Anti-allergic substances from the rhizomes of Dioscorea membranacea

Extracts of five species of Thai medicinal plants, locally known as Hua-Khao-Yen, were screened for anti-allergic activities using RBL-2H3 cells. Of the five species studied, the ethanolic extract of Dioscorea membranacea exhibited potent inhibitory activity against β-hexosaminidase release as a marker of degranulation in RBL-2H3 cells, with an IC₅₀ value of 37.5 μg/mL. Eight compounds were isolated from this crude ethanolic extract, [two naphthofuranoxepins (1, 2), one phenanthraquinone (3), three steroids (4–6), and two steroidal saponins (7, 8)], and tested for their anti-allergic activities. The results showed that dioscorealide B (2) possessed the highest activity with an IC₅₀ value of 5.7 μM, followed by dioscoreanone (3, IC₅₀ = 7.7 μM), dioscorealide A (1, IC₅₀ = 27.9 μM), and diosgenin (9, IC₅₀ = 29.9 μM). Structure–activity relationship studies of naphthofuranoxepins on anti-allergic activity revealed that the hydroxylation at position 8 conferred higher activity than methoxylation. For diosgenin derivatives, the aglycone was found to possess higher activity than the diglucosylated molecule; whereas substitution with rhamnoglucosides apparently results in loss of activity. Furthermore, effects of dioscorealide A, dioscorealide B, and dioscoreanone on antigen-induced release of TNF- and IL-4 in the late phase reaction were also examined.     

Targeting of calcium:calmodulin signals to the cytoskeleton by IQGAP1

Mast cells reorganize their actin cytoskeleton in response to cytosolic calcium signals while in parallel secreting histamine and other inflammatory mediators. The effect of calcium on actin is mediated in large part through calmodulin. EGFP-tagged calmodulin is concentrated in the actin-rich cortex of RBL-2H3 mast cells. Transfection with small interfering RNA directed against the actin and calmodulin-binding protein IQGAP1 dramatically reduced expression of the latter protein and reduced or eliminated the concentration of calmodulin at the actin-rich cortex. Both actin reorganization and secretion were enhanced in IQGAP1 knockdown cells. Our results suggest a model in which calmodulin is targeted to and sequestered at the actin cytoskeleton by IQGAP1. Upon cell stimulation and the subsequent [Ca2+]i increase, it is immediately available to activate local downstream targets.     

Inhibitory effects of geranium essential oil and its major component,citronellol, on degranulation and cytokine production by mast cells

We investigated the effects of geranium essential oil (GEO) on anaphylaxis. GEO can exert antioxidant and anti-inflammatory effects, but its roles in allergic reactions are incompletely understood. Here, we used mouse cells to show that GEO inhibited the degranulation of cultured mast cells (CMCs). Citronellol is the major component of GEO and inhibited CMC degranulation. The l-enantiomer of citronellol more effectively suppressed CMC degranulation than did d-citronellol. We also examined whether citronellol could inhibit the immunoglobulin (Ig) E-induced production of tumor necrosis factor (TNF)-α. Treatment with various concentrations of citronellol before CMC activation with IgE significantly inhibited the induction of TNF-α in a dose-dependent manner. Mechanistically, citronellol suppressed the phosphorylation of mitogen-activated protein kinase (ERK), which is critical for ERK activation and the production of inflammatory cytokines in mast cells. These findings suggest that citronellol may represent a candidate compound for the effective treatment of allergic diseases.     

Inhibitory effects of thunberginols A and B isolated from Hydrangeae Dulcis Folium on mRNA expression of cytokines and on activation of activator protein-1 in RBL-2H3 cells

Previously, thunberginols A and B from the processed leaves of Hydrangeae macrophylla var. thunbergii (Hydrangea dulcis folium) substantially inhibited the degranulation caused by antigen and calcium ionophore A23187, and the release of tumor necrosis factor (TNF)-α and interleukin (IL)-4 by antigen in RBL-2H3 cells. In the present study, we examined the effect of thunberginol B on the expression of mRNA of several cytokines [ILs-2, 3, 4 and 13, TNF-α and granulocyte/macrophage-colony stimulating factor (GM-CSF)] and effects of thunberginols A and B on activator protein (AP)-1 composed of c-jun and c-fos, which is essential for the expression of the cytokine mRNA, in RBL-2H3 cells. Thunberginol B inhibited up-regulated genes of all cytokines, and thunberginols A and B (30 μM) inhibited the phosphorylation of c-jun and expression of c-fos mRNA and phosphorylation of extracellular signal-regulated kinases (ERK1/2). In addition, the profile of gene expression by thunberginol B was similar to that by luteolin, a natural flavone with a potent anti-allergic effect.     

A single cell observation of staurosporine effect on the Ca signals in rat basophilic leukemia cells

A digital imaging fluorescence microscope was used to study the effect of a protein kinase inhibitor staurosporine on the antigen-dependent calcium signals in an individual rat basophilic leukemia cell (RBL-2H3). Although dose dependency of staurosporine was different from one cell to another, staurosporine inhibited, at low concentration, the calcium influx from the external medium into RBL-2H3 cells. At high concentration, however, it inhibited both the removal of calcium ion from internal stores and the calcium influx from the external medium. These results indicated that staurosporine is necessary for the inhibition of the calcium influx from the external medium and that a protein kinase (possibly protein kinase C) is involved in the calcium influx from the external medium into the cytoplasm.     

Eotaxin induces migration of RBL-2H3 mast cells via a Rac-ERK-dependent pathway

Eotaxin is a potent chemokine that acts via CC chemokine receptor 3 (CCR3) to induce chemotaxis, mainly on eosinophils. Here we show that eotaxin also induces chemotactic migration in rat basophilic leukemia (RBL-2H3) mast cells. This effect was dose-dependently inhibited by compound X, a selective CCR3 antagonist, indicating that, as in eosinophils, the effect was mediated by CCR3. Eotaxin-induced cell migration was completely blocked in RBL-RacN17 cells expressing a dominant negative Rac1 mutant, suggesting a crucial role for Rac1 in eotaxin signaling to chemotactic migration. ERK activation also proved essential for eotaxin signaling and it too was absent in RBL-RacN17 cells. Finally, we found that activation of Rac and ERK was correlated with eotaxin-induced actin reorganization known to be necessary for cell motility. It thus appears that Rac1 acts upstream of ERK to signal chemotaxis in these cells, and that a Rac-ERK-dependent cascade mediates the eotaxin-induced chemotactic motility of RBL-2H3 mast cells.     

小鼠孤雌胚、体外培养胚与体内胚H3K9乙酰化模式的比较

孤雌胚胎的发育率比体内体外生成胚胎的发育率要慢,为研究小鼠孤雌胚、体外培养胚H3K9乙酰化(H3K9ac)模式与体内自然胚之间的差异、曲古抑菌素A(Trichostatin,TSA)对孤雌胚H3K9乙酰化模式的影响及表观遗传模式对孤雌胚、体外培养胚发育的影响,文章采用间接免疫荧光法对小鼠植入前各时期孤雌胚、体外培养胚及体内自然胚基因组组蛋白的H3K9乙酰化水平进行检测。结果显示,植入前各时期孤雌胚H3K9乙酰化模式与体内组变化趋势基本一致,但平均荧光强度较体内组普遍偏高;经TSA处理后孤雌胚H3K9乙酰化水平有所提高,原核期至8-细胞期差异显著(P0.05)。体外培养胚H3K9乙酰化荧光强度与体内组变化趋势也基本一致,但平均荧光强度较体内组普遍偏低。以上结果表明,小鼠孤雌胚H3K9乙酰化水平高于体内胚,使植入前胚胎发育过程中本应沉默的基因启动子发生超乙酰化,进而抑制胚胎发育,这可能是造成孤雌胚胎发育能力较差的重要原因之一;TSA处理可以部分弥补体外培养环境对胚胎发育带来的伤害,但TSA提高孤雌胚的发育能力可能并不完全是通过改变H3K9乙酰化水平来实现的。     

活细胞内5-羟色胺可见荧光的多光子激发成像

应用多光子激发激光扫描显微镜对5-羟色胺（5-hydroxytryptamine, 5-HT）孵育的大鼠粘膜型肥大细胞进行自发荧光成像,首次观察到了活细胞内5-HT相关的可见荧光,并对其产生机理进行了初步探讨.实现了对活细胞内5-HT空间分布的高分辨成像,为研究活组织或细胞内5-HT的空间分布和含量与细胞功能状态的关系提供了新的实验方法.     

Inhibition by dexamethasone of interleukin 13 production via glucocorticoid receptor-mediated inhibition of c-Jun phosphorylation

The antigen stimulation of RBL-2H3 cells induced interleukin 13 (IL-13) production, which was inhibited by the steroidal anti-inflammatory drug dexamethasone and by the c-Jun N-terminal kinase (JNK) inhibitor SP600125. Dexamethasone did not inhibit the antigen-induced phosphorylation of JNK but inhibited that of c-Jun. In a cell-free system, the phosphorylation of glutathione S-transferase-fused c-Jun by recombinant JNK was not inhibited by dexamethasone but was inhibited by the addition of recombinant glucocorticoid receptor (GR). These findings suggest that the inhibition of antigen-induced IL-13 production by dexamethasone is due to the GR-mediated inhibition of c-Jun phosphorylation induced by JNK.     

穿膜肽TAT和R9的生物学效应的体内体外研究

本文比较了两种常用穿膜肽TAT和R9的体外穿膜效率、对细胞影响以及小鼠活体内不同器官的穿膜效率。非特异性穿膜肽TAT和R9分别为11个氨基酸和9个氨基酸的短肽,能够有效地穿过细胞膜进入细胞。本研究纯化制备出融合蛋白TAT-EGFP及R9-EGFP多肽,用相同浓度的活性TAT-EGFP和R9-EGFP处理肺癌细胞,观察比较二者的穿膜活性及效率;设置不同浓度梯度检测两种穿膜肽是否对细胞产生影响;通过小鼠腹腔注射TAT-EGFP和R9-EGFP活性肽段,研究二者在活体能够穿膜到达的靶位置,并比较分析二者穿膜的效果器官分布。结果显示制备获得的两种融合蛋白TAT-EGFP和R9-EGFP均能有效穿过实现穿膜功能,两种穿膜肽对乳腺癌细胞影响较小。活体试验证明两种穿膜肽在心、肝、脾、肺、肾器官均有分布,R9主要富集在小鼠的肾和肝脏中,TAT在6种被研究的器官都有富集,两种细胞穿膜肽都能透过血脑屏障到达脑部。相对来说,TAT在小鼠活体中的穿膜效果更好,荧光富集多,R9整体穿膜效果较弱。本研究分析比较了两种穿膜肽的细胞安全性浓度和活体的穿膜能力,为TAT和R9两种穿膜肽在后续试验中的合理有效的选择和使用,提供了基础数据和指导性依据。     

Real-time analysis of ligand-induced cell surface and intracellular reactions of living mast cells using a surface plasmon resonance-based biosensor

Surface plasmon resonance (SPR)-based sensors have been used to detect the binding between interactive molecules. We applied the SPR technology to the analysis of interactions between living cells and molecules reactive to the cells, using mast cells and mast cell-reactive antigens. The exposure of dinitrophenol-human serum albumin (DNP-HSA), an antigen that stimulates mast cells, to IgE-sensitized mast cells induced a robust and long-lasting SPR signal in a dose-dependent manner. The maximal increase in SPR signal induced by 100 ng/ml DNP-HSA was 0.200 +/- 0.120 angle (mean +/- SD, n = 37), about 1000 times larger than the theoretically expected increase for the simple binding of DNP-HSA to Fc(epsilon)RI, the high-affinity IgE receptor. A small, but similarly prolonged signal was observed when the cells were stimulated by an agonist of the adenosine A3 receptor. The signal induced by DNP-HSA was abolished by genistein, and partially inhibited by phorbol 12-myristate 13-acetate and wortmannin. Interestingly, the signal induced by DNP-HSA was only weakly inhibited by DNP-lysine, suggesting that DNP-lysine manifests its action not by inhibiting, but by modulating the crosslinking of Fc(epsilon)RI. We concluded that SPR sensors can detect biologically significant signals in a real-time manner from the interactions between cells and molecules reactive to the cells.     

Long-term effects of tripeptide Ile-Pro-Pro on osteoblast differentiation in vitro

Bone mineralization is a result of the function of bone-forming osteoblasts. Osteoblast differentiation from their precursors is a carefully controlled process that is affected by many signaling molecules. Protein-rich food-derived bioactive peptides are reported to express a variety of functions in vivo. We studied the long-term in vitro effect of bioactive tripeptide Ile-Pro-Pro (IPP) on osteoblasts differentiated from human mesenchymal stem cells. Osteoblast bone alkaline phosphatase activity (bALP), bone-forming capacity and gene expression were investigated. Treatment with 50 microM IPP had no effect on bALP activity, but osteoblast mineralization was increased. Gene expression of beta-catenin, Cbfa1/Runx2, PTHrP, CREB-5, osteoglycin, osteocalcin, caspase-8, osteoprotegerin (OPG) and RANKL was analyzed by quantitative real-time PCR on Days 13, 17 and 20 of culture. The results indicate that IPP increased mineral formation due to enhanced cell survival and matrix formation. In addition, IPP reduced the RANKL/OPG ratio. Bioactive peptides, such as IPP, could be one method by which a protein-rich diet promotes bone integrity.     

Rohitukine inhibits in vitro adipogenesis arresting mitotic clonal expansion and improves dyslipidemia in vivo

We developed a common feature pharmacophore model using known antiadipogenic compounds (CFPMA). We identified rohitukine, a reported chromone anticancer alkaloid as a potential hit through in silico mapping of the in-house natural product library on CFPMA. Studies were designed to assess the antiadipogenic potential of rohitukine. Rohitukine was isolated from Dysoxylum binacteriferum Hook. to ⬧95% purity. As predicted by CFPMA, rohitukine was indeed found to be an antiadipogenic molecule. Rohitukine inhibited lipid accumulation and adipogenic differentiation in a concentration- and exposure-time-dependent manner in 3T3-L1 and C3H10T1/2 cells. Rohitukine downregulated expression of PPARγ, CCAAT/enhancer binding protein α, adipocyte protein 2 (aP2), FAS, and glucose transporter 4. It also suppressed mRNA expression of LPL, sterol-regulatory element binding protein (SREBP) 1c, FAS, and aP2, the downstream targets of PPARγ. Rohitukine arrests cells in S phase during mitotic clonal expansion. Rohitukine was bioavailable, and 25.7% of orally administered compound reached systemic circulation. We evaluated the effect of rohitukine on dyslipidemia induced by high-fat diet in the hamster model. Rohitukine increased hepatic expression of liver X receptor α and decreased expression of SREBP-2 and associated targets. Rohitukine decreased hepatic and gonadal lipid accumulation and ameliorated dyslipidemia significantly. In summary, our strategy to identify a novel antiadipogenic molecule using CFPMA successfully resulted in identification of rohitukine, which confirmed antiadipogenic activity and also exhibited in vivo antidyslipidemic activity.     

Down-regulating protein kinase C alpha: Functional cooperation between the proteasome and the endocytic system

Ubiquitination, proteasome, caveolae and endosomes have been implicated in controlling protein kinase Cα (PKCα) down-regulation. However, the molecular mechanism remained obscure. Here we show that endosomes and proteasome cooperate in phorbol ester 12-O-tetradecanoyl phorbol acetate (TPA)-induced down-regulation of PKCα. We show that following TPA treatment and translocation to the plasma membrane, PKCα undergoes multimonoubiquitination prior to its degradation by the proteasome. However, to reach the proteasome, PKCα must travel through the endocytic system from early to late endosomes. This route requires functional endosomes, whereby endosomal alkalinization, or ablation, abrogates completely PKCα degradation maintaining the enzyme at the plasma membrane. This route also depends on synaptotagmin (Syt) II and the Rab7 GTPase, whereby Syt II knock-down or expression of the GDP-locked Rab7 inactive mutant prevents PKCα degradation. We further show that proteasome plays a dual role, where an active proteasome is required for deubiquitination of PKCα, a step crucial to prevent PKCα targeting to the endocytic recycling compartment. Finally, we show that the association with rafts-localized cell surface proteins that internalize in a clathrin-independent fashion is necessary to allow the trafficking of PKCα from the plasma membrane to the proteasome, its ultimate degradation station.     

RNA干扰抑制人乙型肝炎病毒复制的研究

采用表达shRNA的载体构建了表达针对病毒HBsAgmRNA保守区的shRNA的质粒psiHBs,利用细胞模型和高压注射小鼠模型评价RNA干扰对HBV复制和基因表达的抑制作用。通过Western印迹检测细胞内的HBsAg,用ELISA检测细胞培养上清和血清中的HBsAg,采用Southern印迹检测HBV的复制中间体,最后通过免疫组化的方法检测肝组织切片中HBcAg的表达情况。结果显示pHBV1.3和psiHBs共转染HepG2后,与对照组相比病毒HBsAg和HBeAg的表达和病毒复制中间体的水平下降了90%以上,并且shRNA的作用效率存在序列特异性和剂量依赖性。在高压注射小鼠模型中,psiHBs表达的shRNA使小鼠血清中HBsAg的水平下降了80%以上,免疫组化检测显示,小鼠肝组织内HBcAg阳性细胞数减少了75.1%,而且shRNA的抑制作用至少能持续4d。研究显示载体表达的shRNA无论是在细胞或是在小鼠模型中都能对HBV的复制和基因的表达发挥序列特异性的抑制作用。本研究为我们下一步实现由RNAi介导的基因治疗提供了理论和技术支持。     

Bisorbicillinol inhibits Lyn tyrosine kinase for allergic response on RBL-2H3 cells

Bisorbicillinol, which is isolated from Trichoderma sp. USF2690, is an inhibitor of β-hexosaminidase release and tumor necrosis factor (TNF)-α, and Interleukin (IL)-4 secretion from rat basophilic leukemia (RBL-2H3) cells, with IC₅₀ values of 2.8?μM, 2.9?μM and 2.8?μM respectively. We showed that the inhibitory mechanism of β-hexosaminidase release and TNF-α secretion involved inhibition of Lyn, a tyrosine kinase. The inhibitory activities of bisorbicillinol indicate that this compound is a new candidate anti-allergic agent.     

Identification of anti-allergic effect of Clonorchis sinensis-derived protein venom allergen-like proteins (CsVAL)

Previous studies demonstrated that Clonochis sinensis-derived crude antigens suppress development of allergic responses. We investigated the effects of C. sinensis venom allergen-like (CsVAL) proteins on immune-modulating activities in allergic inflammatory response. Using RBL-2H3 rat mast cells, we demonstrated that CsVAL inhibits antigen-induced β-hexosaminidase release from immunoglobulin E-sensitized RBL-2H3 cells, and this inhibitory activity occurs by suppressing Lyn phosphorylation and intracellular reactive oxygen species production. In addition, CsVAL peptide treatment inhibits activation of protein kinase C-α and extracellular signal-regulated kinase 1/2, which are involved in degranulation of immunoglobulin E-sensitized mast cells. Furthermore, immunization with CsVAL suppressed development of skin inflammation by assessing ear thickness and cutaneous infiltration by eosinophils and mast cells in oxazolone-induced contact hypersensitivity in vivo mouse model. These results suggest that CsVAL is a promising candidate as an effective mast cell inhibitor for allergic and inflammatory diseases.     

Structural transitions involved in a novel amyloid-like beta-sheet assemblage of tripeptide derivatives

Self-assembly of two tripeptide derivatives containing three nonpolar isoleucine moieties and polar oxyethylene groups are studied in methanol. Peptide A [CH3(OCH2CH2)3OCH2CO(Ile)3OCH3] and peptide B [CH3(OCH2CH2)3OCH2CO(Ile)3NH (CH2CH2O)3CH3] take a mixture of unordered and helical conformation at low concentration (8.5 x 10(-4) M). However, at high concentration (2 x 10(-3) M), both the peptide showed significant increase in the helical conformation. An interesting conformational transition of peptides A and B at various methanol contents was observed in the solvated films of these compounds by spectroscopic methods like the far-uv circular dichroism and Fourier transform infrared (FT-IR) techniques. Peptide B, which contains more polar oxyethylene groups than A, showed a highly cooperative conformational transition when the methanol content was decreased. This transition was characterized by a large increase of beta-sheet, retaining a alpha-helical contribution. Peptide A showed a conformational transition resulting in a beta-sheet in the aggregated state. From the CD spectra, the ratio in the ellipticity indicates that peptide B forms twisted antiparallel beta-sheet conformation, whereas peptide A takes a parallel beta-sheet conformation. The results obtained in this work indicates the role of polar derivatization on the conformational preference of peptides having similar sequence.     

New analogues of laminin active fragment YIGSR: synthesis and biological activity in vitro and in vivo.

Eleven analogues of the laminin pentapeptide amide fragment Tyr-Ile-Gly-Ser-Arg-NH2 (YIGSR-NH2) corresponding to a B1 chain fragment of the glycoprotein laminin have been synthesized by the solid phase method, and their biological activity has been studied in vitro by a cell adhesion assay: all of them inhibited the adhesion of LLC tumor cells to laminin. The analogues were found to be more resistant to enzymatic degradation in human serum than YIGSR-NH2 itself. Analogue DatIGSHar-NH2 was selected for an experimental pulmonary metastasis assay in vivo: it had higher antimetastatic activity than YIGSR-NH2.