Effects of Periplocoside P from Periploca sepium on the Midgut Transmembrane Potential of Mythimna separata Larvae.

Periplocoside P (PSP) isolated from the root bark of Periploca sepium contains a pregnane glycoside skeleton and possesses high insecticidal properties. Preliminary studies indicated that PSP disrupts epithelial functions in the midgut of lepidopteran larvae. In the present study, we examined the effects of PSP on the apical and basolateral membrane voltages, Va and Vbl, respectively, of cells from (1) midguts isolated from the larvae of the oriental armyworm Mythimna separata that were in vitro incubated with toxins and (2) midguts isolated from M. separata larvae force-fed with PSP. We compared the effects of PSP with the effects of the Bacillus thuringiensis toxin Cry1Ab and inactive periplocoside E (PSE) on the midgut epithelial cells. The results showed that Va rapidly decreased in the presence of PSP in a time- and dose-dependent manner, similar to the effects of Cry1Ab. By contrast, PSE did not affect the Va and Vbl. Additionally, PSP did not influence the Vbl. Given these results, we speculate that PSP may modulate transport mechanisms at the apical membrane of the midgut epithelial cells by inhibiting the V-type H+ ATPase.

Bioactive C21 Steroidal Glycosides from the Roots of Cynanchum otophyllum That Suppress the Seizure-like Locomotor Activity of Zebrafish Caused by Pentylenetetrazole.

Six new C21 steroidal glycosides, cynotophyllosides A-F (1-6), together with 16 known compounds, were isolated from the roots of Cynanchum otophyllum. The structures of the new compounds were elucidated by spectroscopic analysis and chemical methods. The three major components, otophylloside F (15), otophylloside B (17), and rostratamine 3-O-ß-D-oleandropyranosyl-(1→4)-ß-D-cymaropyranosyl-(1→4)-ß-D-cymaropyranoside (18), suppressed the seizure-like locomotor activity caused by pentylenetetrazole in zebrafish. Preliminary structure-activity relation studies revealed that a pregnene skeleton with a C-12 ester group (ikemaoyl > cinnamoyl > hydroxy > p-hydroxybenzoyl) and a C-3 sugar chain consisting of three 2,6-dideoxysaccharide units is essential for this suppressive activity.

A new pregnane glycoside and oligosaccharide from Parabarium huaitingii.

Two new compounds, along with two known compounds, were isolated from the barks of Parabarium huaitingii, and their structures were determined as 5α-pregn-6-ene-3ß,17α,20(S)-triol-20-O-ß-d-digitoxopyranoside (1), cymaropyranurolactone 4-O-ß-d-digitalopyranosyl-(1 â†’ 4)-O-ß-d-cymaropyranosyl-(1 â†’ 4)-O-ß-d-oleandropyranosyl-(1 â†’ 4)-O-ß-d-cymaropyranoside (2), 3ß,17α,20(S)-trihydroxy-5α-pregn-6-ene (3), and 5α-pregn-6-ene-3ß,17α,20(S)-triol-3-O-ß-d-digitalopyranoside (4) by spectroscopic methods.

Periplocoside E, an effective compound from Periploca sepium Bge, inhibited T cell activation in vitro and in vivo.

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.

Induction of aggregation and augmentation of protein kinase-mediated phosphorylation of purified vimentin intermediate filaments by withangulatin A.

Purified assembly-competent vimentin, an intermediate filament protein, was obtained from bovine lens in this study. The effects of withangulatin A on vimentin assembly with or without phosphorylation were examined by negative-stain electron microscopy. Soluble tetrameric vimentin was assembled into irregular fibrils with lateral associations or short filaments after pretreatment with 50 or 100 microM withangulatin A, respectively. Incubation of assembled vimentin filaments with withangulatin A at 50 or 100 microM resulted in the formation of aggregates, and the degree of aggregation was concentration dependent. The appearance of vimentin filaments was slightly altered after treatment with cAMP-dependent protein kinase or protein kinase C; however, phosphorylation of filamentous vimentin by the protein kinases in the presence of withangulatin A resulted in higher degrees of aggregation of the filaments, compared with those treated only with the drug. Moreover, the level of phosphorylation of filamentous vimentin by the protein kinases was augmented in the presence of withangulatin A. Experimental results indicated that withangulatin A directly and specifically affects the conformation of the vimentin molecules, thereby resulting in alterations in assembly behavior and reactivity toward cAMP-dependent protein kinase and protein kinase C. The data observed further imply that withangulatin A, which directly causes aggregation of vimentin filaments, is a vimentin intermediate filament-targeting drug.

Induction of vimentin modification and vimentin-HSP72 association by withangulatin A in 9L rat brain tumor cells.

Withangulatin A induced cell rounding up and the morphological alteration resulted from the reorganization of all of the major cytoskeletal components, i.e., vimentin, tubulin, and actin, as revealed by immunofluorescence techniques. When the withangulatin A-treated cells changed to a round-up morphology, vimentin intermediate filaments were found to be collapsed and clustered around the nucleus. The alteration was accompanied by characteristic changes of vimentin molecules, including augmentation of phosphorylation, retardation of electrophoretic mobility, and decrease in detergent extractability. The levels of vimentin phosphorylation were augmented by 2.5- and 1.8-fold in cells incubated with 50 microM withangulatin A for 1 and 3 h, respectively. The electrophoretic mobility of vimentin was partially retarded in cells treated with withangulatin A for 1 h at 10 microM and a completely upshift mobility was observed after 5 h treatment at 50 microM. In addition, vimentin molecules became less extractable by nonident P-40 after the cells were treated with withangulatin A and this effect was dose dependent. The decrease in solubility of vimentin was accompanied by the redistribution of HSP72 into the detergent nonextractable fraction and these two events were well correlated. Our results suggest that withangulatin A induced the modification of vimentin, which resulted in the alteration of cell morphology and redistribution of intracellular HSP72, an event that may play an important role in the induction of heat-shock response.

Inhibition of placental 3beta-hydroxy-steroid dehydrogenase by naturally occurring steroids. A potential mechanism regulating oestrogen synthesis from unconjugated precursors.

We have proposed that inhibition of placental steroid 3-sulphatase by endogenous steroids may regulate oestrogen synthesis during human pregnancy. The possibility that an additional regulatory mechanism, involving the placental 3beta-hydroxy-steroid dehydrogenase (SDH), may also be operative has now been examined. Inhibitory effects of naturally occurring steroids on SDH activity were determined from the reduction in initial rate of conversion of 3H-dehydroepiandrosterone to non-digitonin precipitable products by 10 000 x g supernatant from homogenates of human term placentae. The apparent Km for dehydroepiandrosterone was 0.33 x 10(-6) M. delta4-3-Oxo products of SDH action (4-androstene-3,17-dione, app. Ki=0.60x10(-6) M; progesterone, app. Ki=1.5x10(-6) M) were the most potent inhibitors and appeared to act non-competitively. Delta5-3beta-Hydroxy alternative substrates were less inhibitory and in the case of pregnenolone (app. Ki=4.5x10(-6) M) behaved competitively. 11beta-, 16alpha-, 17alpha- or 21-hydroxylation and epimerization of 3beta- or 17beta-hydroxyl functions of inhibitors decreased their activity. It is concluded that inhibition of both sulphatase and SDH by endogenous steroids may provide complementary methods of regulating placental oestrogen synthesis in vivo. The SDH mechanism may regulate oestrogen synthesis from unconjugated precursors, either formed within the placenta or derived from the circulation. The major potential inhibitors appear to be delta4-3-ketones, acting non-competitively, and formed within the placenta. In the sulphatase mechanism alternative substrates of extraplacenta origin, acting competitively, are the major potential inhibitors controlling utilization of conjugated precursors.