Molecular docking and enzyme kinetic studies of dihydrotanshinone on metabolism of a model CYP2D6 probe substrate in human liver microsomes

The effects of Danshen and its active components (tanshinone I, tanshinone IIA, dihydrotanshinone and cryptotanshinone) on CYP2D6 activity was investigated by measuring the metabolism of a model CYP2D6 probe substrate, dextromethorphan to dextrorphan in human pooled liver microsomes. The ethanolic extract of crude Danshen (6.25-100 μg/ml) decreased dextromethorphan O-demethylation in vitro (IC(50)=23.3 μg/ml) and the water extract of crude Danshen (0.0625-1 mg/ml) showed no inhibition. A commercially available Danshen pill (31.25-500 μg/ml) also decreased CYP2D6 activity (IC(50)=265.8 μg/ml). Among the tanshinones, only dihydrotanshinone significantly inhibited CYP2D6 activity (IC(50)=35.4 μM), compared to quinidine, a specific CYP2D6 inhibitor (IC(50)=0.9 μM). Crytotanshinone, tanshinone I and tanshinone IIA produced weak inhibition, with IC(20) of 40.8 μM, 16.5 μM and 61.4 μM, respectively. Water soluble components such as salvianolic acid B and danshensu did not affect CYP2D6-mediated metabolism. Enzyme kinetics studies showed that inhibition of CYP2D6 activity by the ethanolic extract of crude Danshen and dihydrotanshinone was concentration-dependent, with K(i) values of 4.23 μg/ml and 2.53 μM, respectively, compared to quinidine, K(i)=0.41 μM. Molecular docking study confirmed that dihydrotanshinone and tanshinone I interacted with the Phe120 amino acid residue in the active cavity of CYP2D6 through Pi-Pi interaction, but did not interact with Glu216 and Asp301, the key residues for substrate binding. The logarithm of free binding energy of dihydrotanshinone (-7.6 kcal/mol) to Phe120 was comparable to quinidine (-7.0 kcal/mol) but greater than tanshinone I (-5.4 kcal/mol), indicating dihydrotanshinone has similar affinity to quinidine in binding to the catalytic site on CYP2D6.     

Pregnancy-Induced Noncoding RNA (PINC) Associates with Polycomb Repressive Complex 2 and Regulates Mammary Epithelial Differentiation

Pregnancy-induced noncoding RNA (PINC) and retinoblastoma-associated protein 46 (RbAp46) are upregulated in alveolar cells of the mammary gland during pregnancy and persist in alveolar cells that remain in the regressed lobules following involution. The cells that survive involution are thought to function as alveolar progenitor cells that rapidly differentiate into milk-producing cells in subsequent pregnancies, but it is unknown whether PINC and RbAp46 are involved in maintaining this progenitor population. Here, we show that, in the post-pubertal mouse mammary gland, mPINC is enriched in luminal and alveolar progenitors. mPINC levels increase throughout pregnancy and then decline in early lactation, when alveolar cells undergo terminal differentiation. Accordingly, mPINC expression is significantly decreased when HC11 mammary epithelial cells are induced to differentiate and produce milk proteins. This reduction in mPINC levels may be necessary for lactation, as overexpression of mPINC in HC11 cells blocks lactogenic differentiation, while knockdown of mPINC enhances differentiation. Finally, we demonstrate that mPINC interacts with RbAp46, as well as other members of the polycomb repressive complex 2 (PRC2), and identify potential targets of mPINC that are differentially expressed following modulation of mPINC expression levels. Taken together, our data suggest that mPINC inhibits terminal differentiation of alveolar cells during pregnancy to prevent abundant milk production and secretion until parturition. Additionally, a PRC2 complex that includes mPINC and RbAp46 may confer epigenetic modifications that maintain a population of mammary epithelial cells committed to the alveolar fate in the involuted gland.     

基质金属蛋白酶MMP-26能有效促进人绒毛膜上皮癌细胞JEG-3浸润能力

胚胎植入过程中,滋养层细胞浸润与肿瘤的迁移过程非常相似,但显著的区别在于前者是受严格调控的有节制的浸润,基质金属蛋白酶(MMPs)的许多成员在其中起重要的作用.MMP-26是近年来发现的MMPs家族的新成员,它在滋养层细胞中的作用所知甚少.利用国际常用的人滋养层细胞模型——人绒毛膜上皮癌细胞系(JEG-3)作为体外实验模型,探讨MMP-26在人滋养层细胞浸润调节中的作用.将含有MMP-26全长cDNA的pCR3.1质粒转染到JEG-3细胞中,获得过量表达MMP-26基因的稳定细胞系JEG-3/MMP-26;细胞浸润分析表明JEG/MMP-26细胞的浸润能力较母本细胞明显增强;RT-PCR和明胶酶谱分析显示JEG-3/MMP-26细胞中MMP-9的表达和分泌水平提高;双荧光免疫细胞化学进一步显示MMP-26和MMP-9蛋白在细胞中有共定位现象.上述结果表明MMP-26能有效促进人滋养层细胞浸润,其作用可能是通过与其他MMP分子(如MMP-9)的协调来实现的.     

Independent Origins of New Sex-Linked Chromosomes in the <Emphasis Type="Italic">melanica</Emphasis> and <Emphasis Type="Italic">robusta</Emphasis> Species Groups of <Emphasis Type="Italic">Drosophila</Emphasis>

Background  

Recent translocations of autosomal regions to the sex chromosomes represent important systems for identifying the evolutionary forces affecting convergent patterns of sex-chromosome heteromorphism. Additions to the sex chromosomes have been reported in the melanica and robusta species groups, two sister clades of Drosophila. The close relationship between these two species groups and the similarity of their rearranged karyotypes motivates this test of alternative hypotheses; the rearranged sex chromosomes in both groups are derived through a common origin, or the rearrangements are derived through at least two independent origins. Here we examine chromosomal arrangement in representatives of the melanica and the robusta species groups and test these alternative hypotheses using a phylogenetic approach.     

Cannabinoid receptor agonists are mitochondrial inhibitors: a unified hypothesis of how cannabinoids modulate mitochondrial function and induce cell death

Time-lapse microscopy of human lung cancer (H460) cells showed that the endogenous cannabinoid anandamide (AEA), the phyto-cannabinoid Δ-9-tetrahydrocannabinol (THC) and a synthetic cannabinoid HU 210 all caused morphological changes characteristic of apoptosis. Janus green assays of H460 cell viability showed that AEA and THC caused significant increases in OD 595 nm at lower concentrations (10-50 μM) and significant decreases at 100 μM, whilst HU 210 caused significant decreases at all concentrations. In rat heart mitochondria, all three ligands caused significant decreases in oxygen consumption and mitochondrial membrane potential. THC and HU 210 caused significant increases in mitochondrial hydrogen peroxide production, whereas AEA was without significant effect. All three ligands induced biphasic changes in either mitochondrial complex I activity and/or mitochondrial complex II-III activity. These data demonstrate that AEA, THC, and HU 210 are all able to cause changes in integrated mitochondrial function, directly, in the absence of cannabinoid receptors.     

A genetic map of Xenopus tropicalis

We present a genetic map for Xenopus tropicalis, consisting of 2886 Simple Sequence Length Polymorphism (SSLP) markers. Using a bioinformatics-based strategy, we identified unique SSLPs within the X. tropicalis genome. Scaffolds from X. tropicalis genome assembly 2.0 (JGI) were scanned for Simple Sequence Repeats (SSRs); unique SSRs were then tested for amplification and polymorphisms using DNA from inbred Nigerian and Ivory Coast individuals. Thus identified, the SSLPs were genotyped against a mapping cross panel of DNA samples from 190 F2 individuals. Nearly 4000 SSLPs were genotyped, yielding a 2886-marker genetic map consisting of 10 major linkage groups between 73 and 132 cM in length, and 4 smaller linkage groups between 7 and 40 cM. The total effective size of the map is 1658 cM, and the average intermarker distance for each linkage group ranged from 0.27 to 0.75 cM. Fluorescence In Situ Hybridization (FISH) was carried out using probes for genes located on mapped scaffolds to assign linkage groups to chromosomes. Comparisons of this map with the X. tropicalis genome Assembly 4.1 (JGI) indicate that the map provides representation of a minimum of 66% of the X. tropicalis genome, incorporating 758 of the approximately 1300 scaffolds over 100,000 bp. The genetic map and SSLP marker database constitute an essential resource for genetic and genomic analyses in X. tropicalis.     

Cell-based tissue engineering for lung regeneration

Emphysema is a chronic lung disease characterized by alveolar enlargement and tissue loss. Tissue engineering represents an attractive potential for regeneration of several organ systems. The complex three-dimensional architectural structure of lung parenchyma requiring connections of alveolar units to airways and the pulmonary circulation makes this strategy less optimistic. In the present study, we used Gelfoam sponge as a scaffold material, supplemented with fetal rat lung cells as progenitors, to explore the potential application of cell-based tissue engineering for lung regeneration in adult rats. After injection into lung parenchyma, the sponge showed porous structures similar to alveolar units. It did not induce severe local inflammatory response. Fetal lung cells in the sponge were able to survive in the adult lung for at least 35 days, determined by CMTMR [5-(and-6)-{[(4-chloromethyl)benzoyl]amino}tetramethylrhodamine] labeling. Proliferation of cells within the sponge was demonstrated in vivo by bromodeoxyuridine (BrdU) labeling. Cells formed "alveolar-like structures" at the border between the sponge and the surrounding lung tissue with positive immunohistochemical staining for epithelial and endothelial cells. Neovascularization of the sponge was demonstrated with India ink perfusion. The sponge degraded after several months. This study suggests that cell-based tissue engineering possesses the potential to regenerate alveolar-like structures, an important step towards our ultimate goal of lung regeneration.