Identification of 17-methyl-18-norandrosta-5,13(17-dien-3beta-ol, the C19 fragment formed by adrenal side chain cleavage of a 20-aryl analog of (20S)-20-hydroxycholesterol. (1/29)

Incubation of (20R)-20-phenyl-5-pregnene-3beta,20-diol, an aromatic analog of (23S)-20-hydroxycholesterol, with an adrenal mitochondrial preparation leads to the formation of four compounds: pregnenolone, phenol, a C8 ketone, acetophenone, and a nonpolar C19 compound. This latter compound has now been identified by reverse isotope dilution analysis and by gas chromatography/mass spectrometry as 17-methyl-18-norandrosta-5,13(17)-dien-3beta-ol. From these results it is evident that enzymatic fission of the C-17,20 bond of this synthetic derivative occurs. On the other hand, when (20S)-20-hydroxy[21-14C]cholesterol was used as substrate, the analogous cleavage did not take place. Thus, substitution of an aromatic group on C-20 facilitates side chain cleavage between that carbon atom and the nucleus whereas neither of the naturally occuring precursors, cholesterol or its 20-hydroxylated counterpart, are metabolized to a C8 fragment.  (+info)

Stimulation of pregnenolone metabolism and aromatase activity by luteinizing hormone in mouse uterus. (2/29)

In vitro metabolism of pregnenolone (P5) as well as production of 17beta-estradiol (E2) were studied in uteri of untreated and luteinizing hormone (LH)-treated mice that had been ovariectomized (OVX) at late-diestrus stage. In the uteri of untreated mice, [H]pregnenolone was shown to be metabolized to Delta-components such as 17alpha-hydroxypregnenolone (17alpha-P5) and dehydroepiandrosterone (DHEA), whereas LH treatment resulted in significant increases in the formation of progesterone (P4), 17alpha-hydroxyprogesterone (17alpha-P4), androstenedione (AD) and testosterone (T). This was assessed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The content and release of P4 was shown to be stimulated by LH. Trilostane, an inhibitor of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), inhibited LH-induced P4 synthesis and its release in a dose-dependent manner. A considerable increase in [H]estradiol formation from [H]testosterone was recorded in LH-stimulated uterine tissue as compared with the control, indicating the stimulatory effect of LH on aromatase activity. LH-stimulation in the synthesis of P4 and E2 in OVX mouse uteri was mimicked by dbcAMP (cell-permeable cAMP). Incubation with LH was shown to augment the conversion of P4 to various delta-3-oxosteroids. In vitro effects of LH on the synthesis and metabolism of P4, as well as on the stimulation of aromatase activity, were more pronounced in the uterine tissue of LH-primed OVX mice. Thus the results of the present study indicate that, under specific conditions, the uterus of the mouse behaves like steroidogenic tissue. Its prompt response to LH reveals the probable physiological relevance of the existence of LH receptors of high binding affinity in the uterine tissue of the mouse, as reported earlier.  (+info)

Induction of delta-aminolevulinic acid synthetase in isolated rat liver cells by steroids. (3/29)

The role of steroids in regulation of delta-aminolevulinic acid synthetase has been studied in isolated rat liver cell suspensions under conditions previously shown to support inducation of the enzyme by drugs. Addition of a variety of C-19 and C-21 steroids to cell suspensions resulted, after 4 to 6 hours of incubation, in 2- to 5-fold increase in the activity of delta-aminolevulinic acid synthetase as measured in liver cell homogenates. The increase was prevented by cycloheximide. The most active steroid inducers tested were pregnene or pregnane derivatives with keto or hydroxyl groups at C-3 and C-20; in particular a beta-hydroxyl group at C-20 enhanced activity. These C-21 steroids at optimal initial concentrations caused 3- to 5-fold induction over 4 hours. A number of C-19 androstene and androstane compounds caused 2- to 3-fold inducation over the same period. Hydrocortisone had no effect. For a variety of androstane and pregnane derivatives, inducation by 5alphaH steroids was as great as or greater than that by 5betaH compounds, in contrast to previous findings in chick embryo liver. Induction of delta-aminolevulinic acid synthetase by steroids in isolated liver cells was shown to be subject to feedback repression by hemin.  (+info)

Effect of culture conditions on the biosynthesis of gagaminine, a potent antioxidant from the roots of Cynanchum wilfordii. (4/29)

Gagaminine, a steroidal alkaloid isolated from the roots of Cynanchum wilfordii, exhibited potent inhibitory effects on the aldehyde oxidase activity and lipid peroxidation. To find a possible means of mass production of this active component, which will be useful for animal tests, we synthesized it by an in vitro culture method using various growth conditions. Calli were induced from the explants of this medicinal plant and cultivated under culture conditions which varied in light, and the kinds and concentration of plant growth regulators. The production of gagaminine was found to be significantly higher in the dark than in the light. The best gagaminine content (0.960%) was obtained after cultivation of stems on the medium containing 2,4-dichlorophenoxyacetic acid (2,4-D, 2.0 mg/l) only. However, gagaminine was not detected by the mixture of 2,4-D and kinetin, while the mixtures of 2,4-D/zeatin and 2,4-D/6-benzylaminopurine produced a low content of gagaminine (<0.4%). In addition, suspension medium was much better for the formation of gagaminine than solid medium with an increase from 0.960 to 2.227% yield. These results suggest that gagaminine can be produced massively by in vitro culture using stems under the conditions of dark and 2,4-D on liquid medium.  (+info)

Fragments formed by the side chain cleavage of a 20-aryl analog of 20alpha-hydroxycholesterol by adrenal mitochondria. (5/29)

An analog of 20alpha-hydroxycholesterol, (20R)-20-phenyl-5-pregnene-3beta,20-diol, which is completely substituted at C-22 was prepared with radioisotopes at various positions. The analog labeled with 3H at C-M and 14C at C-4 and C-IU was converted into radioactive pregnenolone by an enzyme preparation derived from adrenal mitochondria. Cleavage of the phenyl analog labeled with 3H in the aromatic ring by the same enzyme preparation led to the formation of [3H]phenol. Using the substrate doubly labeled with 14C at C-4 and 3H in the aromatic ring, it appeared that the products of the reactions, pregnenolone and phenol, were formed in equal amounts. During incubation of the side chain labeled substrate, another labeled fragment was formed. It was identified as acetophenone, a product resulting from cleavage of the C17,20 bond. The steroidal fragment corresponding to this C8 ketone was traced using nuclear label analog. From its nonpolar chromatographic properties it appears to be a C-17-deoxy-C19 steroid.  (+info)

Comparative study of the effects of various culture conditions on cell growth and Gagaminine synthesis in suspension culture of Cynanchum wilfordii (MAXIM.) HEMSLEY. (6/29)

Gagaminine, a steroidal alkaloid isolated from the roots of Cynanchum wilfordii, exhibited potent inhibitory effects on aldehyde oxidase activity and lipid peroxidation. To determine whether it would be possible to mass produce this active component, which would be useful for animal tests, we tried to synthesize it using in vitro cell culture methods with various growth conditions. In a previous study it was found that calli were easily induced from the stem of this medicinal plant and cultivated effectively on MS medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) 2 mg/l. In this work we attempted to determine the effects of various culture conditions on cell growth and gagaminine synthesis in suspension culture. Gagaminine production was increased markedly when cell growth proceeded to the death phase. Cell growth was more effective with 5% (v/v) sucrose, in the light (at 38 microE/m(2) x s), on medium containing 2,4-D 2 mg/l, with 2.5 g/10 ml medium as the initial cell concentration. The concentration of gagaminine was optimal with 3% sucrose, in darkness on medium 2,4-D 1 mg/l, with 2.5 g/10 ml medium as an initial cell concentration. However, the highest growth rate was 0.18 d(-1), when the gagaminine concentration was seven- and three-fold (at 140 mu/ml) that of the plant stem and 10 ml of medium respectively, on the 50 ml of medium in suspension culture.  (+info)

Periplocoside E, an effective compound from Periploca sepium Bge, inhibited T cell activation in vitro and in vivo. (7/29)

Periploca sepium Bge, a traditional Chinese herb medicine, is used for treating rheumatoid arthritis in China. Followed the bioactivity-guided isolation, the most potent immunosuppressive compound, periplocoside E (PSE), a pregnane glycoside, had been identified from P. sepium Bge. We investigated the immunosuppressive effects of PSE in vitro and in vivo. The results showed that PSE in a dose-dependent manner significantly inhibited the proliferation of splenocytes induced by concanavalin A and mixed lymphocyte culture reaction at no cytotoxic concentrations (<5 microM). Administration of PSE suppressed a delayed-type hypersensitivity reaction, and ovalbumin (OVA) induced antigen-specific immune responses in mice. In vivo treatment with PSE dose dependently suppressed OVA-induced proliferation and cytokine [interleukin (IL)-2 and interferon (IFN)-gamma] production from splenocytes in vitro. Purified T cells from OVA-immunized mice with PSE treatment showed its low ability for activation by OVA plus normal antigen presenting cell stimulation again in vitro. Further studies showed PSE dose dependently inhibited anti-CD3-induced primary T cell proliferation, activation for IL-2Ralpha (CD25) expression, and cytokine (IFN-gamma and IL-2) production also at the transcriptional level. PSE was highly specific and significantly inhibited the activation of extracellular signal-regulated kinase and Jun N-terminal kinase, whereas activation of p38 was not affected in T cells stimulated with anti-CD3. These results demonstrated that PSE is an immunosuppressive compound in P. sepium Bge, which directly inhibits T cell activation in vitro and in vivo. This study provided evidence to understand the therapeutic effects of P. sepium Bge and indicated that this herb is appropriate for treatment of T cell-mediated disorders, such as autoimmune diseases.  (+info)

Periplocoside E inhibits experimental allergic encephalomyelitis by suppressing interleukin 12-dependent CCR5 expression and interferon-gamma-dependent CXCR3 expression in T lymphocytes. (8/29)

Periplocoside E (PSE) was found to inhibit primary T-cell activation in our previous study. Now we examined the effect and mechanisms of PSE on the central nervous system (CNS) demyelination in experimental allergic encephalomyelitis (EAE). C57BL/6 mice immunized with myelin oligodendrocyte glyco-protein (MOG) were treated with PSE following immunization and continued throughout the study. The effect on the progression of EAE and other relevant parameters were assessed. PSE reduced the incidence and severity of EAE. Spinal cord histopathology analysis showed that the therapeutic effect of PSE was associated with reduced mononuclear cell infiltration and CNS inflammation. As reverse transcription-polymerase chain reaction analysis showed, PSE decreased the CD4(+), CD8(+), and CD11b(+) cell infiltration. T cells from lymph nodes of MOG-immunized mice expressed enhanced levels of CCR5 and CXCR3 mRNA compared with T cells from normal mice. However, CCR5 and CXCR3 expressions were suppressed in T cells from PSE-treated mice. In vitro study also showed PSE inhibited interferon (IFN)-gamma-dependent CXCR3 expression in T cells through suppressing T-cell receptor (TCR) ligation-induced IFN-gamma production, whereas it inhibited interleukin (IL)-12-dependent CCR5 expression through suppressing IL-12 reactivity in TCR-triggered T cells. As a result, the initial influx of T cells into CNS was inhibited in PSE-treated mice. The consequent activation of macrophages/microglia cells was inhibited in spinal cord from PSE-treated mice as determination of chemokine expressions (CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10). Consistently, the secondary influx of CD4(+), CD8(+), and CD11b(+) cells was decreased in spinal cords from PSE-treated mice. These findings suggest the potential therapeutic effect of PSE on multiple sclerosis.  (+info)