Endosome-mediated retrograde axonal transport of P2X3 receptor signals in primary sensory neurons

Neurotrophins and their receptors adopt signaling endosomes to transmit retrograde signals. However, the mechanisms of retrograde signaling for other ligand/receptor systems are poorly understood. Here, we report that the signals of the purinergic (P)2X(3) receptor, an ATP-gated ion channel, are retrogradely transported in dorsal root ganglion (DRG) neuron axons. We found that Rab5, a small GTPase, controls the early sorting of P2X(3) receptors into endosomes, while Rab7 mediates the fast retrograde transport of P2X(3) receptors. Intraplantar injection and axonal application into the microfluidic chamber of α, β-methylene-ATP (α, β-MeATP), a P2X selective agonist, enhanced the endocytosis and retrograde transport of P2X(3) receptors. The α, β-MeATP-induced Ca(2+) influx activated a pathway comprised of protein kinase C, rat sarcoma viral oncogene and extracellular signal-regulated protein kinase (ERK), which associated with endocytic P2X(3) receptors to form signaling endosomes. Disruption of the lipid rafts abolished the α, β-MeATP-induced ERK phosphorylation, endocytosis and retrograde transport of P2X(3) receptors. Furthermore, treatment of peripheral axons with α, β-MeATP increased the activation level of ERK and cAMP response element-binding protein in the cell bodies of DRG neurons and enhanced neuronal excitability. Impairment of either microtubule-based axonal transport in vivo or dynein function in vitro blocked α, β-MeATP-induced retrograde signals. These results indicate that P2X(3) receptor-activated signals are transmitted via retrogradely transported endosomes in primary sensory neurons and provide a novel signaling mechanism for ligand-gated channels.     

Global Changes in the Mammary Epigenome Are Induced by Hormonal Cues and Coordinated by Ezh2

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Highlights? The mammary epigenome is highly sensitive to steroid hormones at specific developmental stages ? Ezh2 links hormonal cues to changes in chromatin structure and gene expression ? Progesterone is a key in vivo regulator of Ezh2 ? Hormone-induced chromatin changes likely play a role in the initiation of breast cancer     

Observer-based tracking control of abnormal oscillations in demyelination symptom

Fast axonal conduction of action potentials in mammals relies on myelin insulation. Demyelination can cause slowed, blocked, desynchronized, or paradoxically excessive spiking that underlies the symptoms observed in demyelination diseases. Feedback control via functional electrical stimulation (FES) seems to be a promising treatment modality in such diseases. However, there are challenges to implementing such method for neurons: high nonlinearity, biological tissue constrains and unobservable ion channel states. To address this problem, we propose an estimating and tracking control strategy for systems based on Kalman filter, in order to enhance the action potential propagation reliability of demyelinated neuron via FES. Unscented Kalman filter (UKF) is employed to estimate the unobservable states and parameters in the demyelination neuron model from membrane potential dynamics. Our method could promote the design of new closed-loop electrical stimulation systems for patients suffering from different nerve system dysfunctions.     

miR-185 is an independent prognosis factor and suppresses tumor metastasis in gastric cancer

miR-185 has been identified as an important factor in several cancers such as breast cancer, ovarial cancer, and prostate cancer. However, its effect and prognostic value in gastric cancer are still poorly known. In this study, we found that the expression levels of miR-185 were strongly downregulated in gastric cancer and associated with clinical stage and the presence of lymph node metastases. Moreover, miR-185 might independently predict OS and RFS in gastric cancer. We further found that upregulation of miR-185 inhibited the proliferation and metastasis of gastric cancer cells in vitro and in vivo. Taken together, our findings demonstrate that the miR-185 is important for gastric cancer initiation and progression and holds promise as a prognostic biomarker to predict survival and relapse in gastric cancer. It is also a potential therapeutic tool to improve clinical outcomes in the above disease.     

Production of influenza H1N1 vaccine from MDCK cells using a novel disposable packed-bed bioreactor

A process for human influenza H1N1 virus vaccine production from Madin–Darby canine kidney (MDCK) cells using a novel packed-bed bioreactor is described in this report. The mini-bioreactor was used to study the relationship between cell density and glucose consumption rate and to optimize the infection parameters of the influenza H1N1 virus (A/New Caledonia/20/99). The MDCK cell culture and virus infection were then monitored in a disposable perfusion bioreactor (AmProtein Current Perfusion Bioreactor) with proportional–integral–derivative control of pH, dissolved O₂ (DO), agitation, and temperature. During 6 days of culture, the total cell number increased from 2.0?×?10⁹ to 3.2?×?10¹⁰ cells. The maximum virus titers of 768 hemagglutinin units/100 μL and 7.8?×?10⁷ 50 % tissue culture infectious doses/mL were obtained 3 days after infection. These results demonstrate that using a disposable perfusion bioreactor for large-scale cultivation of MDCK cells, which allows for the control of DO, pH, and other conditions, is a convenient and stable platform for industrial-scale production of influenza vaccines.     

Amplification, analysis and chromosome mapping of novel homeobox-containing and homeobox-flanking sequences in rice

Homeobox genes, widely distributed among animal and plant kingdoms, play an important role in developmental process. Several homeobox conserved fragments were amplified by PCR and the flanking regions were also obtained by an LM-PCR procedure. Sequencing and Southern analysis showed that they belong to a homeobox gene family of rice. Six homeobox-containing fragments were mapped on the molecular linkage map of rice. They were located on chromosomes 3, 4 and 7 respectively. It is noteworthy that there are 4 homeobox fragments located on rice chromosome 3 and the result is also consistent with the comparative genomics between rice and maize.     

Establishment of Smad2 conditional gene targeting mice based on the Cre-LoxP system

Smads is a new gene family in transforming growth factor-β (TGF- β) signaling pathway. Smad2 mutated in multiple human tumors and may be a candidate tumor suppressor gene. Targeted disruption of murine Smad2 gene resulted in embryonic lethality at E6.5. To study the function of Smad2 in vertebrate organgenesis and tumorigenesis, we constructed the Smad2 conditional targeting vector in which two LoxP sequences were placed to flank the sequences encoding the C terminal functional domain of Smad2. The validity of the LoxP sites in the targeting construct was tested in E. coli that express the Cre recombinase constitutively. The vector was electroporated into ES cells and 3 targeted ES cell clones were obtained by Southern blot screening. Targeted ES cells were introduced into C57BL/6J blastocysts by microinjection to generate germ-line chimeras. Genotyping analysis showed that 2 progeny among these chimeras carried the Smad2 conditional targeted allele. The establishment of Smad2 conditional gene targeting mouse has laid a solid foundation for producing the tissue specific Smad2 gene knockout mice.     

Factors Influencing the Differentiation of Dopaminergic Traits in Transplanted Neural Stem Cells

1. Our previous studies demonstrated that when neural stem cells (NSCs) of the C17.2 clonal line are transplanted into the intact or 6-hydroxydopamine (6-OHDA) lesioned rat striatum, in most, but not all grafts, cells spontaneously express the dopamine (DA) biosynthetic enzymes, tyrosine hydroxylase (TH), and aromatic L-amino acid decarboxylase (Yang, M., Stull, N. D., Snyder, E. Y., Berk, M. A., and Iacovitti, L. (2002). Exp. Neurol.).2. These results suggested that there were certain conditions which were more conducive to the development of DA traits in NSCs and possibly other neurotransmitter phenotypes.3. In the present study, we modified a number of variables in vitro (i.e. passage number, confluence) and/or in vivo (degree, type, and site of injury) before assessing the survival, migration, and differentiation of engrafted NSCs.4. We found that low confluence cultures were comprised exclusively of flattened polygonal cells, which when transplanted, migrated widely in the brain but did not express TH.5. In contrast, high confluence cultures contained both polygonal cells and an overlying bed of fusiform cells.6. When these NSCs were maintained for 12–20 passages and then transplanted, virtually all engrafted cells in 65% of the grafts expressed TH but not markers of other neurotransmitter systems.7. Importantly, all TH⁺ grafts were accompanied by significant physical damage to the brain while TH^– grafts were not, suggesting that local injury-related factors were also important.8. Of no apparent influence on TH expression, regardless of how cells were grown prior to implantation, was the site of transplantation (cortex or striatum) or the degree of chemical lesion (intact, partial or full).9. We conclude that transplanted NSCs can express traits specifically associated with DA neurons but only when cells are grown under certain conditions in vitro and then transplanted in proximity to injury-induced factors present in vivo.