Regulation and dysregulation of astrocyte activation and implications in tumor formation.

Astrocytic activation is a cellular response to disturbances of the central nervous system (CNS). Recent advances in cellular and molecular biology have demonstrated the remarkable changes in molecular signaling, morphology, and metabolism that occur during astrocyte activation. Based on these studies, it has become clear that the astrocyte activation process is regulated by a variety of signaling pathways, which result in metabolic support, wound healing and scar formation. While normal astrocyte activation pathways drive homeostasis and/or repair in the CNS, dysregulation of these pathways can lead to astrocyte abnormalities, including glioma formation with similar phenotypes as reactive astrocytes. We review the principle pathways responsible for astrocytic activation, as well as their potential contribution to tumor formation in the CNS.

PACSIN1, a Tau-interacting protein, regulates axonal elongation and branching by facilitating microtubule instability.

Tau is a major member of the neuronal microtubule-associated proteins. It promotes tubulin assembly and stabilizes axonal microtubules. Previous studies have demonstrated that Tau forms cross-bridges between microtubules, with some particles located on cross-bridges, suggesting that some proteins interact with Tau and might be involved in regulating Tau-related microtubule dynamics. This study reports that PACSIN1 interacts with Tau in axon. PACSIN1 blockade results in impaired axonal elongation and a higher number of primary axonal branches in mouse dorsal root ganglia neurons, which is induced by increasing the binding ability of Tau to microtubules. In PACSIN1-blocked dorsal root ganglia neurons, a greater amount of Tau is inclined to accumulate in the central domain of growth cones, and it promotes the stability of the microtubule network. Taken together, these results suggest that PACSIN1 is an important Tau binding partner in regulating microtubule dynamics and forming axonal plasticity.

ß-Catenin signaling initiates the activation of astrocytes and its dysregulation contributes to the pathogenesis of astrocytomas.

Astrocytes are the most abundant cell of the CNS and demonstrate contact inhibition in which a nonproliferative, nonmotile cellular state is achieved once stable intercellular contacts are formed between mature cells. Cellular injury disrupts these intercellular contacts, causing a loss of contact inhibition and the rapid initiation of healing. Dysregulation of the molecular pathways involved in this process is thought to lead to an aggressive cellular state associated with neoplasia. We investigated whether a comparable correlation exists between the response of astrocytes to injury and the malignant phenotype of astrocytomas. We discovered that the loss of contact inhibition plays a critical role in the initiation and regulation of reactive astrocytes in the healing of wounds. In particular, injury of the astrocytes interrupts and destabilizes the cadherin-catenin complexes at the cell membrane leading to nuclear translocation of ß-catenin and characteristic changes associated with the activation of astrocytes. Similar signaling pathways are found to be active--but dysregulated--in astrocytomas. Inhibition of ß-catenin signaling diminished both the response of astrocytes to injury and induction of the malignant phenotype of astrocytomas. The findings shed light on a unique mechanism associated with the pathogenesis of astrocytomas and provide a model for the loss of contact inhibition that may broadly apply to understanding the mechanisms of tissue repair and tumorigenesis in the brain.

Cep57, a NEDD1-binding pericentriolar material component, is essential for spindle pole integrity.

Formation of a bipolar spindle is indispensable for faithful chromosome segregation and cell division. Spindle integrity is largely dependent on the centrosome and the microtubule network. Centrosome protein Cep57 can bundle microtubules in mammalian cells. Its related protein (Cep57R) in Xenopus was characterized as a stabilization factor for microtubule-kinetochore attachment. Here we show that Cep57 is a pericentriolar material (PCM) component. Its interaction with NEDD1 is necessary for the centrosome localization of Cep57. Depletion of Cep57 leads to unaligned chromosomes and a multipolar spindle, which is induced by PCM fragmentation. In the absence of Cep57, centrosome microtubule array assembly activity is weakened, and the spindle length and microtubule density decrease. As a spindle microtubule-binding protein, Cep57 is also responsible for the proper organization of the spindle microtubule and localization of spindle pole focusing proteins. Collectively, these results suggest that Cep57, as a NEDD1-binding centrosome component, could function as a spindle pole- and microtubule-stabilizing factor for establishing robust spindle architecture.

Characterization of kinesin-like proteins in silkworm posterior silk gland cells.

Kinesins are microtubule-based motors involved in various intracellular transports. Neurons, flagellated cells, and pigment cells have been traditionally used as model systems to study the cellular functions of kinesins. Here, we report silkworm posterior silkgland (PSG), specialized cells with an extensive endomembrane system for intracellular transport and efficient secretion of fibroin, as a novel model for kinesin study. To investigate kinesin-driven intracellular transport in PSG cells, we cloned five silkworm kinesin-like proteins (KLPs), BmKinesin-1, BmKinesin-6, BmKinesin-7, BmKinesin-13, and BmKinesin-14A. We determined their expression patterns by relative real-time PCR and western blotting. Immunofluorescence microscopy verified their colocalization with microtubules. By combining pull-down assays, LC-MS/MS, and western blotting analysis, we identified many potential cargoes of BmKinesin-1 in PSG, including fibroin-containing granules and exuperantia-associated ribonucleoprotein (RNP) complexes. Moreover, BmKinesin-13 overexpression disrupted the microtubule network in BmN cells, which is consistent with a role of Kinesin-13 in regulating microtubule dynamics in other organisms. On the basis of these results, we concluded that PSG might have advantages in elucidating mechanisms of intracellular transport in secretory tissues and could serve as a potential model for kinesin studies.

The Neuroscience Research Institute at Peking University: a place for the solution of pain and drug abuse.

Neuroscience research in China has undergone rapid expansion since 1980. The Neuroscience Research Institute of Peking University, one of the most active neuroscience research groups in China, was founded in 1987. Currently, the institute is overseeing four research areas, i.e., (1) pain and analgesia, (2) drug abuse and acupuncture treatment for drug addiction, (3) the mechanism of neurological degenerative disorders, and (4) the role of neuroglia in central nervous system injury. The institute is simultaneously investigating both theoretical and clinical studies. Acupuncture remains the core of research, while pain and drug abuse form the two disciplines.

Sinomenine inhibits activation of rat retinal microglia induced by advanced glycation end products.

Diabetic retinopathy involves an inflammatory response in the retina characterized by an increase in inflammatory cytokines and activation of microglia. The degree of microglia activation may influence the extent of retina injury following an inflammatory stimulus. Cytokines, released by activated microglia, regulate the influx of inflammatory cells to the damaged area. Thus, therapeutic strategy to reduce cytokine expression in microglia would be neuroprotective. Sinomenine, an alkaloid isolated from the stem and root of Sinomenium acutum, has long been recognized as an anti-inflammatory drug for rheumatoid arthritis and also inhibits macrophage activation. In this study, we activated retinal microglia in culture with advanced glycation end products (AGEs) treatment and attempted to determine whether sinomenine could reduce the production of cytokines from the activated microglia at both gene and protein levels. Changes in inflammatory cytokines, TNF alpha, IL-1 beta and IL-6, were measured by semi-quantitative RT-PCR and enzyme-linked immunosorbent assay (ELISA) both in the presence and absence of AGEs. The effect of sinomenine on levels of reactive oxygen species (ROS) and the nuclear translocation of NF-kB p65 were studied with a laser confocal scanning microscope. AGEs treatment induced a significant release of TNF alpha, IL-1 beta, and IL-6 from retinal microglia. Sinomenine could inhibit release of these cytokines. Sinomenine attenuated ROS production in a dose-dependent fashion and reduced the nuclear translocation of NF-kB p65 in AGEs-activated retinal microglia in culture.

AGEs mediated expression and secretion of TNF alpha in rat retinal microglia.

Diabetic retinopathy induces an inflammatory response in the retina characterized by an increase in inflammatory cytokines and the activation of microglia. The degree of microglia activation may influence the extent of retina injury following retinal metabolic stress. We have previously shown that DR rats have elevated levels of advanced glycation end products (AGEs) in their blood. We have also suggested that AGEs might be involved in microglial activation and production of tumor necrosis factor alpha (TNF alpha). In this study, we attempted to confirm that AGEs induce the release of TNF alpha from rat retinal microglia using an in vitro microglia culture system, and concurrently to explore the mediating mechanisms. AGEs increased the protein secretion and mRNA expression of TNF alpha in cultured rat retinal microglia. These effects of AGEs were primarily mediated by reactive oxygen species (ROS). Furthermore, the inhibitors for mitogen-activated protein kinases (MAPK; p38, JNK and ERK 1/2) and nuclear factor-kB (NF-kB) could significantly decrease AGEs-induced TNF alpha release. AGEs-activated microglia showed an increase of NF-kB p65 nuclear translocation. These observations indicated that pathophysiological levels of AGEs may alter rat retinal microglia function by up-regulating TNF alpha expression and release via enhanced formation of intracellular ROS. AGEs-induced ROS subsequently activates MAPK (p38, JNK and ERK1/2) and NF-kB.

Nucleoside transporter expression and function in cultured mouse astrocytes.

Uptake of purine and pyrimidine nucleosides in astrocytes is important for several reasons: (1) uptake of nucleosides contributes to nucleic acid synthesis; (2) astrocytes synthesize AMP, ADP, and ATP from adenosine and GTP from guanosine; and (3) adenosine and guanosine function as neuromodulators, whose effects are partly terminated by cellular uptake. It has previously been shown that adenosine is rapidly accumulated by active uptake in astrocytes (Hertz and Matz, Neurochem Res 14:755-760, 1989), but the ratio between active uptake and metabolism-driven uptake of adenosine is unknown, as are uptake characteristics for guanosine. The present study therefore aims at providing detailed information of nucleoside transport and transporters in primary cultures of mouse astrocytes. Reverse transcription-polymerase chain reaction identified the two equilibrative nucleoside transporters, ENT1 and ENT2, together with the concentrative nucleoside transporter CNT2, whereas CNT3 was absent, and CNT1 expression could not be investigated. Uptake studies of tritiated thymidine, formycin B, guanosine, and adenosine (3-s uptakes at 1-4 degrees C to study diffusional uptake and 1-60-min uptakes at 37 degrees C to study concentrative uptake) demonstrated a fast diffusional uptake of all four nucleosides, a small, Na(+)-independent and probably metabolism-driven uptake of thymidine (consistent with DNA synthesis), larger metabolism-driven uptakes of guanosine (consistent with synthesis of DNA, RNA, and GTP) and especially of adenosine (consistent with rapid nucleotide synthesis), and Na(+)-dependent uptakes of adenosine (consistent with its concentrative uptake) and guanosine, rendering neuromodulator uptake independent of nucleoside metabolism. Astrocytes are accordingly well suited for both intense nucleoside metabolism and metabolism-independent uptake to terminate neuromodulator effects of adenosine and guanosine.

Minocycline inhibits LPS-induced retinal microglia activation.

Retinal neurodegenerative disease involves an inflammatory response in the retina characterized by an increase in inflammatory cytokines and activation of microglia. The degree of microglia activation may influence the extent of retinal injury following an inflammatory stimulus. Cytokines released by activated microglia regulate the influx of inflammatory cells to the damaged area. Thus, a therapeutic strategy to reduce cytokine expression in microglia would be neuroprotective. Minocycline, a semisynthetic tetracycline derivative, is known to protect rodent brain from ischemia and to inhibit microglial activation. In this study, we activated retinal microglia in culture with lipopolysaccharide (LPS) and attempted to determine whether minocycline could reduce the production of cytokines from activated microglia at both gene and protein levels. Changes in inflammatory cytokines, TNF-alpha and IL-1beta, were measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) in the presence or absence of LPS. We also measured the levels of nitric oxide (NO) by the nitrate reductase method under similar conditions. LPS treatment induced a significant upregulation of the mRNA and release of TNF-alpha, IL-1beta, and NO from retinal microglia. Minocycline inhibited these releases. Thus, minocycline might exert its antiinflammatory effect on microglia by inhibiting the expression and release of TNF-alpha, IL-1beta, and NO.

Nucleic acid sequence-based amplification methods to detect avian influenza virus.

Infection of poultry with highly pathogenic avian influenza virus (AIV) can be devastating in terms of flock morbidity and mortality, economic loss, and social disruption. The causative agent is confined to certain isolates of influenza A virus subtypes H5 and H7. Due to the potential of direct transfer of avian influenza to humans, continued research into rapid diagnostic tests for influenza is therefore necessary. A nucleic acid sequence-based amplification (NASBA) method was developed to detect a portion of the haemagglutinin gene of avian influenza A virus subtypes H5 and H7 irrespective of lineage. A further NASBA assay, based on the matrix gene, was able to detect examples of all known subtypes (H1-H15) of avian influenza virus. The entire nucleic acid isolation, amplification, and detection procedure was completed within 6h. The dynamic range of the three AIV assays was five to seven orders of magnitude. The assays were sensitive and highly specific, with no cross-reactivity to phylogenetically or clinically relevant viruses. The results of the three AIV NASBA assays correlated with those obtained by viral culture in embryonated fowl's eggs.

Parentage testing anomalies in Hong Kong SAR of China.

OBJECTIVE: To determine if there are any differences in the number of exclusions from paternity of men using an anonymous parentage testing service compared with that of men using an in-person parentage testing service provided by the same company in Hong Kong SAR of China. METHODS: Comparable numbers of consecutive anonymous and in-person parentage tests conducted by the same company were analyzed. RESULTS: Men using an anonymous parentage testing service were excluded from paternity at a significantly greater rate (P < 0.001), compared with men using an in-person parentage test service. CONCLUSIONS: The results obtained from anonymous parentage testing indicate that the number of families containing children of doubtful parentage is much greater than expected previously. As illegitimate children are known to suffer greater degrees of abuse and neglect, this finding poses serious social questions regarding the welfare of families, which the relevant authorities should acknowledge and address.

Alpha-adrenergic stimulation of ERK phosphorylation in astrocytes is alpha(2)-specific and may be mediated by transactivation.

The highly specific alpha(2)-adrenergic agonist, dexmedetomidine, has hypnotic-sedative, anesthetic-sparing and analgesic effects, and it protects neurons against ischemia. The alpha(1)-adrenergic agonist, phenylephrine, does not share dexmedetomidine's pharmacological properties, although both dexmedetomidine and phenylephrine increase free cytosolic Ca(2+) ([Ca(2+)](i)) in astrocytes, and most of dexmedetomidine's actions in the brain are exerted on postjunctional receptors. alpha(2)-Adrenergic receptors are abundant on astrocytes. Dexmedetomidine-mediated 'down-streamn' signal transduction was therefore investigated in primary cultures of mouse astrocytes and contrasted with that of phenylephrine. The cultures were incubated with dexmedetomidine concentrations known to be pharmacologically active and to act specifically on alpha(2)-adrenergic receptors (25-100 nM). ERK(1/2) phosphorylation was measured using specific antibodies. Peak increases of ERK(1/2) phosphorylation occurred at 50 nM dexmedetomidine, with less effect at higher concentrations. Phenylephrine caused ERK phosphorylation only at a concentration high enough to exert non subtype-specific effects (10 microM), and this effect was counteracted by the alpha(2)-adrenergic antagonist atipamezole. The phosphorylation of ERK was reduced by tyrphostin AG1478, an inhibitor of phosphorylation of the epidermal growth factor receptor (EGFR), and by heparin, which neutralizes heparin-binding epithelial growth factor (HB-EGF), suggesting the involvement of a transactivation process, in which alpha(2)-adrenergic stimulation leads to proteolytic shedding of HB-EGF (and perhaps other EGFR agonists) from transmembrane-spanning precursors.

Pulsatilloside A and anemoside A3 protect PC12 cells from apoptosis induced by sodium cyanide and glucose deprivation.

Using sodium cyanide (NaCN) and glucose deprivation induced cell injury in PC12 as an injury model, we investigated the protective effects of pulsatilloside A and anemoside A 3 on neurons. The results showed that PC12 cells under the NaCN-injury and glucose deprivation would undergo apoptosis. Additions of pulsatilloside A and anemoside A 3, at dosages ranging from 0.1, 1 and 10 microg/ml, protected PC12 cells from apoptosis determined by MTT, LDH release analysis, and flow cytometry measurement.

A method to detect major serotypes of foot-and-mouth disease virus.

Nucleic acid sequence-based amplification (NASBA) is an isothermal technique that allows the rapid amplification of specific regions of nucleic acid obtained from a diverse range of sources. It is especially suitable for amplifying RNA sequences. A rapid and specific NASBA technique was developed, allowing the detection of foot-and-mouth disease virus genetic material in a range of sample material, including preserved skin biopsy material from infected animals, vaccines prepared from denatured cell-free material, and cell-free antigen-based detection kits. A single pair of DNA oligonucleotide primers was able to amplify examples of all major FMD virus subtypes. The amplified viral RNA was detected by electrochemiluminescence. The method was at least as sensitive as existing cell-free antigen detection methods.

Up-regulation of 5-HT2B receptor density and receptor-mediated glycogenolysis in mouse astrocytes by long-term fluoxetine administration.

The effects were studied of short-term (1 week) versus long-term (2-3 weeks) fluoxetine treatment of primary cultures of mouse astrocytes, differentiated by treatment with dibutyryl cyclic AMP. From previous experiments it is known that acute treatment with fluoxetine stimulates glycogenolysis and increases free cytosolic Ca2+ concentration ([Ca2+]i]) in these cultures, whereas short-term (one week) treatment with 10 microM down-regulates the effects on glycogen and [Ca2+]i, when fluoxetine administration is renewed (or when serotonin is administered). Moreover, antagonist studies have shown that these responses are evoked by activation of a 5-HT2, receptor that is different from the 5-HT2A receptor and therefore at that time tentatively were interpreted as being exerted on 5-HT2C receptors. In the present study the cultures were found by RT-PCR to express mRNA for 5-HT2A and 5-HT2B receptors, but not for the 5-HT2C receptor, identifying the 5-HT2 receptor activated by fluoxetine as the 5-HT2B receptor, the most recently cloned 5-Ht2 receptor and a 5-HT receptor known to be more abundant in human, than in rodent, brain. Both short-term and long-term treatment with fluoxetine increased the specific binding of [3H]mesulergine, a ligand for alL three 5-HT2 receptors. Long-term treatment with fluoxetine caused an agonist-induced up-regulation of the glycogenolytic response to renewed administration of fluoxetine, whereas short-term treatment abolished the fluoxetine-induced hydrolysis of glycogen. Thus, during a treatment period similar to that required for fluoxetine's clinical response to occur, 5-HT2B-mediated effects are initially down-regulated and subsequently up-regulated.