for the quantitative analysis of five active physalins. The transformation products were also detected and identified for the first time. OBJECTIVE: To establish a LC-MS/MS method to quantify five physalins in Chinese lantern for the purpose of quality control, and to identify the transformation products of 4,7-didehydrophysalin B. METHODOLOGY: The separation was carried out on an Acquity UPLC BEH Shield RP C18-column with water and acetonitrile as the mobile phase under gradient conditions. ESI-MS/MS was used as the detector to quantify the five physalins. The transformation products of 4,7-didehydroneophysalin B were detected by UPLC-PDA-ESI-MS/MS and identified through comparing their HRMS and MS² ion fragmentations with corresponding references. RESULTS: All the compounds showed good linearity (R² > 0.998). The recoveries, measured at three concentration levels, varied from 98.8 to 101.4% with RSDs < 4.5%. The total contents of the five physalins in Chinese lantern varied significantly. Three transformation products of 4,7-didehydroneophysalin B were detected and tentatively identified. CONCLUSION: The present study developed a highly effective analytical method for the quality control of Chinese lantern, and it could provide comprehensive information for quality evaluation and new drug development of Chinese lantern.

A highly sensitive LC-ESI-MS/MS method for the quantification of cholesterol ozonolysis products secosterol-A and secosterol-B after derivatization with 2-hydrazino-1-methylpyridine.

Cholesterol ozonolysis products, 3ß-hydroxy-5-oxo-5,6-secocholestan-6-al (secosterol-A) and its aldolization product 3ß-hydroxy-5ß-hydroxy-B-norcholestane-6ß-carboxaldehyde (secosterol-B) have been found in atherosclerosis plaques and the brain tissues of Alzheimer's disease patients, implicating them in the pathogenesis of cardiovascular and neurodegenerative diseases. We have recently reported that when cholesterol is oxidized with an ozone-like oxidant generated by activated mouse neutrophils, secosterol-A is generated which is then converted to secosterol-B by an aldol reaction. To investigate further pathophysiological roles of secosterols, we have developed a highly sensitive method to detect secosterol-A and -B as derivatives with 2-hydrazino-1-methylpyridine (HMP) by LC-ESI-MS/MS. The limits of detection for the HMP derivatives of secosterol-A and secosterol-B were 0.05 and 0.01fmol, respectively, which were approximately 400 and 2000 times better than those for underivatized secosterol-A and -B. We also developed a highly reproducible and accurate method to extract, purify and derivatize secosterol in small volumes of biological specimens. Using this method, we determined the levels of secosterol-A and -B as 1.4 ± 0.7 and 4.3 ± 0.8 nM, respectively, in the plasma of normal C57BL/6 mice, and in the range of 10.4 ± 16.3 to 40.7 ± 20.1 pmol/g and 110.9 ± 10.6 to 161.5 ± 56.3 pmol/g, respectively, in the brain, liver and lung tissues.

Formation of cholesterol ozonolysis products in vitro and in vivo through a myeloperoxidase-dependent pathway.

3ß-Hydroxy-5-oxo-5,6-secocholestan-6-al (secosterol-A) and its aldolization product 3ß-hydroxy-5ß-hydroxy-B-norcholestane-6ß-carboxaldehyde (secosterol-B) were recently detected in human atherosclerotic tissues and brain specimens, and they may play pivotal roles in the pathogenesis of atherosclerosis and neurodegenerative diseases. However, as their origin remains unidentified, we examined the formation mechanism, the stability, and the fate of secosterols in vitro and in vivo. About 40% of secosterol-A remained unchanged after 3 h incubation in the FBS-free medium, whereas 20% and 40% were converted to its aldehyde-oxidation product, 3ß-hydroxy-5-oxo-secocholestan-6-oic acid, and secosterol-B, respectively. In the presence of FBS, almost all secosterol-A was converted immediately to these compounds. Secosterol-B in the medium, with and without FBS, was relatively stable, but ∼30% was converted to its aldehyde-oxidation product, 3ß-hydroxy-5ß-hydroxy-B-norcholestane-6-oic acid (secoB-COOH). When neutrophil-like differentiated human leukemia HL-60 (nHL-60) cells activated with PMA were cultured in the FBS-free medium containing cholesterol, significantly increased levels of secosterol-A and its aldehyde-oxidation product, but not secosterol-B, were formed. This secosterol-A formation was decreased in the culture of PMA-activated nHL-60 cells containing several reactive oxygen species (ROS) inhibitors and scavengers or in the culture of PMA-activated neutrophils isolated from myeloperoxidase (MPO)-deficient mice. Our results demonstrate that secoterol-A is formed by an ozone-like oxidant generated with PMA-activated neutrophils through the MPO-dependent mechanism.

TRO40303, a new cardioprotective compound, inhibits mitochondrial permeability transition.

3,5-Seco-4-nor-cholestan-5-one oxime-3-ol (TRO40303) is a new cardioprotective compound coming from a chemical series identified initially for neuroprotective properties. TRO40303 binds specifically to the mitochondrial translocator protein 18 kDa (TSPO) at the cholesterol site. After intravenous administration, TRO40303 tissue distribution was comparable to that of TSPO, and, in particular, the drug accumulated rapidly in the heart. In a model of 35 min of myocardial ischemia/24 h of reperfusion in rats, TRO40303 (2.5 mg/kg) reduced infarct size by 38% (p < 0.01 versus control), when administered 10 min before reperfusion, which was correlated with reduced release of apoptosis-inducing factor from mitochondria to the cytoplasm in the ischemic area at risk. Although TRO40303 had no effect on the calcium retention capacity of isolated mitochondria, unlike cyclosporine A, the drug delayed mitochondrial permeability transition pore (mPTP) opening and cell death in isolated adult rat cardiomyocytes subjected to 2 h of hypoxia followed by 2 h of reoxygenation and inhibited mPTP opening in neonatal rat cardiomyocytes treated with hydrogen peroxide. The effects of TRO40303 on mPTP in cell models of oxidative stress are correlated with a significant reduction in reactive oxygen species production and subsequent calcium overload. TRO40303 is a new mitochondrial-targeted drug and inhibits mPTP triggered by oxidative stress. Its mode of action differs from that of other mPTP inhibitors such as cyclosporine A, thus providing a new pharmacological approach to study mPTP regulation. Its efficacy in an animal model of myocardial infarctions makes TRO40303 a promising new drug for the reduction of cardiac ischemia-reperfusion injury.

Crystal structure of CYP24A1, a mitochondrial cytochrome P450 involved in vitamin D metabolism.

Cytochrome P450 (CYP) 24A1 catalyzes the side-chain oxidation of the hormonal form of vitamin D. Expression of CYP24A1 is up-regulated to attenuate vitamin D signaling associated with calcium homeostasis and cellular growth processes. The development of therapeutics for disorders linked to vitamin D insufficiency would be greatly facilitated by structural knowledge of CYP24A1. Here, we report the crystal structure of rat CYP24A1 at 2.5 A resolution. The structure exhibits an open cleft leading to the active-site heme prosthetic group on the distal surface that is likely to define the path of substrate access into the active site. The entrance to the cleft is flanked by conserved hydrophobic residues on helices A' and G', suggesting a mode of insertion into the inner mitochondrial membrane. A docking model for 1alpha,25-dihydroxyvitamin D(3) binding in the open form of CYP24A1 that clarifies the structural determinants of secosteroid recognition and validates the predictive power of existing homology models of CYP24A1 is proposed. Analysis of CYP24A1's proximal surface identifies the determinants of adrenodoxin recognition as a constellation of conserved residues from helices K, K'', and L that converge with an adjacent lysine-rich loop for binding the redox protein. Overall, the CYP24A1 structure provides the first template for understanding membrane insertion, substrate binding, and redox partner interaction in mitochondrial P450s.

Cholesterol secoaldehyde induces apoptosis in J774 macrophages via mitochondrial pathway but not involving reactive oxygen species as mediators.

Cholesterol secoaldehyde (3beta-hydroxy-5-oxo-5,6-secocholestan-6-al or ChSeco) is an oxysterol known to be formed in reactions of ozone with cholesterol and also when cholesterol-5alpha-hydroperoxide undergoes Hock cleavage. In view of its widespread occurrence and atherogenic potential, we examined the effects of ChSeco on mouse J774 macrophage viability and events associated with apoptosis. A dose-dependent decrease in cell viability, disruptions in mitochondrial transmembrane potential (64+/-5.5%; mean+/-SD, n=3), increased levels of cytosolic cytochrome c (8.8+/-0.84 ng/ml; mean+/-SD, n=3), activation of caspase-3 (ca. 3.6-fold) and caspase-9 (ca.1.8-fold), and increased DNA fragmentation (ca. 5-fold), all indicative of apoptosis, were observed in response to exposure to ChSeco. The apoptotic nature of cell death in macrophages was confirmed by dual staining with acridine orange and ethidium bromide. However, unlike the case with cardiomyoblasts and neuronal cells, the apoptotic process in these immune cells was not mediated by increased levels of reactive oxygen species as indicated by a minimal or no increase in 2',7'-dichlorofluorescein fluorescence. It is suggested that the apoptotic process is mediated via the mitochondrial pathway and that ChSeco formed in biological environments contributes to the initiation, progression, and culmination of atherosclerotic plaque formation, as these processes are critically dependent on macrophage apoptosis.

Intracellular oxidative stress and cytotoxicity in rat primary cortical neurons exposed to cholesterol secoaldehyde.

Cholesterol secoaldehyde (ChSeco or 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al) has been shown to induce Abeta aggregation and apoptosis in GT1-7 hypothalamic neurons. The present study was undertaken to evaluate the effects of ChSeco on rat primary cortical neuronal cells. ChSeco was cytotoxic at concentrations ranging from 5 to 20 microM, while cholesterol of comparable concentrations showed little or no toxicity. In ChSeco-exposed neuronal cells, there was an increased formation of intracellular peroxide or peroxide-like substance(s), the levels of which were comparable to those found in typical menadione exposures. There was a loss in the mitochondrial transmembrane potential, the extent of which was dependent on concentration of ChSeco employed. Pre-treatment with N-acetyl-L-cysteine (5 mM; 1 h) offered protection against the cytotoxicity and the generation of intracellular oxidants. Cytotoxicity of ChSeco was evidenced by the loss of axonal branches and also condensed apoptotic nuclei in these cells. Immunohistochemical analysis revealed a decreased intracellular Abeta42 staining proportional to the loss in the axonal out growth and dendritic branches. The observed decrease in Abeta42 has been suggested to be due to loss of integrity of dendrites and the plasma membrane, possibly resulting from increased production of reactive oxygen species.

Sequential metabolism of 7-dehydrocholesterol to steroidal 5,7-dienes in adrenal glands and its biological implication in the skin.

Since P450scc transforms 7-dehydrocholesterol (7DHC) to 7-dehydropregnenolone (7DHP) in vitro, we investigated sequential 7DHC metabolism by adrenal glands ex vivo. There was a rapid, time- and dose-dependent metabolism of 7DHC by adrenals from rats, pigs, rabbits and dogs with production of more polar 5,7-dienes as detected by RP-HPLC. Based on retention time (RT), UV spectra and mass spectrometry, we identified the major products common to all tested species as 7DHP, 22-hydroxy-7DHC and 20,22-dihydroxy-7DHC. The involvement of P450scc in adrenal metabolic transformation was confirmed by the inhibition of this process by DL-aminoglutethimide. The metabolism of 7DHC with subsequent production of 7DHP was stimulated by forscolin indicating involvement of cAMP dependent pathways. Additional minor products of 7DHC metabolism that were more polar than 7DHP were identified as 17-hydroxy-7DHP (in pig adrenals but not those of rats) and as pregna-4,7-diene-3,20-dione (7-dehydroprogesterone). Both products represented the major identifiable products of 7DHP metabolism in adrenal glands. Studies with purified enzymes show that StAR protein likely transports 7DHC to the inner mitochondrial membrane, that 7DHC can compete effectively with cholesterol for the substrate binding site on P450scc and that the catalytic efficiency of 3betaHSD for 7DHP (V(m)/K(m)) is 40% of that for pregnenolone. Skin mitochondria are capable of transforming 7DHC to 7DHP and the 7DHP is metabolized further by skin extracts. Finally, 7DHP, its photoderivative 20-oxopregnacalciferol, and pregnenolone exhibited biological activity in skin cells including inhibition of proliferation of epidermal keratinocytes and melanocytes, and melanoma cells. These findings define a novel steroidogenic pathway: 7DHC-->22(OH)7DHC-->20,22(OH)(2)7DHC-->7DHP, with potential further metabolism of 7DHP mediated by 3betaHSD or CYP17, depending on mammalian species. The 5-7 dienal intermediates of the pathway can be a source of biologically active vitamin D3 derivatives after delivery to or production in the skin, an organ intermittently exposed to solar radiation.

Cholesterol secoaldehyde, an ozonation product of cholesterol, induces amyloid aggregation and apoptosis in murine GT1-7 hypothalamic neurons.

Aldehydic products from ozonation of cholesterol and peroxidation of phospholipids have been shown to accelerate aggregation of amyloid-beta (Abeta) in vitro. Here, we show that 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al (ChSeco), an ozonation product of cholesterol, induces Abeta aggregation, generation of reactive oxygen species (ROS), and cytotoxicity in murine GT1-7 hypothalamic neurons. The formation of Abeta aggregates in situ was dose-dependent at ChSeco concentrations ranging from 1 to 20 microM. The increase in insoluble Abeta aggregates at increasing concentrations of ChSeco was accompanied by a decrease in soluble Abeta as evidenced by Western blot analysis. The formation of ROS in neuronal cells was found to be dose- and time-dependent with the magnitude being higher at 20 microM compared to 10 microM ChSeco or untreated controls. The increase in ROS was associated with depletion of GSH. The cytotoxicity induced by ChSeco involved changes in phosphatidylserine translocation, DNA fragmentation, and caspase 3/7 activity that are characteristic of apoptosis. Pretreatment of neuronal cells with Trolox, a water-soluble analog of alpha-tocopherol offered partial, but significant protection against ChSeco-induced cell death, whereas, N-acetyl-L-cysteine (NAC) completely prevented the cytotoxic effects of ChSeco. NAC and Trolox were without any effects on ChSeco-induced Abeta aggregation. Fibrillogenesis inhibitors, which inhibited Abeta aggregation, did not inhibit cell death induced by ChSeco, implying that ROS generation, and not Abeta aggregation, plays a major role in the observed cytotoxicity. However, since Alzheimer's and other neurodegenerative diseases are slow and progressive, the formation of Abeta aggregates in vivo by ChSeco may have long-term pathological consequences.

A product of ozonolysis of cholesterol alters the biophysical properties of phosphatidylethanolamine membranes.

There is evidence that some products of the reaction of ozone with cholesterol contribute to atherosclerosis. One of these compounds is 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al. We have synthesized this compound and have demonstrated that it reacts with phosphatidylethanolamine to form a Schiff base. The 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al also affects the physical properties of phosphatidylethanolamines. We show by both DSC and X-ray diffraction that it increases the negative curvature of the membrane. In addition, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al causes the lamellar phase to become disorganized, resulting in the loss of lamellar periodicity. The chemical and physical interactions of 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al with phosphatidylethanolamines may contribute to damaging effects of this lipid on cell membranes, resulting in pathology.

A new bacterial steroid degradation gene cluster in Comamonas testosteroni TA441 which consists of aromatic-compound degradation genes for seco-steroids and 3-ketosteroid dehydrogenase genes.

In Comamonas testosteroni TA441, testosterone is degraded via aromatization of the A ring, which is cleaved by the meta-cleavage enzyme TesB, and further degraded by TesD, the hydrolase for the product of TesB. TesEFG, encoded downstream of TesD, are probably hydratase, aldolase, and dehydrogenase for degradation of 2-oxohex-4-enoicacid, one of the products of TesD. Here we present a new and unique steroid degradation gene cluster in TA441, which consists of ORF18, ORF17, tesI, tesH, ORF11, ORF12, and tesDEFG. TesH and TesI are 3-ketosteroid-Delta(1)-dehydrogenase and 3-ketosteroid-Delta(4)(5alpha)-dehydrogenase, respectively, which work in the early steps of steroid degradation. ORF17 probably encodes the reductase component of 9alpha-hydroxylase for 1,4-androstadiene-3,17-dione, which is the product of TesH in testosterone degradation. Gene disruption experiments showed that these genes are necessary for steroid degradation and do not have any isozymes in TA441. By Northern blot analysis, these genes were shown to be induced when TA441 was incubated with steroids (testosterone and cholic acid) but not with aromatic compounds [phenol, biphenyl, and 3-(3-hydroxyphenyl)propionic acid], indicating that these genes function exclusively in steroid degradation.

Feeding stimulative activity of steroidal and secoiridoid glucosides and their hydrolysed derivatives toward the olive weevil (Dyscerus perforatus).

Beta-sitosteryl-D-glucoside and oleuropein isolated from the olive tree (Olea europaea) and their hydrolysed derivatives were tested by a feeding stimulative activity bioassay using the olive weevil (Dyscerus perforatus). Although the steroidal glucoside showed potent feeding stimulative activity, the activity of the aglycone (beta-sitosterol) was significantly lower than that of the glucoside. On the other hand, the difference in the activity between oleuropein, a secoiridoid glucoside, and the hydrolysed derivatives was not significant.