Journal: The Journal of steroid biochemistry and molecular biology

Article Title: TGFβ1 alters androgenic metabolites and hydroxysteroid dehydrogenase enzyme expression in human prostate reactive stromal primary cells: Is steroid metabolism altered by prostate reactive stromal microenvironment?

doi: 10.1016/j.jsbmb.2013.05.016

Figure Lengend Snippet: DHEA metabolic pathways in the prostate and in cultured 6S cells. Metabolism of DHEA in the prostate toward androgens or estrogens depends on activity of steroid metabolism enzymes including: hydroxysteroid dehydrogenase (HSD), alpha-keto reductase (AKR), steroid reductase dehydrogenase 5 alpha 1,2 (SRD5a1,2), sulfatase (STS), sulfonotransferase (SULT2A1) and aromatase. DHEA androgenic or estrogenic metabolites include: dehydroepiandrosterone (DHEA), DHEA sulphate (DHEA-S), androstenediol (5-diol), 4-androstenedione (4-dione), testosterone (T), estrone (E1), estradiol (E2), 5α-androstanedione (A-dione), dihydrotestosterone (DHT), androsterone (ADT), 3α-andostanediol (3α-diol), 3β-androstanediol (3β-diol).

Article Snippet: DHEA metabolites were detected by using ELISA kits for free testosterone (cat # 11-FTSHU-E01-ALPCO, Salem, NH) and androstenedione (cat # DSL-10-3800-Beckman Coulter DSLabs, Webster, TX) according to the manufacturer’s instructions.

Techniques: Cell Culture, Activity Assay

Journal: The Journal of steroid biochemistry and molecular biology

Article Title: TGFβ1 alters androgenic metabolites and hydroxysteroid dehydrogenase enzyme expression in human prostate reactive stromal primary cells: Is steroid metabolism altered by prostate reactive stromal microenvironment?

doi: 10.1016/j.jsbmb.2013.05.016

Figure Lengend Snippet: DHEA metabolism in prostate stromal (6S) cells: TGFβ1 induced increase in 4-dione and T production in DHEA-treated prostate stromal 6S cells over time. Cells were plated onto 60-mm plates at a density of 6.0 × 105 cells/plate with normal medium. After 24 h, normal medium was replaced by 3 mL treatment medium (see Section 2) and the cells were cultured for further 24 h before stimulation. The medium was replaced with 2.5 mL of new treatment medium containing 100 nM DHEA plus or minus 100 pM TGFβ1, and the cells were cultured for 6, 12, 24, 48 and 72 h before the media were collected. The amount of 4-dione (A) and T (B) in the media was analyzed by ELISA. Control values of stromal metabolism are represented by hatched bars. Solid bars represent induction of 4-dione or T by TGFβ1. The results represent mean ± SD obtained from three independent experiments, with triplicate samples in each. *P < 0.05, ***P < 0.001; treated compared to control at each time point.

Article Snippet: DHEA metabolites were detected by using ELISA kits for free testosterone (cat # 11-FTSHU-E01-ALPCO, Salem, NH) and androstenedione (cat # DSL-10-3800-Beckman Coulter DSLabs, Webster, TX) according to the manufacturer’s instructions.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Control

Journal: The Journal of steroid biochemistry and molecular biology

Article Title: TGFβ1 alters androgenic metabolites and hydroxysteroid dehydrogenase enzyme expression in human prostate reactive stromal primary cells: Is steroid metabolism altered by prostate reactive stromal microenvironment?

doi: 10.1016/j.jsbmb.2013.05.016

Figure Lengend Snippet: Effect of increasing doses of TGFβ1 on 4-dione and T production in prostate stromal 6S cells. 6S cells were plated onto 60-mm plates at a density of 6.0 × 105 cells/plate with normal medium. After 24 h the medium was replaced with 2.5 mL new treatment medium containing 100 nM DHEA and 10–200 pM TGFβ1, and the cells were cultured for 48 h before the media was collected for ELISA of 4-dione (A) and T (B). To distinguish action of TGFβ1 on the conversion of 4-dione to T in 6S cells (3C), cells were treated as above then final treatment consisted of 10 nM 4-dione and 100 pM TGFβ1, for 48 h. Conditioned media were collected and T concentration was measured by ELISA. All results shown in A–C represent mean ± SD obtained from three independent experiments, with triplicate in each. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: DHEA metabolites were detected by using ELISA kits for free testosterone (cat # 11-FTSHU-E01-ALPCO, Salem, NH) and androstenedione (cat # DSL-10-3800-Beckman Coulter DSLabs, Webster, TX) according to the manufacturer’s instructions.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: The Journal of steroid biochemistry and molecular biology

Article Title: TGFβ1 alters androgenic metabolites and hydroxysteroid dehydrogenase enzyme expression in human prostate reactive stromal primary cells: Is steroid metabolism altered by prostate reactive stromal microenvironment?

doi: 10.1016/j.jsbmb.2013.05.016

Figure Lengend Snippet: Effects of TGFβ1 on DHEA metabolism in PrSC and LAPC4 cells. Normal primary stromal cells (PrSC) cells were plated onto 60-mm plates at a density of 6.0 × 105 cells/plate (or 2 × 106 for LAPC4 cells) with normal medium and allowed to attach for 24 h (PrSC; A and B) or 48 h (LAPC4 cells; C and D). Then, normal medium was replaced by 3 mL treatment medium, and the cells were cultured for further 24 h. Cells were then treated with 2.5 mL of new treatment medium consisting of 100 nM DHEA or DHEA plus 100 pM TGFβ1 for 48 h. The media were collected and the concentrations of 4-dione (A and C) and T (B and D) were accessed by ELISA. All results shown represent mean ± SD obtained from three independent experiments, with triplicate in each ***P < 0.001.

Article Snippet: DHEA metabolites were detected by using ELISA kits for free testosterone (cat # 11-FTSHU-E01-ALPCO, Salem, NH) and androstenedione (cat # DSL-10-3800-Beckman Coulter DSLabs, Webster, TX) according to the manufacturer’s instructions.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: The Journal of steroid biochemistry and molecular biology

Article Title: TGFβ1 alters androgenic metabolites and hydroxysteroid dehydrogenase enzyme expression in human prostate reactive stromal primary cells: Is steroid metabolism altered by prostate reactive stromal microenvironment?

doi: 10.1016/j.jsbmb.2013.05.016

Figure Lengend Snippet: Regulation of HSD gene expression by TGFβ1. 6S cells were plated at a density of 6.0 × 105 cells/plate onto 60-mm plates with normal medium. After 24 h, medium were replaced by 3 ml treatment medium and the cells were cultured for further 24 h. Then the cells were stimulated by replacing the medium with 2.5 ml of new treatment medium containing 100 nM DHEA and 10–200 pM TGFβ1. After treatment for 48 h, total RNAs were isolated from the cells using TRIzol reagent. Performance of reverse transcription is described in Section 2. For expression of 3βHSD1(A), 0.5 μl of cDNA was used for each real-time PCR reaction. For expression of 17HSD5 (B) and 17HSD2 (C) 1.0 μl of cDNA was used for each real-time PCR reaction. The fold changes in the amount of mRNAs are represented as mean ± SD obtained from three independent experiments, with triplicate in each. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: DHEA metabolites were detected by using ELISA kits for free testosterone (cat # 11-FTSHU-E01-ALPCO, Salem, NH) and androstenedione (cat # DSL-10-3800-Beckman Coulter DSLabs, Webster, TX) according to the manufacturer’s instructions.

Techniques: Gene Expression, Cell Culture, Isolation, Reverse Transcription, Expressing, Real-time Polymerase Chain Reaction

Journal: iScience

Article Title: Lemon-derived nanovesicles achieve antioxidant and anti-inflammatory effects activating the AhR/Nrf2 signaling pathway

doi: 10.1016/j.isci.2023.107041

Figure Lengend Snippet: Antioxidant effects of prophylactic treatment with LNVs on innate immunity of zebrafish embryo 48 hpf embryos were prophylactically treated with 25 μg/mL of LNVs and then undergone Pa-LPS -induced (A–E) local inflammatory stimulus. (A) Schematic representation of LPS-induced stimulus model; (B) representative images at 4 hpi of neutrophils recruitment and ROS production (DHE) at the site of Pa- LPS intramuscular injection, in the whole embryo (upper) and the region of interest of the trunk (lower panels), in embryos pre-treated or not with LNVs; white asterisks indicate mpx + DHE + cells; (C–E) quantitative analysis at 4 hpi of DHE + cell count (C), mpx + cell count (D) and mpx + DHE + cell count (E) at the region of interest. Mean and SEM of at least two independent experiments are shown; dots represent cell count in a single embryo. Statistical significance was assessed by unpaired Student’s t test followed by Welch’s correction for C and D (the Gaussian data distribution were assessed by Kolmogorov-Smirnov normality test) or by non-parametric test (Mann-Whitney test) for E.

Article Snippet: The commercial kit DHE (dihydroethidium, Santa Cruz biotechnology, Dallas, Texas, USA) was used: 0.5 μl of 30 mM DHE stock solution was diluted in E3+PTU to obtain a final concentration of 5 μM.

Techniques: Injection, Cell Counting, MANN-WHITNEY

Journal: iScience

Article Title: Lemon-derived nanovesicles achieve antioxidant and anti-inflammatory effects activating the AhR/Nrf2 signaling pathway

doi: 10.1016/j.isci.2023.107041

Figure Lengend Snippet:

Article Snippet: The commercial kit DHE (dihydroethidium, Santa Cruz biotechnology, Dallas, Texas, USA) was used: 0.5 μl of 30 mM DHE stock solution was diluted in E3+PTU to obtain a final concentration of 5 μM.

Techniques: Recombinant, SYBR Green Assay, Bradford Assay, Viability Assay, Isolation, Reverse Transcription, Transgenic Assay, Software

Journal: Endocrinology

Article Title: 3beta-hydroxysteroid dehydrogenase is a possible pharmacological target in the treatment of castration-resistant prostate cancer.

doi: 10.1210/en.2010-0138

Figure Lengend Snippet: FIG. 2. DHEA metabolism to AD occurs in LNCaP cells and is blocked by chemical inhibition of 3HSD. Cells were treated with [3H]-DHEA (100 nM) in the presence or absence of 4-MA and trilostane (10 M), and steroids were extracted and analyzed by TLC at the indicated time points. Experiments were done in triplicate, twice repeated indepen- dently, and a representative experiment is shown. Steroid products were confirmed by HPLC.

Article Snippet: Before treatment, cells were cultured in phenol-red free and serum-free medium for 24 h. DHEA, A5diol, and trilostane were purchased from Steraloids (Newport, RI) and 17 -N,N-diethylcarbamoyl-4-methyl-4-aza-5- -androstan-3-one (4-MA) was obtained courtesy of Dr. David Russell (University of Texas Southwestern Medical Center, Dallas, TX).

Techniques: Inhibition

Journal: Endocrinology

Article Title: 3beta-hydroxysteroid dehydrogenase is a possible pharmacological target in the treatment of castration-resistant prostate cancer.

doi: 10.1210/en.2010-0138

Figure Lengend Snippet: FIG. 1. Metabolic pathways of DHEA and A5diol and resulting androgen response in prostate cancer. A, DHEA and A5diol are interconverted by 17-hydroxysteroid dehydrogenases. Both steroids are substrates for 3-hydroxysteroid dehydrogenase, which metabolizes them to AD and T, respectively. B, AR chromatin occupancy on the PSA and TMPRSS2 enhancers is induced by treatment of LNCaP cells with the indicated concentrations (in nM) of both DHEA and A5diol for 24 h. AR chromatin immunoprecipitation for each treatment was normalized to input DNA and vehicle-treated cells. C, Treatment of LNCaP cells for 24 h with DHEA or A5diol induces expression of PSA, TMPRSS2, and FKBP5 mRNA in a dose-dependent manner. Expression of the indicated transcript was normalized to RPLP0 and vehicle-treated cells. Experiments were conducted in triplicate, and error bars indicate the SE of the mean. Experiments were independently repeated twice, and a representative experiment is shown.

Article Snippet: Before treatment, cells were cultured in phenol-red free and serum-free medium for 24 h. DHEA, A5diol, and trilostane were purchased from Steraloids (Newport, RI) and 17 -N,N-diethylcarbamoyl-4-methyl-4-aza-5- -androstan-3-one (4-MA) was obtained courtesy of Dr. David Russell (University of Texas Southwestern Medical Center, Dallas, TX).

Techniques: Chromatin Immunoprecipitation, Expressing

Journal: Endocrinology

Article Title: 3beta-hydroxysteroid dehydrogenase is a possible pharmacological target in the treatment of castration-resistant prostate cancer.

doi: 10.1210/en.2010-0138

Figure Lengend Snippet: FIG. 3. Induction of AR nuclear localization, AR chromatin occupancy, expression of androgen-regulated genes, and cell proliferation in LNCaP by DHEA and A5diol are reversible by inhibition of 3HSD. A, AR nuclear localization by DHEA (20 nM) or A5diol (20 nM) is suppressed by pre- treatment with 4-MA (1 M). After treatment for 24 h, protein was separated into nuclear and cytoplasmic fractions, analyzed by Western blot, quantitated using the LI-COR Odyssey IR Imaging System. The signal for nuclear AR protein was normalized to lamin B. The fold nuclear AR signal normalized to untreated cells is 2.9 for DHEA, 1.2 for DHEA 4-MA, 3.5 for A5diol, and 1.1 for A5diol 4-MA. B, AR chromatin occupancy induced by DHEA and A5diol on the PSA and TMPRSS2 enhancers in LNCaP is reversed by 4-MA. Cells were treated with 20 nM DHEA or A5diol with and without 1-h pretreatment with 4-MA, and AR ChIP was performed in triplicate using an anti-AR antibody and assessed by qPCR. AR ChIP for each treatment was normalized to input DNA and vehicle-treated cells. C, Expression of FKBP5, TMPRSS2, and PSA mRNA in LNCaP is reversed by 4-MA in a dose-dependent manner. Cells were treated with 20 nM DHEA or A5diol alone for 24 h, or after 1-h pretreatment with the indicated concentration of 4-MA, and gene expression was assessed by qPCR, in triplicate. D, 4-MA inhibits LNCaP growth induced by DHEA and A5diol. Cells were treated with 500 nM DHEA and 20 nM A5diol with or without 1 M 4-MA, and cell density was assessed on the indicated days by DNA absorption using Hoechst 33258 stain, with four replicates. Error bars indicate the SD of the mean. *, P 0.05 (Student’s t test) for comparisons of 4-MA-treated groups with the same treatments without 4-MA. All experiments were independently repeated at least once, and a representative experiment is shown.

Article Snippet: Before treatment, cells were cultured in phenol-red free and serum-free medium for 24 h. DHEA, A5diol, and trilostane were purchased from Steraloids (Newport, RI) and 17 -N,N-diethylcarbamoyl-4-methyl-4-aza-5- -androstan-3-one (4-MA) was obtained courtesy of Dr. David Russell (University of Texas Southwestern Medical Center, Dallas, TX).

Techniques: Expressing, Inhibition, Western Blot, Imaging, Concentration Assay, Gene Expression, Staining

Journal: Endocrinology

Article Title: 3beta-hydroxysteroid dehydrogenase is a possible pharmacological target in the treatment of castration-resistant prostate cancer.

doi: 10.1210/en.2010-0138

Figure Lengend Snippet: FIG. 4. 3HSD inhibition reverses DHEA and A5diol induction of transcription by wild-type AR and other AR mutants. A, 4-MA pretreatment (1 M) blocks PSA induction in LAPC4 cells 24 h after addition of DHEA (20 nM) and A5diol (20 nM) but not R1881 (20 nM), which does not undergo metabolism by 3HSD. B, Delay of 3HSD inhibition restores response to DHEA and A5diol in LAPC4 cells. The same concentrations of each compound were used as in A, for a total duration of 48 h. 4-MA was added 1 h before, 6 h after, or 24 h after DHEA and A5diol treatment. C, 4-MA inhibits induction of FKBP5 by DHEA and A5diol, but not by R1881, in 22Rv1 cells. Cells were treated with DHEA, A5diol, or R1881 (20 nM) for 24 h with or without 1-h pretreatment with the indicated concentrations (in M) of 4-MA. FKBP5 expression is normalized to RPLP0 and to vehicle-treated cells. All experiments were done in triplicate, and error bars indicate the SE of the mean. *, P 0.05 (Student’s t test) for compari- sons of 4-MA-treated groups with the same treatments without 4-MA. All experiments were independently repeated, and a representative experiment is shown.

Article Snippet: Before treatment, cells were cultured in phenol-red free and serum-free medium for 24 h. DHEA, A5diol, and trilostane were purchased from Steraloids (Newport, RI) and 17 -N,N-diethylcarbamoyl-4-methyl-4-aza-5- -androstan-3-one (4-MA) was obtained courtesy of Dr. David Russell (University of Texas Southwestern Medical Center, Dallas, TX).

Techniques: Inhibition, Expressing

Journal: Molecules

Article Title: Dried Urine Microsampling Coupled to Liquid Chromatography—Tandem Mass Spectrometry (LC–MS/MS) for the Analysis of Unconjugated Anabolic Androgenic Steroids

doi: 10.3390/molecules25143210

Figure Lengend Snippet: Chemical structures of the analytes (first part).

Article Snippet: Certified analytical standards of testosterone, mesterolone, danazol, DHT, methandrostenolone, and clostebol as pure powders; standard solutions at the concentration of 1 mg/mL in methanol of epitestosterone, 1-androstenedione, DHEA, norethandrolone, nandrolone, fluoxymesterone, stanozolol and ISs as stable isotope-labelled analogues solutions at 100 µg/mL in methanol of DHEA-D5 (IS1), testosterone-D3 (IS2), DHT-D3 (IS3) and stanozolol-D3 (IS4) were acquired from Cerilliant (Round Rock, TX, USA).

Techniques:

Journal: Molecules

Article Title: Dried Urine Microsampling Coupled to Liquid Chromatography—Tandem Mass Spectrometry (LC–MS/MS) for the Analysis of Unconjugated Anabolic Androgenic Steroids

doi: 10.3390/molecules25143210

Figure Lengend Snippet: Liquid chromatography–tandem mass spectrometry (LC-MS/MS) chromatogram of a blank VAMS sample spiked with the analytes and ISs. 1: fluoxymesterone, 2: 1-androstenedione, 3: nandrolone, 4: dehydroepiandrosterone (DHEA), 5: testosterone, 6: epitestosterone, 7: clostebol, 8: dihydrotestosterone (DHT), 9: methandrostenolone, 10: norethandrolone, 11: mesterolone, 12: danazol, 13: stanozolol.

Article Snippet: Certified analytical standards of testosterone, mesterolone, danazol, DHT, methandrostenolone, and clostebol as pure powders; standard solutions at the concentration of 1 mg/mL in methanol of epitestosterone, 1-androstenedione, DHEA, norethandrolone, nandrolone, fluoxymesterone, stanozolol and ISs as stable isotope-labelled analogues solutions at 100 µg/mL in methanol of DHEA-D5 (IS1), testosterone-D3 (IS2), DHT-D3 (IS3) and stanozolol-D3 (IS4) were acquired from Cerilliant (Round Rock, TX, USA).

Techniques: Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

Journal: Molecules

Article Title: Dried Urine Microsampling Coupled to Liquid Chromatography—Tandem Mass Spectrometry (LC–MS/MS) for the Analysis of Unconjugated Anabolic Androgenic Steroids

doi: 10.3390/molecules25143210

Figure Lengend Snippet: Linearity, limit of quantification (LOQ), limit of detection (LOD), method detection limit (MDL).

Article Snippet: Certified analytical standards of testosterone, mesterolone, danazol, DHT, methandrostenolone, and clostebol as pure powders; standard solutions at the concentration of 1 mg/mL in methanol of epitestosterone, 1-androstenedione, DHEA, norethandrolone, nandrolone, fluoxymesterone, stanozolol and ISs as stable isotope-labelled analogues solutions at 100 µg/mL in methanol of DHEA-D5 (IS1), testosterone-D3 (IS2), DHT-D3 (IS3) and stanozolol-D3 (IS4) were acquired from Cerilliant (Round Rock, TX, USA).

Techniques:

Journal: Molecules

Article Title: Dried Urine Microsampling Coupled to Liquid Chromatography—Tandem Mass Spectrometry (LC–MS/MS) for the Analysis of Unconjugated Anabolic Androgenic Steroids

doi: 10.3390/molecules25143210

Figure Lengend Snippet: Extraction yield and precision.

Article Snippet: Certified analytical standards of testosterone, mesterolone, danazol, DHT, methandrostenolone, and clostebol as pure powders; standard solutions at the concentration of 1 mg/mL in methanol of epitestosterone, 1-androstenedione, DHEA, norethandrolone, nandrolone, fluoxymesterone, stanozolol and ISs as stable isotope-labelled analogues solutions at 100 µg/mL in methanol of DHEA-D5 (IS1), testosterone-D3 (IS2), DHT-D3 (IS3) and stanozolol-D3 (IS4) were acquired from Cerilliant (Round Rock, TX, USA).

Techniques: Concentration Assay

Journal: Molecules

Article Title: Dried Urine Microsampling Coupled to Liquid Chromatography—Tandem Mass Spectrometry (LC–MS/MS) for the Analysis of Unconjugated Anabolic Androgenic Steroids

doi: 10.3390/molecules25143210

Figure Lengend Snippet: Matrix effect and stability.

Article Snippet: Certified analytical standards of testosterone, mesterolone, danazol, DHT, methandrostenolone, and clostebol as pure powders; standard solutions at the concentration of 1 mg/mL in methanol of epitestosterone, 1-androstenedione, DHEA, norethandrolone, nandrolone, fluoxymesterone, stanozolol and ISs as stable isotope-labelled analogues solutions at 100 µg/mL in methanol of DHEA-D5 (IS1), testosterone-D3 (IS2), DHT-D3 (IS3) and stanozolol-D3 (IS4) were acquired from Cerilliant (Round Rock, TX, USA).

Techniques: Concentration Assay

Journal: Molecules

Article Title: Dried Urine Microsampling Coupled to Liquid Chromatography—Tandem Mass Spectrometry (LC–MS/MS) for the Analysis of Unconjugated Anabolic Androgenic Steroids

doi: 10.3390/molecules25143210

Figure Lengend Snippet: Results of method application to real samples.

Article Snippet: Certified analytical standards of testosterone, mesterolone, danazol, DHT, methandrostenolone, and clostebol as pure powders; standard solutions at the concentration of 1 mg/mL in methanol of epitestosterone, 1-androstenedione, DHEA, norethandrolone, nandrolone, fluoxymesterone, stanozolol and ISs as stable isotope-labelled analogues solutions at 100 µg/mL in methanol of DHEA-D5 (IS1), testosterone-D3 (IS2), DHT-D3 (IS3) and stanozolol-D3 (IS4) were acquired from Cerilliant (Round Rock, TX, USA).

Techniques: Concentration Assay

Journal: Molecules

Article Title: Dried Urine Microsampling Coupled to Liquid Chromatography—Tandem Mass Spectrometry (LC–MS/MS) for the Analysis of Unconjugated Anabolic Androgenic Steroids

doi: 10.3390/molecules25143210

Figure Lengend Snippet: LC-MS/MS chromatogram from a real urine sample spiked with ISs. 1: 1-androstenedione, 2: DHEA, 3: testosterone, 4: epitestosterone, 5: clostebol, 6: DHT.

Article Snippet: Certified analytical standards of testosterone, mesterolone, danazol, DHT, methandrostenolone, and clostebol as pure powders; standard solutions at the concentration of 1 mg/mL in methanol of epitestosterone, 1-androstenedione, DHEA, norethandrolone, nandrolone, fluoxymesterone, stanozolol and ISs as stable isotope-labelled analogues solutions at 100 µg/mL in methanol of DHEA-D5 (IS1), testosterone-D3 (IS2), DHT-D3 (IS3) and stanozolol-D3 (IS4) were acquired from Cerilliant (Round Rock, TX, USA).

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: Molecules

Article Title: Dried Urine Microsampling Coupled to Liquid Chromatography—Tandem Mass Spectrometry (LC–MS/MS) for the Analysis of Unconjugated Anabolic Androgenic Steroids

doi: 10.3390/molecules25143210

Figure Lengend Snippet: MRM transitions, cone voltage, collision energy and retention time for each analyte and IS.

Article Snippet: Certified analytical standards of testosterone, mesterolone, danazol, DHT, methandrostenolone, and clostebol as pure powders; standard solutions at the concentration of 1 mg/mL in methanol of epitestosterone, 1-androstenedione, DHEA, norethandrolone, nandrolone, fluoxymesterone, stanozolol and ISs as stable isotope-labelled analogues solutions at 100 µg/mL in methanol of DHEA-D5 (IS1), testosterone-D3 (IS2), DHT-D3 (IS3) and stanozolol-D3 (IS4) were acquired from Cerilliant (Round Rock, TX, USA).

Techniques:

Journal: Advances in Pharmacological Sciences

Article Title: Neurosteroid Binding Sites on the GABA A Receptor Complex as Novel Targets for Therapeutics to Reduce Alcohol Abuse and Dependence

doi: 10.1155/2011/926361

Figure Lengend Snippet: The amount of DHEA (ng/g) present in the hypothalamus, hippocampus, and frontal cortex of rats over a six-hour time period after a single acute intraperitoneal injection. Adult male Long-Evans rats received either 56 mg/kg of DHEA ( n = 6) or an equal volume of cyclodextrin vehicle ( n = 5). A DHEA-treated subject was sacrificed with a vehicle-treated control at 15, 30, 60, 120, and 180 minutes after injection, while the final DHEA-treated subject was sacrificed 360 minutes after injection. Brains were collected, flash frozen, and later dissected using the Glowinski technique . Steroids were extracted from the hypothalamus, hippocampus, and frontal cortex of each subject using the solid-phase extraction method described and validated by Newman et al. . Briefly, tissue from each region was prepared in an aqueous matrix and steroids were extracted from each sample using a C18 column primed with ethanol and equilibrated with water. Each sample was eluted, dried, and resuspended in deionized water, and DHEA levels were determined using ELISA.

Article Snippet: Steroids were extracted using the solid-phase technique established and validated by Newman et al. [ ] and then analyzed using a commercially-available ELISA (DHEA Saliva ELISA kit, IBL International, Hamburg, Germany).

Techniques: Injection, Control, Extraction, Enzyme-linked Immunosorbent Assay

Journal: Advances in Pharmacological Sciences

Article Title: Neurosteroid Binding Sites on the GABA A Receptor Complex as Novel Targets for Therapeutics to Reduce Alcohol Abuse and Dependence

doi: 10.1155/2011/926361

Figure Lengend Snippet: Mean number of GABA A alpha-4 subunit transcript copies per cell in the hypothalamus (top panel) and frontal cortex (bottom panel) of rats administered 56 mg/kg of DHEA or vehicle. Drug-naïve male rats received either DHEA ( n = 12) or an equal volume of cyclodextrin vehicle ( n = 12) for ten consecutive days; on the tenth day, subjects were sacrificed and their brains were collected. Brains were dissected using the Glowinski technique , and each brain region was pooled and homogenized. Due to the high lipid content of the samples, a spin column technique was utilized for the RNA extraction. RNA analysis was performed using TaqMan assay kits (Applied Biosystems, Foster City, Calif, USA). Approximately 1 to 2 μ L of each sample were used to determine the RNA concentration in each sample using Nanodrop. Values are expressed as a fraction of a normalizing gene, ribosomal 18S RNA.

Article Snippet: Steroids were extracted using the solid-phase technique established and validated by Newman et al. [ ] and then analyzed using a commercially-available ELISA (DHEA Saliva ELISA kit, IBL International, Hamburg, Germany).

Techniques: RNA Extraction, TaqMan Assay, Concentration Assay

Journal: Advances in Pharmacological Sciences

Article Title: Neurosteroid Binding Sites on the GABA A Receptor Complex as Novel Targets for Therapeutics to Reduce Alcohol Abuse and Dependence

doi: 10.1155/2011/926361

Figure Lengend Snippet: Expression of the α 4, α 3, and δ subunits of GABA A receptors in the hypothalamus of rats administered 56 mg/kg of DHEA for 10 consecutive days as measured by Western blot analysis. 100 μ g of tissue from each area was resuspended in lysis buffer (20 mM Tris pH 8.0, 137 mM NaCl, 0.5 mM sodium orthovanadate, 2 mM okadaic acid, 10% glycerol, 1% Nonidet P40, 2% protease inhibitor) and processed for protein extraction using MicroRoto for Lysis Kit (Bio-Rad, Hercules, Calif, USA). The Bradford Method was used to determine protein concentration, and then samples were diluted, separated by SDS-PAGE, and transferred to nitrocellulose PDVF membranes (Amersham Biosciences, Piscataway, NJ, USA). The membranes were immunoblotted for two hours at room temperature with two specific antibodies, a rabbit anti- α 4 antibody at a 1 : 500 dilution (Santa Cruz Biotechnology, Santa Cruz, Calif, USA), and a mouse anti- β -actin diluted in a proportion of 1 : 2000 (Santa Cruz Biotechnology). A specific secondary antibody (PerkinElmer Life Sciences, Waltham, Mass, USA) followed at a dilution of 1 : 2000. Expression was visualized using ECL Plus (PerkinElmer) and a Fuji Film luminescent image analyzer (LAS-1000 Plus, Fuji Photo Film Co. Ltd., Tokyo, Japan). The images were then quantified by densitometry using the Image Gauge program , and the expression value of each subunit was normalized to β -actin values.

Article Snippet: Steroids were extracted using the solid-phase technique established and validated by Newman et al. [ ] and then analyzed using a commercially-available ELISA (DHEA Saliva ELISA kit, IBL International, Hamburg, Germany).

Techniques: Expressing, Western Blot, Lysis, Protease Inhibitor, Protein Extraction, Protein Concentration, SDS Page

Journal: Advances in Pharmacological Sciences

Article Title: Neurosteroid Binding Sites on the GABA A Receptor Complex as Novel Targets for Therapeutics to Reduce Alcohol Abuse and Dependence

doi: 10.1155/2011/926361

Figure Lengend Snippet: Effects of intraperitoneal administration of DHEA on rats ( n = 5) responding under an FR-10 schedule for 0.1 mL of 18% (v/v) ethanol. The dependent measures were response rate in responses/min and the dose of ethanol presented in g/kg. The points and vertical lines above “V” indicate the means and standard error of the mean (SEM) for sessions in which vehicle was administered (control). The points with vertical lines in the dose-effect data indicate the mean ± SEM for sessions in which DHEA was administered. The points without vertical lines indicate instances in which the SEM is encompassed by the point.

Article Snippet: Steroids were extracted using the solid-phase technique established and validated by Newman et al. [ ] and then analyzed using a commercially-available ELISA (DHEA Saliva ELISA kit, IBL International, Hamburg, Germany).

Techniques: Control