Cimicifuga racemosa extract BNO 1055 inhibits proliferation of the human prostate cancer cell line LNCaP

Extracts from black cohosh (Cimicifuga racemosa, CR) exert an anti-proliferative action in human breast cancer cell cultures, which has been attributed to an anti-estrogenic effect. However, CR constituents do not bind to either of the known estrogen receptors. Thus, the anti-tumor effect of CR me be mediated by mechanisms not involving these receptors. Polycyclic aromatic hydrocarbons are toxic environmental pollutants, which indirectly act as anti-estrogens by activating the aryl hydrocarbon receptor (AhR). The AhR is widely expressed in mammalian tissues and tumors. A recent screening study demonstrated activation of the AhR by a variety of herbal extracts, among others, CR. Since activation of the AhR causes inhibition of growth of prostate cancer cells, we addressed the question, whether CR may not only inhibit growth of breast cancer--but also of prostate cancer cells. In the AhR ligand assay, the CR extract BNO 1055 reduced tracer binding to 71% of the control demonstrating interaction of constituents of this extract with the receptor. Under basal as well as under estradiol- and dihydrotestosterone stimulated conditions, the CR extract dose dependently inhibited proliferation of LNCaP cells. A significant reduction of cell growth was observed at a concentration as low as 50 ng/ml. Thus, it is demonstrated for the first time that CR compounds potently inhibit the growth of human prostate cancer cells in vitro. This anti-proliferative effect may be mediated via the AhR.     

Influence of pulsed electromagnetic field with different pulse duty cycles on neurite outgrowth in PC12 rat pheochromocytoma cells

The influence of low frequency (50 Hz repetition rate) pulsed electromagnetic field (EMF) on PC12 cell neurite outgrowth in vitro was investigated in this study. We studied the percentage of neurite bearing cells, average length of neurites, and directivity of neurite outgrowth in PC12 cells cultured for 96 h in the presence of nerve growth factor (NGF). PC12 cells were exposed in one incubator to pulsed EMF at 1.36 mT (peak value) generated by a pair of Helmholtz coils, and the control samples were placed in another identical incubator. We found that the pulse duty cycle had significant effect on neurite outgrowth. Low (10%) pulse on-time significantly inhibited the percentage of neurite bearing cells, but at the same time increased the average length of neurites, while 100% on-time (DC) had exactly the opposite effects. Furthermore, we found that neurites were prone to extend along the direction of pulsed EMF with 10% pulse on-time. Our studies show that neurite outgrowth in PC12 cells is sensitive to the pulse duty and this sensitivity was associated with NGF concentration.     

The class A macrophage scavenger receptor type I (SR-AI) recognizes complement iC3b and mediates NF-κB activation

The macrophage scavenger receptor SR-AI binds to host tissue debris to perform clearance and it binds to bacteria for phagocytosis. In addition, SR-AI modulates macrophage activation through cell signaling. However, investigation of SR-AI signaling on macrophages is complicated due to its promiscuous ligand specificity that overlaps with other macrophage receptors. Therefore, we expressed SR-AI on HEK 293T cells to investigate its ligand binding and signaling. On 293T cells, SR-AI could respond to E. coli DH5α, leading to NF-κB activation and IL-8 production. However, this requires E. coli DH5α to be sensitized by fresh serum that is treated with heat-inactivation or complement C3 depletion. Anti-C3 antibody inhibits the binding of SR-AI to serum-sensitized DH5α and blocks DH5α stimulation of SR-AI signaling. Further analysis showed that SR-AI can directly bind to purified iC3b but not C3 or C3b. By mutagenesis, The SRCR domain of SR-AI was found to be essential in SR-AI binding to serum-sensitized DH5α. These results revealed a novel property of SR-AI as a complement receptor for iC3b-opsonized bacteria that can elicit cell signaling.     

免疫活性细胞中的非神经元型胆碱能系统

本文对淋巴细胞、巨噬细胞、粒细胞、肥大细胞及胶质细胞等免疫活性细胞中非神经元型胆碱能系统的特点、药理学和病理生理学意义进行了综述。免疫活性细胞,尤其是淋巴细胞中存在完整、独立的胆碱能系统。与神经元型胆碱能系统相比,该系统具有不同特点,如乙酰胆碱(ACh)的合成存在于整个细胞内;ACh可能储存于细胞中某种储存结构,也可能不需储存,而是按需合成并直接释放。免疫活性细胞中的非神经元型胆碱能系统对机体免疫功能及炎症过程可能发挥重要的调控作用。对该系统的深入研究将有助于进一步阐明免疫性疾病、炎症/感染性疾病和神经退行性疾病等的病理生理和发现药物治疗的新靶标。     

Generation of functional gut-like organ from mouse induced pluripotent stem cells

Induced pluripotent stem (iPS) cells have the pluripotency to differentiate into broad spectrum derivatives of all three embryonic germ layers. However, the in vitro organ differentiation potential of iPS cells to organize a complex and functional “organ” has not yet been demonstrated. Here, we demonstrate that mouse iPS cells have the ability to organize a gut-like organ with motor function in vitro by a hanging drop culture system. This “induced gut (iGut)” exhibited spontaneous contraction and highly coordinated peristalsis accompanied by a transportation of contents. Ultrastructural analysis identified that the iGut had large lumens surrounded by three distinct layers (epithelium, connective tissue and musculature). Immunoreactivity for c-Kit, a marker of interstitial cells of Cajal (ICCs, enteric pacemaker cells), was observed in the wall of the lumen and formed a distinct and dense network. The neurofilament immunoreactivity was identified to form large ganglion-like structures and dense neuronal networks. The iGut was composed of all the enteric components of three germ layers: epithelial cells (endoderm), smooth muscle cells (mesoderm), ICCs (mesoderm), and enteric neurons (ectoderm). This is the first report to demonstrate the in vitro differentiation potential of iPS cells into particular types of functional “organs.” This work not only contributes to understanding the mechanisms of incurable gut disease through disease-specific iPS cells, but also facilitates the clinical application of patient-specific iPS cells for novel therapeutic strategies such as patient-specific “organ” regenerative medicine in the future.     

Scale‐up of hydrophobin‐assisted recombinant protein production in tobacco BY‐2 suspension cells

Plant suspension cell cultures are emerging as an alternative to mammalian cells for production of complex recombinant proteins. Plant cell cultures provide low production cost, intrinsic safety and adherence to current regulations, but low yields and costly purification technology hinder their commercialization. Fungal hydrophobins have been utilized as fusion tags to improve yields and facilitate efficient low‐cost purification by surfactant‐based aqueous two‐phase separation (ATPS) in plant, fungal and insect cells. In this work, we report the utilization of hydrophobin fusion technology in tobacco bright yellow 2 (BY‐2) suspension cell platform and the establishment of pilot‐scale propagation and downstream processing including first‐step purification by ATPS. Green fluorescent protein‐hydrophobin fusion (GFP‐HFBI) induced the formation of protein bodies in tobacco suspension cells, thus encapsulating the fusion protein into discrete compartments. Cultivation of the BY‐2 suspension cells was scaled up in standard stirred tank bioreactors up to 600 L production volume, with no apparent change in growth kinetics. Subsequently, ATPS was applied to selectively capture the GFP‐HFBI product from crude cell lysate, resulting in threefold concentration, good purity and up to 60% recovery. The ATPS was scaled up to 20 L volume, without loss off efficiency. This study provides the first proof of concept for large‐scale hydrophobin‐assisted production of recombinant proteins in tobacco BY‐2 cell suspensions.     

Epac1 increases migration of endothelial cells and melanoma cells via FGF2‐mediated paracrine signaling

Fibroblast growth factor (FGF2) regulates endothelial and melanoma cell migration. The binding of FGF2 to its receptor requires N‐sulfated heparan sulfate (HS) glycosamine. We have previously reported that Epac1, an exchange protein activated by cAMP, increases N‐sulfation of HS in melanoma. Therefore, we examined whether Epac1 regulates FGF2‐mediated cell–cell communication. Conditioned medium (CM) of melanoma cells with abundant expression of Epac1 increased migration of human umbilical endothelial cells (HUVEC) and melanoma cells with poor expression of Epac1. CM‐induced increase in migration was inhibited by antagonizing FGF2, by the removal of HS and by the knockdown of Epac1. In addition, knockdown of Epac1 suppressed the binding of FGF2 to FGF receptor in HUVEC, and in vivo angiogenesis in melanoma. Furthermore, knockdown of Epac1 reduced N‐sulfation of HS chains attached to perlecan, a major secreted type of HS proteoglycan that mediates the binding of FGF2 to FGF receptor. These data suggested that Epac1 in melanoma cells regulates melanoma progression via the HS–FGF2‐mediated cell–cell communication.     

Computational modelling and analysis of mechanical conditions on cell locomotion and cell–cell interaction

Between other parameters, cell migration is partially guided by the mechanical properties of its substrate. Although many experimental works have been developed to understand the effect of substrate mechanical properties on cell migration, accurate 3D cell locomotion models have not been presented yet. In this paper, we present a novel 3D model for cells migration. In the presented model, we assume that a cell follows two main processes: in the first process, it senses its interface with the substrate to determine the migration direction and in the second process, it exerts subsequent forces to move. In the presented model, cell traction forces are considered to depend on cell internal deformation during the sensing step. A random protrusion force is also considered which may change cell migration direction and/or speed. The presented model was applied for many cases of migration of the cells. The obtained results show high agreement with the available experimental and numerical data.