[Chemical constituents from Pulsatillae Radix].

Chemical constituents of ethanol extract of Pulsatillae Radix were investigated. The n-butanol fraction of ethanol extract of Pulsatillae Radix was isolated and purified by macroporous resin and silica gel column chromatography and semi-preparative high performance liquid chromatography. The triterpenoid glycosides were identified by multiple spectral methods. Six compounds were obtained from the n-butanol fraction of ethanol extract of Pulsatillae Radix and identified as 23-aldehyde-cussosaponin C(1), cussosaponin C(2), anemoside B4(3), akebia saponin D(4), pulchinenoside E3(5), and hederacoside C(6). Among them, compound 1 was a new compound.

Ultrasonic-assisted extraction and adsorption separation: Large-scale preparation of trans-ε-Viniferin, suffruficosol B and trans-Gnetin H for the first time.

In this study, a Standard Operating Procedure (SOP) for the large-scale extraction, enrichment, and separation of suffruticosol B (SB), trans-ε-Viniferin (TV), trans-gnetin H (TG) from oil tree peony seeds shell (PSS) was successfully constructed. The ultrasonic-assisted extraction (UAE), macroporous adsorption resin (MAR), and column chromatography (CC) were employed to extract, enrich and separate SB, TV and TG from PSS, and the conditions were optimized. The results implied that SB (1.6937 g), TV (0.5884 g) and TG (3.8786 g) with the purity of 99.67 %, 99.32 % and 98.54 %, respectively, were obtained after the extraction, enrichment and separation. The total yields of the SB, TV and TG were 0.61 mg/g, 0.02 mg/g and 6.64 mg/g with the total extraction rates at 70.55 %, 69.77 % and 78.36 %, respectively. This is the first report on the large-scale extraction, enrichment and separation of oligostilbenes. The SOP in this paper could produce high purity SB, TV and TG, and provide a new idea for PSS as a new oligostilbene resource. The study expands the new development and research field of PSS and provides theoretical support for the green utilization of oil tree peony.

Type-3 ryanodine receptors mediate hypoxia-, but not neurotransmitter-induced calcium release and contraction in pulmonary artery smooth muscle cells.

In this study we examined the expression of RyR subtypes and the role of RyRs in neurotransmitter- and hypoxia-induced Ca2+ release and contraction in pulmonary artery smooth muscle cells (PASMCs). Under perforated patch clamp conditions, maximal activation of RyRs with caffeine or inositol triphosphate receptors (IP3Rs) with noradrenaline induced equivalent increases in [Ca2+]i and Ca2+-activated Cl- currents in freshly isolated rat PASMCs. Following maximal IP3-induced Ca2+ release, neither caffeine nor chloro-m-cresol induced a response, whereas prior application of caffeine or chloro-m-cresol blocked IP3-induced Ca2+ release. In cultured human PASMCs, which lack functional expression of RyRs, caffeine failed to affect ATP-induced increases in [Ca2+]i in the presence and absence of extracellular Ca2+. The RyR antagonists ruthenium red, ryanodine, tetracaine, and dantrolene greatly inhibited submaximal noradrenaline- and hypoxia-induced Ca2+ release and contraction in freshly isolated rat PASMCs, but did not affect ATP-induced Ca2+ release in cultured human PASMCs. Real-time quantitative RT-PCR and immunofluorescence staining indicated similar expression of all three RyR subtypes (RyR1, RyR2, and RyR3) in freshly isolated rat PASMCs. In freshly isolated PASMCs from RyR3 knockout (RyR3-/-) mice, hypoxia-induced, but not submaximal noradrenaline-induced, Ca2+ release and contraction were significantly reduced. Ruthenium red and tetracaine can further inhibit hypoxic increase in [Ca2+]i in RyR3-/- mouse PASMCs. Collectively, our data suggest that (a) RyRs play an important role in submaximal noradrenaline- and hypoxia-induced Ca2+ release and contraction; (b) all three subtype RyRs are expressed; and (c) RyR3 gene knockout significantly inhibits hypoxia-, but not submaximal noradrenaline-induced Ca2+ and contractile responses in PASMCs.

[LIM protein KyoT2 interacts with human tight junction protein ZO-2-i3].

It was reported that LIM protein KyoT2 negatively regulated transcription by association with the RBP-J DNA-binding protein. Using yeast two-hybrid system with LIM protein KyoT2 as a bait, we have isolated an alternatively spliced form of human tight junction protein 2--ZO-2-i3. Sequence analysis indicated that ZO-2-i3 is composed of 19 exons, and selected usage of exons led to an alteration in the region following the kinase domain as compared with the published sequence. To identify the interaction between LIM protein KyoT2 and ZO-2-i3, yeast two-hybrid system, purification of KyoT2 protein, and GST pull-down assay were performed in the experiments. After KyoT2 and ZO-2-i3 changed vectors, positive two-hybrid yeast was obtained. Using KyoT2 protein and antibody in GST pull-down assay positive result was also obtained. Therefore we conformed KyoT2 interacted ZO-2-i3 in vitro. Furthermore it was identified in yeast that KyoT2 associated with ZO-2-i3 through its LIM2 domain.