Effect of cadmium on the regulatory mechanism of steroidogenic pathway of Leydig cells during spermatogenesis.

Cadmium is a male reproductive toxicant that interacts with a variety of pathogenetic mechanisms. However, the effect of cadmium on the regulatory mechanism of the steroidogenic pathway of Leydig cells during spermatogenesis is still ambiguous. Light microscopy, Western blot, immunohistochemistry, immunofluorescence, and quantitative polymerase chain reaction were performed to study the regulatory mechanism of the steroidogenic pathway of Leydig cells during spermatogenesis. The results indicated that in the control group, Leydig cells showed dynamic immunoreactivity and immunosignaling action with a strong positive significant secretion of 3ß-hydroxysteroid hydrogenase (3ß-HSD) in the interstitial compartment of the testis. Leydig cells showed a high active regulator mechanism of the steroidogenic pathway with increased the proteins and genes expression level of steroidogenic acute regulatory protein (STAR), cytochrome P450 cholesterol (CYP11A1), cytochrome P450 cholesterol (CYP17A1), 3ß-hydroxysteroid hydrogenase (3ß-HSD) 17ß-hydroxysteroid hydrogenase (17ß-HSD), and androgen receptor (AR) that maintained the healthy and vigorous progressive motile spermatozoa. However, on treatment with cadmium, Leydig cells were irregularly dispersed in the interstitial compartment of the testis. Leydig cells showed reduced immunoreactivity and immunosignaling of 3ß-HSD protein. Meanwhile, cadmium impaired the regulatory mechanism of the steroidogenic process of the Leydig cells with reduced protein and gene expression levels of STAR, CYP11A1, CYP17A1, 3ß-HSD, 17ß-HSD, and AR in the testis. Additionally, treatment with cadmium impaired the serum LH, FSH, and testosterone levels in blood as compared to control. This study explores the hazardous effect of cadmium on the regulatory mechanism of the steroidogenic pathway of Leydig cells during spermatogenesis.

Neutralizing the adverse effects of cyclophosphamide on the mouse testis and sperm parameters through pentoxifylline: A molecular and stereological study.

Cyclophosphamide (CP) is one of the chemotherapeutic drugs, which plays its role by interfering with all rapidly proliferating tissues like cancer and testis. The aim of this study was to investigate the protective effect of pentoxifylline (PTX) on the sperm parameters, spermatogenesis indices, biochemical alterations and gene expressions, in adult male mice treated with CP. A total of 24 male NMRI mice were divided into four groups: control, CP group (15 mg/kg weekly), PTX (100 mg/kg daily) and CP + PTX and treated for 35 days with the intraperitoneal injection. A significant decrease in the spermatogenesis indices, Leydig cells, sperm motility, viability, count, tail length and daily sperm production was found in the CP group compared to the control group. The results of this study indicate that PTX prevented these adverse effects of CP and decreased the number of apoptotic cells. Moreover, the CP group showed decreased levels of total antioxidant capacity, testosterone, lipid peroxidation and the expression of cytochrome P450 and 3ß-hydroxysteroid, all of which were neutralized in the CP + PTX group. It seems that PTX has the potential to be used in therapeutic regimens of cancer patients to reduce the side effects of CP. However, more research is needed to evaluate this prevention in mice models of cancer.

Characterization of aldo-keto reductase 1C subfamily members encoded in two rat genes (akr1c19 and RGD1564865). Relationship to 9-hydroxyprostaglandin dehydrogenase.

Rat genes, akr1c19 and RGD1564865, encode members (R1C19 and 20HSDL, respectively) of the aldo-keto reductase (AKR) 1C subfamily, whose functions, however, remain unknown. Here, we show that recombinant R1C19 and 20HSDL exhibit NAD+-dependent dehydrogenase activity for prostaglandins (PGs) with 9α-hydroxy group (PGF2α, its 13,14-dihydro- and 15-keto derivatives, 9α,11ß-PGF2 and PGD2). 20HSDL oxidized the PGs with much lower Km (0.3-14 µM) and higher kcat/Km values (0.064-2.6 min-1µM-1) than those of R1C19. They also differed in other properties: R1C19, but not 20HSDL, oxidized some 17ß-hydroxysteroids (5ß-androstane-3α,17ß-diol and 5ß-androstan-17ß-ol-3-one). 20HSDL was specifically inhibited by zomepirac, but not by R1C19-selective inhibitors (hexestrol, flavonoids, ibuprofen and flufenamic acid), although the two enzymes were sensitive to indomethacin and cis-unsaturated fatty acids. The mRNA for 20HSDL was expressed abundantly in rat kidney and at low levels in the liver, testis, brain, heart and colon, in contrast to ubiquitous expression of R1C19 mRNA. The comparison of enzymic features of R1C19 and 20HSDL with rat PG dehydrogenases and other AKRs suggests not only a close relationship of 20HSDL with 9-hydroxy-PG dehydrogenase in rat kidney, but also roles of R1C19 and rat AKRs (1C16 and 1C24) in the metabolism of PGF2α, PGD2 and 9α,11ß-PGF2 in other tissues.

The Distribution of Asterosaponins, Polyhydroxysteroids and Related Glycosides in Different Body Components of the Far Eastern Starfish Lethasterias fusca.

Glycoconjugated and other polar steroids of starfish have unique chemical structures and show a broad spectrum of biological activities. However, their biological functions remain not well established. Possible biological roles of these metabolites might be indicated by the studies on their distribution in the organism-producer. In order to investigate the localization of polar steroids in body components of the Far Eastern starfish Lethasterias fusca, chemical constituents of body walls, gonads, stomach, pyloric caeca, and coelomic fluid were studied by nanoflow liquid chromatography/mass spectrometry with captive spray ionization (nLC/CSI-QTOF-MS). It has been shown that the levels of polar steroids in the studied body components are qualitatively and quantitatively different. Generally, the obtained data confirmed earlier made assumptions about the digestive function of polyhydroxysteroids and protective role of asterosaponins. The highest level of polar steroids was found in the stomach. Asterosaponins were found in all body components, the main portion of free polyhydroxysteroids and related glycosides were located in the pyloric caeca. In addition, a great inter-individual variability was found in the content of most polar steroids, which may be associated with the peculiarities in their individual physiologic status.

Production of 14α-hydroxysteroids by a recombinant Saccharomyces cerevisiae biocatalyst expressing of a fungal steroid 14α-hydroxylation system.

The 14α-hydroxysteroids have specific anti-gonadotropic and carcinolytic biological activities and can be produced by microbial biotransformation. The steroid 11ß-/14α-hydroxylase P-450lun from Cochliobolus lunatus is the only fungal cytochrome P450 enzyme identified to date with steroid C14 hydroxylation ability. Previous work has mainly revealed the 11ß-hydroxylation activity of the P-450lun towards cortexolone (RSS) substrate; however, the potential steroid 14α-hydroxylation activity of this enzyme, especially for androstenedione (AD) substrate, has not yet conducted in-depth testing. In this work, we further tested the steroid 14α-hydroxylation activity of the P-450lun towards RSS and AD in the Saccharomyces cerevisiae system. We demonstrated that P-450lun functions as the specific 14α-hydroxylase towards the AD substrate (regiospecificity > 99%); however, it showed a poor C14-hydroxylation regiospecificity (around 40%) for the RSS substrate. In addition, through transcriptome analysis combined with gene functional characterizations, we also identified and cloned the gene for the P-450lun-associated redox partner CPRlun. Finally, through codon optimization, knockout of genes for the side reactions related enzymes GCY1 and YPR1, and increasing copies of the P-450lun and CPRlun, we developed a recombinant S. cerevisiae biocatalyst based on the C. lunatus steroid 14α-hydroxylation system to produce 14α-hydroxysteroids. Initial production of 14α-OH-AD (150 mg/L day productivity, 99% regioisomeric purity, and 60% w/w yield) and 14α-OH-RSS (64 mg/L day productivity, 40% regioisomeric purity, and 26% w/w yield) were separately achieved in shake flasks; these results represent the highest level of 14α-hydroxysteroid production in the current yeast system.

The 7α-hydroxysteroid dehydratase Hsh2 is essential for anaerobic degradation of the steroid skeleton of 7α-hydroxyl bile salts in the novel denitrifying bacterium Azoarcus sp. strain Aa7.

Bile salts are steroid compounds from the digestive tract of vertebrates and enter the environment via defecation. Many aerobic bile-salt degrading bacteria are known but no bacteria that completely degrade bile salts under anoxic conditions have been isolated so far. In this study, the facultatively anaerobic Betaproteobacterium Azoarcus sp. strain Aa7 was isolated that grew with bile salts as sole carbon source under anoxic conditions with nitrate as electron acceptor. Phenotypic and genomic characterization revealed that strain Aa7 used the 2,3-seco pathway for the degradation of bile salts as found in other denitrifying steroid-degrading bacteria such as Sterolibacterium denitrificans. Under oxic conditions strain Aa7 used the 9,10-seco pathway as found in, for example, Pseudomonas stutzeri Chol1. Metabolite analysis during anaerobic growth indicated a reductive dehydroxylation of 7α-hydroxyl bile salts. Deletion of the gene hsh2 Aa7 encoding a 7-hydroxysteroid dehydratase led to strongly impaired growth with cholate and chenodeoxycholate but not with deoxycholate lacking a hydroxyl group at C7. The hsh2 Aa7 deletion mutant degraded cholate and chenodeoxycholate to the corresponding C19 -androstadienediones only while no phenotype change was observed during aerobic degradation of cholate. These results showed that removal of the 7α-hydroxyl group was essential for cleavage of the steroid skeleton under anoxic conditions.

SULFATION PATHWAYS: Expression of SULT2A1, SULT2B1 and HSD3B1 in the porcine testis and epididymis.

In the porcine testis, in addition to estrogen sulfates, the formation of numerous sulfonated neutral hydroxysteroids has been observed. However, their functions and the underlying synthetic pathways are still widely unclear. To obtain further information on their formation in postpubertal boars, the expression of sulfotransferases considered relevant for neutral hydroxysteroids (SULT2A1, SULT2B1) was investigated in the testis and defined segments of the epididymis applying real-time RT-qPCR, Western blot and immunohistochemistry (IHC). Sulfotransferase activities were assessed in tissue homogenates or cytosolic preparations applying dehydroepiandrosterone and pregnenolone as substrates. A high SULT2A1 expression was confirmed in the testis and localized in Leydig cells by IHC. In the epididymis, SULT2A1 expression was virtually confined to the body. SULT2B1 expression was absent or low in the testis but increased significantly along the epididymis. Immunohistochemical observations indicate that both enzymes are secreted into the ductal lumen via an apocrine mechanism. The results from the characterization of expression patterns and activity measurements suggest that SULT2A1 is the prevailing enzyme for the sulfonation of hydroxysteroids in the testis, whereas SULT2B1 may catalyze the formation of sterol sulfates in the epididymis. In order to obtain information on the overall steroidogenic capacity of the porcine epididymis, the expression of important steroidogenic enzymes (CYP11A1, CYP17A1, CYP19, HSD3B1, HSD17B3, SRD5A2) was monitored in the defined epididymal segments applying real-time RT-qPCR. Surprisingly, in addition to a high expression of SRD5A2 in the epididymal head, a substantial expression of HSD3B1 was detected, which increased along the organ.

Inherent steroid 17α,20-lyase activity in defunct cytochrome P450 17A enzymes.

Cytochrome P450 (P450) 17A1 catalyzes the oxidations of progesterone and pregnenolone and is the major source of androgens. The enzyme catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction, and several mechanisms have been proposed for the latter step. Zebrafish P450 17A2 catalyzes only the 17α-hydroxylations. We previously reported high similarity of the crystal structures of zebrafish P450 17A1 and 17A2 and human P450 17A1. Five residues near the heme, which differed, were changed. We also crystallized this five-residue zebrafish P450 17A1 mutant, and the active site still resembled the structure in the other proteins, with some important differences. These P450 17A1 and 17A2 mutants had catalytic profiles more similar to each other than did the wildtype proteins. Docking with these structures can explain several minor products, which require multiple enzyme conformations. The 17α-hydroperoxy (OOH) derivatives of the steroids were used as oxygen surrogates. Human P450 17A1 and zebrafish P450s 17A1 and P450 17A2 readily converted these to the lyase products in the absence of other proteins or cofactors (with catalytically competent kinetics) plus hydroxylated 17α-hydroxysteroids. The 17α-OOH results indicate that a "Compound I" (FeO3+) intermediate is capable of formation and can be used to rationalize the products. We conclude that zebrafish P450 17A2 is capable of lyase activity with the 17α-OOH steroids because it can achieve an appropriate conformation for lyase catalysis in this system that is precluded in the conventional reaction.

Reaction mechanism of sterol hydroxylation by steroid C25 dehydrogenase - Homology model, reactivity and isoenzymatic diversity.

Steroid C25 dehydrogenase (S25DH) is a molybdenum-containing oxidoreductase isolated from the anaerobic Sterolibacterium denitrificans Chol-1S. S25DH is classified as 'EBDH-like' enzyme (EBDH, ethylbenzene dehydrogenase) and catalyzes the introduction of an OH group to the C25 atom of a sterol aliphatic side-chain. Due to its regioselectivity, S25DH is proposed as a catalyst in production of pharmaceuticals: calcifediol or 25-hydroxycholesterol. The aim of presented research was to obtain structural model of catalytic subunit α and investigate the reaction mechanism of the O2-independent tertiary carbon atom activation. Based on homology modeling and theoretical calculations, a S25DH α subunit model was for the first time characterized and compared to other S25DH-like isoforms. The molecular dynamics simulations of the enzyme-substrate complexes revealed two stable binding modes of a substrate, which are stabilized predominantly by van der Waals forces in the hydrophobic substrate channel. However, H-bond interactions involving polar residues with C3=O/C3-OH in the steroid ring appear to be responsible for positioning the substrate. These results may explain the experimental kinetic results which showed that 3-ketosterols are hydroxylated 5-10-fold faster than 3-hydroxysterols. The reaction mechanism was studied using QM:MM and QM-only cluster models. The postulated mechanism involves homolytic CH cleavage by the MoO ligand, giving rise to a radical intermediate with product obtained in an OH rebound process. The hypothesis was supported by kinetic isotopic effect (KIE) experiments involving 25,26,26,26-[2H]-cholesterol (4.5) and the theoretically predicted intrinsic KIE (7.0-7.2). Finally, we have demonstrated that the recombinant S25DH-like isoform catalyzes the same reaction as S25DH.

Regio- and stereoselective reduction of 17-oxosteroids to 17ß-hydroxysteroids by a yeast strain Zygowilliopsis sp. WY7905.

The reduction of 17-oxosteroids to 17ß-hydroxysteroids is one of the important transformations for the preparation of many steroidal drugs and intermediates. The strain Zygowilliopsis sp. WY7905 was found to catalyze the reduction of C-17 carbonyl group of androst-4-ene-3,17-dione (AD) to give testosterone (TS) as the sole product by the constitutive 17ß-hydroxysteroid dehydrogenase (17ß-HSD). The optimal conditions for the reduction were pH 8.0 and 30°C with supplementing 10g/l glucose and 1% Tween 80 (w/v). Under the optimized transformation conditions, 0.75g/l AD was reduced to a single product TS with >90% yield and >99% diastereomeric excess (de) within 24h. This strain also reduced other 17-oxosteroids such as estrone, 3ß-hydroxyandrost-5-en-17-one and norandrostenedione, to give the corresponding 17ß-hydroxysteroids, while the C-3 and C-20 carbonyl groups were intact. The absence of by-products in this microbial 17ß-reduction would facilitate the product purification. As such, the strain might serve as a useful biocatalyst for this important transformation.

An unexplored pathway for degradation of cholate requires a 7α-hydroxysteroid dehydratase and contributes to a broad metabolic repertoire for the utilization of bile salts in Novosphingobium sp. strain Chol11.

Bile salts such as cholate are surface-active steroid compounds with functions for digestion and signaling in vertebrates. Upon excretion into soil and water bile salts are an electron- and carbon-rich growth substrate for environmental bacteria. Degradation of bile salts proceeds via intermediates with a 3-keto-Δ1,4 -diene structure of the steroid skeleton as shown for e.g. Pseudomonas spp. Recently, we isolated bacteria degrading cholate via intermediates with a 3-keto-7-deoxy-Δ4,6 -structure of the steroid skeleton suggesting the existence of a second pathway for cholate degradation. This potential new pathway was investigated with Novosphingobium sp. strain Chol11. A 7α-hydroxysteroid dehydratase encoded by hsh2 was identified, which was required for the formation of 3-keto-7-deoxy-Δ4,6 -metabolites. A hsh2 deletion mutant could still grow with cholate but showed impaired growth. Cholate degradation of this mutant proceeded via 3-keto-Δ1,4 -diene metabolites. Heterologous expression of Hsh2 in the bile salt-degrading Pseudomonas sp. strain Chol1 led to the formation of a dead-end steroid with a 3-keto-7-deoxy-Δ4,6 -diene structure. Hsh2 is the first steroid dehydratase with an important function in a metabolic pathway of bacteria that use bile salts as growth substrates. This pathway contributes to a broad metabolic repertoire of Novosphingobium strain Chol11 that may be advantageous in competition with other bile salt-degrading bacteria.

Biotransformations of Bile Acids with Bacteria from Cayambe Slaughterhouse (Ecuador): Synthesis of Bendigoles.

The biotransformations of cholic acid (1a), deoxycholic acid (1b), and hyodeoxycholic acid (1c) to bendigoles and other metabolites with bacteria isolated from the rural slaughterhouse of Cayambe (Pichincha Province, Ecuador) were reported. The more active strains were characterized, and belong to the genera Pseudomonas and Rhodococcus. Various biotransformation products were obtained depending on bacteria and substrates. Cholic acid (1a) afforded the 3-oxo and 3-oxo-4-ene derivatives 2a and 3a (45% and 45%, resp.) with P. mendocina ECS10, 3,12-dioxo-4-ene derivative 4a (60%) with Rh. erythropolis ECS25, and 9,10-secosteroid 6 (15%) with Rh. erythropolis ECS12. Bendigole F (5a) was obtained in 20% with P. fragi ECS22. Deoxycholic acid (1b) gave 3-oxo derivative 2b with P. prosekii ECS1 and Rh. erythropolis ECS25 (20% and 61%, resp.), while 3-oxo-4-ene derivative 3b was obtained with P. prosekii ECS1 and P. mendocina ECS10 (22% and 95%, resp.). Moreover, P. fragi ECS9 afforded bendigole A (8b; 80%). Finally, P. mendocina ECS10 biotransformed hyodeoxycholic acid (1c) to 3-oxo derivative 2c (50%) and Rh. erythropolis ECS12 to 6α-hydroxy-3-oxo-23,24-dinor-5ß-cholan-22-oic acid (9c, 66%). Bendigole G (5c; 13%) with P. prosekii ECS1 and bendigole H (8c) with P. prosekii ECS1 and Rh. erythropolis ECS12 (20% and 16%, resp.) were obtained.

11-ß hydroxysteroid type 1 knockout mice display an antidepressant-like phenotype in the forced swim test.

OBJECTIVE: 11ß-dehydroxysteroid dehydrogenase (HSD) types 1 and 2, enzymes are involved in the activation and inactivation of glucocorticoids in vivo, respectively. Indirect evidence implicates two enzymes in the aetiology of depression but no study has directly assessed the potential role of 11 ß-HSD1 in animal tests. METHODS: We assessed 11 ß-HSD1 knockout mice in the forced swim test (FST), tail suspension test (TST) and for locomotor activity. RESULTS: Genetic ablation of the 11ß-HSD1 gene results in an antidepressant-like phenotype in the FST; the most widely utilised animal test of antidepressant activity, but not in the related TST. This may be related to the different biological substrates underlying these tests. The decreased FST immobility was not due to alterations in general activity. CONCLUSIONS: Taken together these results suggest that 11ß-HSD1 may play an important role in depression-related behaviours and further studies are necessary to fully characterise its role in such behaviour.

5Alpha-Reduced Steroids Are Major Metabolites in the Early Equine Embryo Proper and Its Membranes.

Steroid production and metabolism by early conceptuses are very important for the establishment and maintenance of pregnancy in horses. Our earlier work suggested the possible formation of 5alpha-reduced steroids in equine conceptuses. We have now demonstrated the formation of 5alpha-reduced metabolites of androstenedione, testosterone, and progesterone by the embryo and its membranes. A total of 44 conceptuses were collected from 26 mares between 20 and 31 days of pregnancy. Tissues from the embryo proper and from the separated components of the conceptus (bilaminar and trilaminar trophoblast, allantois) were incubated with tritium-labeled substrates. 5Alpha-reduced metabolites (5alpha-dihydro- and 3beta,5alpha-tetrahydro- steroids) as radiolabeled products were identified from a series of chromatographic steps using four solvent systems for high-performance liquid chromatography. Use of a 5alpha-reductase inhibitor confirmed the metabolites were indeed 5alpha-reduced steroids. For the embryo, the only products from androstenedione were 5alpha-dihydroandrostenedione and 3beta,5alpha-tetrahydroandrostenedione, with no evidence of more polar metabolites; there was some 3beta,5alpha-tetrahydrotestosterone but no 5alpha-dihydrotestosterone from testosterone, and formation of androstenedione was followed by the production of 5alpha-dihydroandrostenedione and 3beta,5alpha-tetrahydroandrostenedione. The major 5alpha-reduced product from progesterone was 3beta,5alpha-tetrahydroprogesterone, with lesser amounts of 5alpha-dihydroprogesterone. For the membranes, reductions to tetrahydro, 5alpha-reduced steroids were prominent in most instances, but also present were considerable amounts of products more polar than the substrates. The well-recognized activity of some 5alpha-reduced steroids--for example, 5alpha-dihydrotestosterone in male sexual differentiation--provokes interest in their even earlier appearance, as seen in this study, and suggests a possible role for them in early embryonic development in horses and, more generally, in other species.

Regioselective Acetylation of C21 Hydroxysteroids by the Bacterial Chloramphenicol Acetyltransferase I.

Chloramphenicol acetyltransferase I (CATI) detoxifies the antibiotic chloramphenicol and confers a corresponding resistance to bacteria. In this study we identified this enzyme as a steroid acetyltransferase and designed a new and efficient Escherichia-coli-based biocatalyst for the regioselective acetylation of C21 hydroxy groups in steroids of pharmaceutical interest. The cells carried a recombinant catI gene controlled by a constitutive promoter. The capacity of the whole-cell system to modify different hydroxysteroids was investigated, and NMR spectroscopy revealed that all substrates were selectively transformed into the corresponding 21-acetoxy derivatives. The biotransformation was optimized, and the reaction mechanism is discussed on the basis of a computationally modeled substrate docking into the crystal structure of CATI.

Substrate specificity and inhibitor sensitivity of rabbit 20α-hydroxysteroid dehydrogenase.

In this study, we examined the substrate specificity and inhibitor sensitivity of rabbit 20α-hydroxysteroid dehydrogenase (AKR1C5), which plays a role in the termination of pregnancy by progesterone inactivation. AKR1C5 moderately reduced the 3-keto group of only 5α-dihydrosteroids with 17ß- or 20α/ß-hydroxy group among 3-ketosteroids. In contrast, the enzyme reversibly and efficiently catalyzed the reduction of various 17- and 20-ketosteroids, including estrogen precursors (dehydroepiandrosterone, estrone and 5α-androstan-3ß-ol-17-one) and tocolytic 5ß-pregnane-3,20-dione. In addition to the progesterone inactivation, the formation of estrogens and metabolism of the tocolytic steroid by AKR1C5 may be related to its role in rabbit parturition. AKR1C5 also reduced various non-steroidal carbonyl compounds, including isatin, an antagonist of the C-type natriuretic peptide receptor, and 4-oxo-2-nonenal, suggesting its roles in controlling the bioactive isatin and detoxification of cytotoxic aldehydes. AKR1C5 was potently and competitively inhibited by flavonoids such as kaempferol and quercetin, suggesting that its activity is affected by ingested flavonoids.

GC-MS-based quantitative signatures of cytochrome P450-mediated steroid oxidation induced by rifampicin.

BACKGROUND: Drug-induced cytochrome P450 (CYP) activity affects endocrine function and drug clearance rates, leading to the development of unpredictable pathologic and toxicologic risks. METHODS: Urinary steroid profiling based on gas chromatography-mass spectrometry (GC-MS) was used for simultaneous quantification of CYP-mediated regioselective hydroxysteroids and their substrates, including 26 androgens, 9 estrogens, 5 progestins, and 7 corticoids. The quantitative data were visualized using a hierarchically clustered heat map to allow identification of CYP-mediated steroid signatures. Twelve healthy subjects were orally administered 600 mg of rifampicin a day for 7 days, and their CYP enzyme activity was evaluated. RESULTS: Using GC-MS, all 47 steroids were well separated with good peak shapes. This assay had good linearity (r > 0.994) in a dynamic range, and the interassay imprecision (% CV) and inaccuracy (% bias) were 3.0%-15.6% and 98.0%-109.2%, respectively. Administration of the CYP3A4 inducer rifampicin produced distinct differences in CYP3A4 and CYP11B1, CYP19A1, HSD11B, and HSD17B, which were indicated by their heat map-visualized steroid signatures. CONCLUSIONS: This CYP-mediated steroid signature profile allows simultaneous assessment of CYP1A, CYP1B, CYP2C, CYP3A, CYP11B, CYP17A, CYP19A, and CYP21A in urine samples. This method could therefore be a useful tool for assessing drug efficacy.

B cell localization: regulation by EBI2 and its oxysterol ligand.

During antibody responses, B cells undergo a series of migratory events that guide them to the appropriate microenvironments for activation and differentiation. Epstein-Barr virus-induced molecule 2 [EBI2; also known as G-protein-coupled receptor (GPR)183] is a key chemotactic receptor guiding B cell localization. EBI2 and its ligand, 7α,25-dihydroxycholesterol, direct the migration of activated B cells to interfollicular and outer follicular regions of secondary lymphoid tissues. Moreover, modulation of EBI2 expression is crucial for the generation of extrafollicular plasma cell responses and germinal center formation. Here, we review the current findings that have delineated the function of EBI2 and its ligand and discuss how they collaborate with conventional lymphoid chemokine systems to position B cells optimally during immune responses.

Activity-guided screening of bioactive natural compounds implementing a new glucocorticoid-receptor-translocation assay and detection of new anti-inflammatory steroids from bacteria.

Using an in vitro cell-based assay in a flow-design, we have applied activity-guided screening to search for new bioactive compounds isolated from microorganisms. A first assay employs the stable expression of nuclear factor kappa B (NF-κB) while a second assay utilizes the glucocorticoid receptor (GR) coupled to green fluorescent protein. A specialized assay was implemented for both the translocation of NF-κB and to inhibit the translocation of cytokine-mediated NF-κB. In addition, we developed in a wide palette of cell lines used for a highly specialized GR-translocation assay to detect anti-inflammatory effects. This approach demonstrates the straight-forward combination of cell-based assays arranged with an automated fluorescence microscope. This allows for the direct sorting of extracts which are acting in a pharmaceutically interesting way. Initial results using this technique have led to the detection of new anti-inflammatory steroids from bacterial crude extracts.

Phosphatidylethanolamine of Helicobacter pylori functions as a steroid-binding lipid in the assimilation of free cholesterol and 3ß-hydroxl steroids into the bacterial cell membrane.

One of the unique features of Helicobacter pylori is its ability to assimilate free-cholesterol (FC) into its membranes. Via FC assimilation, H. pylori strengthens the membrane lipid barrier and/or evades the host immune system. No previous studies, however, have investigated the FC uptake mechanisms of the H. pylori cell. Phosphatidylethanolamine (PE) is the most prevalent lipid component of bacteria, including H. pylori, but the function of PE remains unclear. We were therefore interested in H. pylori PE (HpPE) and investigated the interaction of its PE with cholesterols. The PE isolated from H. pylori underwent a unique molecular interaction with FC, cholesterol ester (CE), and 2,6-di-O-methyl-ß-cyclodextrin (dMßCD), a sterol solubilizer. HpPE interacted not only with the FC molecule, but also with the FC-dMßCD inclusion complex. In contrast, Escherichia coli PE (EcPE), prepared as a reference PE, seemed to bind only FC, and only via a hydrophobic interaction, without binding dMßCD. HpPE was clearly more potent than EcPE in binding FC. Intriguingly, HpPE had a negligible affinity for CE, while EcPE had a high affinity for CE, comparable to its affinity for FC. Further, HpPE interacted with 3ß-OH steroids, pregnenolone and dehydroepiandrosterone, in the absence of dMßCD. Gas chromatogram-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) analyses revealed that the fatty acid compositions of HpPE were quite distinct from those of EcPE, and the C(14:0) fatty acid in the HpPE molecule was found to be significant in binding FC selectively. These results indicate that PE is a key candidate of nonesterified steroid-binding lipids in H. pylori.