Cellular target identification of Withangulatin A using fluorescent analogues and subsequent chemical proteomics.

Withangulatin A (WA) has been reported to exhibit potent antitumor activity. However, its possible mechanism and direct proteomic targets remain unknown. Herein we report the subcellular localization of WA by designing and synthesizing its fluorescent analogues with coumarin moieties. Furthermore, sarco/endoplasmic reticulum calcium-ATPase (SERCA)2 was identified as the potential target of WA for its antitumor activity by chemical proteomics.

Measurement of cortisol, cortisone, prednisolone, dexamethasone and 11-deoxycortisol with ultra high performance liquid chromatography-tandem mass spectrometry: Application for plasma, plasma ultrafiltrate, urine and saliva in a routine laboratory.

We describe an ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC MS/MS) method suitable for a routine laboratory to determine endogenous and exogenous glucocorticoids in plasma, plasma ultrafiltrate, urine and saliva in a single analytical run. After addition of a multi-analyte internal standard, a standardised sample preparation procedure with solid phase extraction followed, before injecting into a tandem mass spectrometer with positive mode electron spray ionisation and multiple reactant monitoring acquisition. The chromatography time was 3min. The limit of quantitation for cortisol and cortisone in plasma was 3.75nmol/L and linearity extended to 2000nmol/L. The limit of quantitation for cortisol in plasma ultrafiltrate and saliva was 0.6nmol/L. The limit of quantitation for 11-deoxycortisol and prednisolone was 5nmol/L and for dexamethasone 1nmol/L. The intra-assay CV was <5% and the inter-assay CV <10% for all analytes in all matrices. Comparison with an immuno-assay (IA) plasma cortisol method resulted in a regression equation of UHPLC=0.79×IA+31.12 with R(2)=0.960 (p<0.0001). Comparison with a high performance liquid chromatography (HPLC) cortisol method yielded a regression equation of UHPLC=1.06×HPLC+9.82, R(2)=0.992 (p<0.0001). The simultaneous measurement of endogenous and exogenous glucocorticoids contributed to patient care in cases with dexamethasone and metyrapone dynamic tests and unsuspected therapeutic glucocorticoid use.

[Synthesis and spectral analysis of three kinds of epoxy pregnene derivatives].

Three kinds of derivatives, 16alpha, 17alpha-epoxy-6-methylene-pregn-4-ene-3,20-dione(EMPD I), 16alpha, 17alpha-epoxy-6-methyl-pregn-4,6-diene-3,20-dione (EMPD II) and 16alpha, 17alpha-epoxy-6-methyl-pregn-4-ene-3, 20-dione(EMPD III) were synthesized and their FTIR, UV and 1H NMR spectra were described. EMPD II is a novel compound. In the UV spectra, the maximum absorptions peaks of EMPD I, EMPD II and EMPD III are at 259, 287 and 239 nm respectively. Their IR spectra are also obviously different. For EMPD I, there are two absorption peaks, which are respectively at 3084 cm(-1) originated from ==CH2 and 3 030 cm(-1) from C==CH--. For EMPD II and III, there is only one absorption peak at 3054 cm(-1). From 1710-1660 cm(-1), only one absorption peak appears for EMPD III, but two appear for EMPD I and EMPD II. In 1H NMR spectrum, the peaks at 4.92 (s, 1H), 5.06 (s, 1H) and 5.88 (s, 1H) were observed for EMPD I; the peaks at 1.83 (s, 3H), 5.87 (s, 1H) and 5.85 (s, 1H) for EMPD II; while the peaks at 1.06-1.07 (d, 3H), 5.78 ppm (s, 1H, 1 ppm=l nicrog x mL(-1)), for EMPD III.

The influence of temperature on the high performance liquid chromatographic separation of steroids using mobile phases modified with beta-cyclodextrin.

The effect of temperature on the retention and multiple separation of hydrocortisone, testosterone, 17 alpha-methyltestosterone, prednisone, cortisone and 17 alpha-hydroxyprogesterone in reversed-phase liquid chromatography has been studied. Capacity factors (k') of the steroids were measured using a mobile phase modified with different concentrations of beta-cyclodextrin (from 0-16 mM), a fixed solvent composition (acetonitrile-water) and a wide range of column temperatures (from 5-80 degrees C). The plots of capacity factors vs. reciprocal of absolute temperature are nonlinear in every case when mobile phase modified with beta-cyclodextrin was used. Particularly strong nonlinearity was observed at lower temperature and at higher beta-cyclodextrin concentration. The complex chromatograms were evaluated using optimization parameters such as capacity factor of the last-eluted peak (k'max), the smallest resolution between adjacent peaks (Rs; min) and relative resolution product (r). The results presented describe precisely the role of temperature in high performance liquid chromatography systems in which mobile phases modified with cyclodextrin were used.

Concentrations of progestagens and oestrogens in the faeces of pregnant Lipizzan, trotter and thoroughbred mares.

Faecal samples were collected at weekly intervals from pregnant Lipizzan mares during Weeks 7-16 following mating and from Lipizzan, Trotter and Thoroughbred mares during the last 3 months of gestation. After parturition, samples were taken daily from the Thoroughbred mares for another 6 days. Non-pregnant mares served as controls. The concentrations of unconjugated oestrogens (Eg), 20 alpha-OH-progestagens (20 alpha-G) and 20 beta-OH-progestagens (20 beta-G) were measured by enzyme immunoassay. In the faeces of Lipizzan mares, immunoreactive progestagens were significantly (P less than 0.01) elevated above the levels in non-pregnant mares by Week 11, and Eg by Week 13 of pregnancy onwards. During the last 3 months of gestation, concentrations of Eg were significantly higher in Trotter mares than in Lipizzan and Thoroughbred mares. Concentrations of 20 alpha-G and 20 beta-G increased to maximal values in the last month of gestation. There was no significant difference among the 3 breeds with respect to 20 alpha-G but, during the 10 weeks before parturition, concentrations of 20 beta-G in the Lipizzan mares were significantly lower (P less than 0.05) than those in the Thoroughbred mares. They were also significantly lower than those of the Trotter mares during the last 4 weeks of gestation. After parturition, the concentrations of Eg and progestagens had declined to baseline values by Days 3 and 4 respectively. From these results we conclude that high concentrations of progestagens with 20 alpha- and 20 beta-hydroxyl groups are present in the faeces of pregnant mares, especially during the last month of gestation.

High performance liquid chromatography of steroids in bovine adrenal glands: changes during the postpartum interval.

High performance liquid chromatography was utilized to characterize changes in the content of various steroids in adrenal glands with time postpartum. Adrenal glands were obtained from 32 beef cows on either days, 7, 14, 28, 42 or 56 after parturition (n = 6-8/group). Of the major glucocorticoids, only cortisone significantly changed with time postpartum. Adrenal content of cortisone was 56% lower (P less than 0.05) on day 14 than on days 7, 28, 42 and 56. Average adrenal content (microgram/g) of cortisol, cortisone, corticosterone and 11-dehydrocorticosterone was 3.3, 2.4, 1.8 and 1.8, respectively. All other adrenal steroids measured were less than 0.8 micrograms/g. Correlation coefficients among the adrenal steroids measured indicated that terminal products (e.g. 11-dehydrocorticosterone and cortisone) of glucocorticoidogenesis were positively correlated (r = 0.56-0.64) with each other (P less than 0.05), whereas cortisone was negatively correlated (P less than 0.05) with both 17 alpha-hydroxyprogesterone (r = -0.49) and progesterone (r = -0.58). The latter suggests that a product (cortisone) inhibition mechanism may exist in bovine adrenal glucocorticoidogenesis. Collectively, these data suggest that cortisone should be considered a potential factor in the regulation of physiologic functions in postpartum cattle.

Structures and concentrations of fifteen different steroid sulfates in human breast cyst fluids.

By applying capillary gas chromatography (GC) and gas chromatography mass spectrometry (GC-MS), a simultaneous quantitation of all important steroid sulfates present in a number of breast cyst fluids, has been obtained. The fact that prevailing androgen sulfate structures in the cyst fluids are different from those in blood suggests at least intracystic metabolism of blood-born precursors. Particularly greater amounts of 5 alpha-reduced steroids are found in breast cysts. 5 alpha-Androstane-3 alpha,17 beta-diol is a major androgen sulfate of breast cyst fluids, its concentration being some 2000-fold that of blood. After prolonged topical application of progesterone on the breast, an accumulation of the sulfates of several pregnanediol isomers could be observed.

Steroid concentrations in the outer and inner zones of the adrenal cortex of the guinea pig.

The outer (glomerulosa and fasciculata) and inner (reticularis) zones of the adrenal cortex of the guinea pig were separated and their steroid content determined. It was found that the concentration of 21-hydroxypregnenolone, deoxycorticosterone, corticosterone, aldosterone, 11-deoxycortisol, and cortisol was significantly higher in the outer cortical region, while the concentration of pregnenolone, 17-hydroxypregnenolone, and dehydroepiandrosterone was significantly higher in the inner zone. The concentration of progesterone, 17-hydroxyprogesterone, and androstenedione was not different in the two zones. Examination of specific steroid ratios suggested the following: (1) 3 beta-ol dehydrogenase/isomerase and 21-hydroxylase activities are reduced in the inner zone, (2) 17-hydroxylase and C17 20 lyase activities appear to be equally active in the two zones (3) 11 beta-hydroxylase activity appears to be more active in the inner zone (4) 21- hydroxypregnenolone , deoxycorticosterone, corticosterone, 11-deoxycortisol, and cortisol along with aldosterone are produced principally in the outer zone.

Follicular fluid steroid levels and ovarian steroid secretion in polycystic ovarian disease.

Comparison of the plasma levels of steroids between ovarian and peripheral venous blood in polycystic ovarian disease revealed that polycystic ovaries secreted androstenedione, testosterone, dihydrotestosterone, dehydroepiandrosterone and 17-hydroxyprogesterone significantly more than healthy ovaries at the early follicular phase of the cycle, whereas the levels of estradiol and progesterone did not differ from the control values. Follicular cyst fluid from polycystic ovaries showed significantly higher concentrations of testosterone, androstenedione jand dehydroepiandrosterone and much lower levels of estrone, estradiol, progesterone and 17-hydroxyprogesterone than follicular fluid from healthy preovulatory follicles. Testosterone and estradiol did not, however, show any significant difference when follicles in polycystic ovaries were compared to those in healthy ovaries at early follicular phase of the cycle. According to these findings follicle development in polycystic ovaries is arrested at a stage corresponding to the early follicular phase of the cycle. Increased androgen and 17-hydroxyprogesterone secretion by polycystic ovaries seems to originate mainly from the hyperplastic theca interna cells.

Suppression of progesterone secretion in lactating rats by administration of ergocornine and the effect of prolactin replacement.

In order to assess the stimulatory effect of prolactin on the secretion of progesterone from corpora lutea in lactating rats, ergocornine meleate (ECO; 1 mg/day), an inhibitor of prolactin secretion, was administered subcutaneously on days 6 and 7 of lactation in primiparous rats. By day 8 of lactation, the concentration of progesterone in ovarian venous blood fell to a undetectable level in the ECO-treated animals, while the concentration in the control animals was very high at this stage of lactation. The level of 20-alpha-hydroxypregn-4-en-3-one was significantly higher on day 8 of lactation in ECO-treated than in control rats. Lactational dioestrus was interrupted by treatment with ECO and vaginal oestrus appeared 3-4 days after the start of treatment. Administration of ECO caused deleterious depression of milk production and of food intake of mother rats. In the pair-fed control animals, lactation continued almost normally throughout the experimental period. Prolactin (1 mg/day) administered simultaneously with ECO increased progesterone to levels even higher than those in control rats and restored 20-alpha-hydroxypregn-4-en-3-oen levels to those of the controls. The effect of the drug on milk production was alleviated. The results strongly suggest that prolactin is the most important factor in maintaining the function of corpora lutea in the lactating rats.