Glutamate receptors play major roles in excitatory transmission in the vertebrate brain. Among ionotropic glutamate receptors (AMPA, kainate, NMDA), AMPA receptors mediate fast synaptic transmission and require TARP auxiliary subunits. NMDA receptors and kainate receptors play roles in synaptic transmission, but it remains uncertain whether these ionotropic glutamate receptors also have essential subunits. Using a proteomic screen, we have identified NETO2, a brain-specific protein of unknown function, as an interactor with kainate-type glutamate receptors. NETO2 modulates the channel properties of recombinant and native kainate receptors without affecting trafficking of the receptors and also modulates kainate-receptor-mediated mEPSCs. Furthermore, we found that kainate receptors regulate the surface expression of NETO2 and that NETO2 protein levels and surface expression are decreased in mice lacking the kainate receptor GluR6. The results show that NETO2 is a kainate receptor subunit with significant effects on glutamate signaling mechanisms in brain.

The ligandin activity of specific glutathione S-transferases (GSTs) is necessary for the transport of anthocyanins from the cytosol to the plant vacuole. Five GSTs were purified from Vitis vinifera L. cv. Gamay Fréaux cell suspension cultures by glutathione affinity chromatography. These proteins underwent Edman sequencing and mass spectrometry fingerprinting, with the resultant fragments aligned with predicted GSTs within public databases. The corresponding coding sequences were cloned, with heterologous expression in Escherichia coli used to confirm GST activity. Transcriptional profiling of these candidate GST genes and key anthocyanin biosynthetic pathway genes (PAL, CHS, DFR, and UFGT) in cell suspensions and grape berries against anthocyanin accumulation demonstrated strong positive correlation with two sequences, VvGST1 and VvGST4, respectively. The ability of VvGST1 and VvGST4 to transport anthocyanins was confirmed in the heterologous maize bronze-2 complementation model, providing further evidence for their function as anthocyanin transport proteins in grape cells. Furthermore, the differential induction of VvGST1 and VvGST4 in suspension cells and grape berries suggests functional differences between these two proteins. Further investigation of these candidate ligandins may identify a mechanism for manipulating anthocyanin accumulation in planta and in vitro suspension cells.

CD147 plays a critical role in the invasive and metastatic activity of hepatocellular carcinoma (HCC) cells by stimulating the surrounding fibroblasts to express matrix metalloproteinases (MMPs). Tumor cells adhesion to extracellular matrix (ECM) proteins is the first step to the tumor metastasis. MMPs degrade the ECM to promote tumor metastasis. The aim of this study is to investigate the effects of small interfering RNA (siRNA) against CD147 (si-CD147) on hepatocellular carcinoma cells' (SMMC-7721) architecture and functions.

Expression of neutrophil gelatinase-associated lipocalin (NGAL)/lipocalin2, a recently recognized iron regulatory protein that binds to matrix metalloproteinase-9 (MMP9), is increased in a spectrum of cancers, including those of the colorectum. Using colon carcinoma cell lines stably transfected with NGAL or antisense NGAL, we showed that NGAL overexpression altered subcellular localization of E-cadherin and catenins, decreased E-cadherin-mediated cell-cell adhesion, enhanced cell-matrix attachment, and increased cell motility and in vitro invasion. Conversely, a decrease in NGAL enhanced more aggregated growth pattern and decreased in vitro invasion. We further showed that NGAL exerted these effects through the alteration of the subcellular localization of Rac1 in an extracellular matrix-dependent, but MMP9-independent, manner. Furthermore, we observed that the NGAL-overexpressing cells tolerated increased iron levels in the culture environment, whereas the NGAL-underexpressing cells showed significant cell death after prolonged incubation in high-iron condition. Thus, overexpressing NGAL in colon carcinomas is an important regulatory molecule that integrates extracellular environment cues, iron metabolism, and intracellular small GTPase signaling in cancer migration and invasion. NGAL may therefore be a new target for therapeutic intervention in colorectal carcinoma.

Unlike other members of HSP70 family, GRP78 manifests multifaceted subcellular distribution and forms complex with different signals, resulting in its close correlation with various tumors. However, its expression profile and function in glioma remain less well defined. In this study, normal brain tissue and astrocytic tumor specimens were evaluated for GRP78 expression, which was shown to be up-regulated in astrocytoma compared with normal tissue, increased markedly as astrocytoma pathologic grade escalates, and can still be enhanced for disease recurrence. By employing Cox regression analyses, high GRP78 expression was correlated with a poorer outcome for recurrent glioblastoma patients. In addition, immunofluorescence microscopy detected cell surface positioning of GRP78 on human glioma cells. After transfection with siRNA or antibody ligation of surface GRP78, phosphorylation of Akt and ERK was attenuated. These findings indicate that GRP78 plays an important role in astrocytoma malignancy, whereas its cell surface localization may be attractive for clinical utilization.

Boron neutron capture therapy (BNCT) is an alternative treatment modality for patients with glioma. The aim of this study was to determine whether induction of apoptosis contributes to the main therapeutic efficacy of BNCT and to compare the relative biological effect (RBE) of BNCT, γ-ray and reactor neutron irradiation.

Cytokinesis, the final step of cell division, usually ends with the abscission of the two daughter cells. In some tissues, however, daughter cells never completely separate and remain interconnected by intercellular bridges or ring canals. In this paper, we report the identification and analysis of a novel ring canal component, Nessun Dorma (Nesd), isolated as an evolutionarily conserved partner of the centralspindlin complex, a key regulator of cytokinesis. Nesd contains a pectin lyase-like domain found in proteins that bind to polysaccharides, and we present evidence that it has high affinity for β-galactosides in vitro. Moreover, nesd is an essential gene in Drosophila melanogaster, in which it is required for completion of cytokinesis during male meiosis and possibly in female germline cells. Our findings indicate that Nesd is a novel carbohydrate-binding protein that functions together with centralspindlin in late cytokinesis, thus highlighting the importance of glycosylation in this process.

CD1d is an MHC class I-like molecule comprised of a transmembrane glycoprotein (heavy chain) associated with β(2)-microglobulin (β(2)m) that presents lipid antigens to NKT cells. Initial folding of the heavy chain involves its glycan-dependent association with calreticulin (CRT), calnexin (CNX), and the thiol oxidoreductase ERp57, and is followed by assembly with β(2)m to form the heterodimer. Here we show that in CRT-deficient cells CD1d heavy chains convert to β(2)m-associated dimers at an accelerated rate, indicating faster folding of the heavy chain, while the rate of intracellular transport after assembly is unaffected. Unlike the situation with MHC class I molecules, antigen presentation by CD1d is not impaired in the absence of CRT. Instead, there are elevated levels of stable and functional CD1d on the surface of CRT-deficient cells. Association of the heavy chains with the ER chaperones Grp94 and Bip is observed in the absence of CRT, and these may replace CRT in mediating CD1d folding and assembly. ER retention of free CD1d heavy chains is impaired in CRT-deficient cells, allowing their escape and subsequent expression on the plasma membrane. However, these free heavy chains are rapidly internalized and degraded in lysosomes, indicating that β(2)m association is required for the exceptional resistance of CD1d to lysosomal degradation that is normally observed.

Bacterial pathogens often show significant intraspecific variations in ecological fitness, host preference and pathogenic potential to cause infectious disease. The species of Listeria monocytogenes, a facultative intracellular pathogen and the causative agent of human listeriosis, consists of at least three distinct genetic lineages. Two of these lineages predominantly cause human sporadic and epidemic infections, whereas the third lineage has never been implicated in human disease outbreaks despite its overall conservation of many known virulence factors.

Several functional MRI (fMRI) activation studies have highlighted specific differences in brain response in social anxiety disorder (SAD) patients. Little is known, so far, about the changes in the functional architecture of resting state networks (RSNs) in SAD during resting state. We investigated statistical differences in RSNs on 20 SAD and 20 controls using independent component analysis. A diffuse impact on widely distributed RSNs and selective changes of RSN intrinsic functional connectivity were observed in SAD. Functional connectivity was decreased in the somato-motor (primary and motor cortices) and visual (primary visual cortex) networks, increased in a network including medial prefrontal cortex which is thought to be involved in self-referential processes, and increased or decreased in the default mode network (posterior cingulate cortex/precuneus, bilateral inferior parietal gyrus, angular gyrus, middle temporal gyrus, and superior and medial frontal gyrus) which has been suggested to be involved in episodic memory, and self-projection, the dorsal attention network (middle and superior occipital gyrus, inferior and superior parietal gyrus, and middle and superior frontal gyrus) which is thought to mediate goal-directed top-down processing, the core network (insula-cingulate cortices) which is associated with task control function, and the central-executive network (fronto-parietal cortices). A relationship between functional connectivity and disease severity was found in specific regions of RSNs, including medial and lateral prefrontal cortex, as well as parietal and occipital regions. Our results might supply a novel way to look into neuro-pathophysiological mechanisms in SAD patients.

The tumor suppressor gene CDKN2A generates at least three different transcriptional variants, each of which is thought to encode a tumor suppressor. However, the inhibitory activities of these variants have not yet been compared in the same cells. Protein therapy is known to have several advantages over gene therapy. Thus, investigation of the exogenous protein molecule of the most effective suppressor may yield meaningful information regarding protein-based cancer therapy.

Transforming growth factor β (TGF-β) signaling plays an important role in the pathogenesis of cardiac hypertrophy. However, the molecular mechanism of TGF-β signaling during the process of cardiac remodeling remains poorly understood. In the present study, by employing single-molecule fluorescence imaging approach, we demonstrated that in neonatal rat cardiomyocytes, TGF-β type II receptors (TβRII) existed as monomers at the low expression level, and dimerized upon TGF-β1 stimulation. Importantly, for the first time, we found the increased dimerization of TβRII in hypertrophic cardiomyocytes comparing to the normal cardiomyocytes. The enhanced TβRII dimerization was correlated with the enhanced Smad3 phosphorylation levels. These results provide new information on the mechanism of TGF-β signaling in cardiac remodeling.

Cancer is a multifaceted disease that results from dysregulated normal cellular signaling networks caused by genetic, genomic and epigenetic alterations at cell or tissue levels. Uncovering the underlying protein signaling network changes, including cell cycle gene networks in cancer, aids in understanding the molecular mechanism of carcinogenesis and identifies the characteristic signaling network signatures unique for different cancers and specific cancer subtypes. The identified signatures can be used for cancer diagnosis, prognosis, and personalized treatment. During the past several decades, the available technology to study signaling networks has significantly evolved to include such platforms as genomic microarray (expression array, SNP array, CGH array, etc.) and proteomic analysis, which globally assesses genetic, epigenetic, and proteomic alterations in cancer. In this review, we compared Pathway Array analysis with other proteomic approaches in analyzing protein network involved in cancer and its utility serving as cancer biomarkers in diagnosis, prognosis and therapeutic target identification. With the advent of bioinformatics, constructing high complexity signaling networks is possible. As the use of signaling network-based cancer diagnosis, prognosis and treatment is anticipated in the near future, medical and scientific communities should be prepared to apply these techniques to further enhance personalized medicine.

Activating mutations in one allele of an oncogene (heterozygous mutations) are widely believed to be sufficient for tumorigenesis. However, mutant allele specific imbalance (MASI) has been observed in tumors and cell lines harboring mutations of oncogenes.

LSVT LOUD (Lee Silverman Voice Treatment) is efficacious in the treatment of speech disorders in idiopathic Parkinson's disease (IPD), particularly hypophonia. Functional imaging in patients with IPD has shown abnormalities in several speech regions and changes in these areas immediately following treatment. This study serves to extend the analysis by correlating changes of regional neural activity with the main behavioral change following treatment, namely, increased vocal intensity. Ten IPD participants with hypophonia were studied before and after LSVT LOUD. Cerebral blood flow during rest and reading conditions were measured by H(2)(15)O-positron emission tomography. Z-score images were generated by contrasting reading with rest conditions for pre- and post-LSVT LOUD sessions. Neuronal activity during reading in the pre- versus post-LSVT LOUD contrast was correlated with corresponding change in vocal intensity to generate correlation images. Behaviorally, vocal intensity for speech tasks increased significantly after LSVT LOUD. The contrast and correlation analyses indicate a treatment-dependent shift to the right hemisphere with modification in the speech motor regions as well as in prefrontal and temporal areas. We interpret the modification of activity in these regions to be a top-down effect of LSVT LOUD. The absence of an effect of LSVT LOUD on the basal ganglion supports this argument. Our findings indicate that the therapeutic effect of LSVT LOUD in IPD hypophonia results from a shift in cortical activity to the right hemisphere. These findings demonstrate that the short-term changes in the speech motor and multimodal integration areas can occur in a top-down manner.

Perifosine belongs to the class of alkylphospholipid analogues, which act primarily at the cell membrane, thereby targeting signal transduction pathways. In phase I/II clinical trials, perifosine has induced tumour regression and caused disease stabilisation in a variety of tumour types. The genetic determinants responsible for its cytotoxicity have not been comprehensively studied, however. We performed a genome-wide analysis to identify genes whose expression levels or genotypic variation were correlated with the cytotoxicity of perifosine, using public databases on the US National Cancer Institute (NCI)-60 human cancer cell lines. For demonstrating drug specificity, the NCI Standard Agent Database (including 171 drugs acting through a variety of mechanisms) was used as a control. We identified agents with similar cytotoxicity profiles to that of perifosine in compounds used in the NCI drug screen. Furthermore, Gene Ontology and pathway analyses were carried out on genes more likely to be perifosine specific. The results suggested that genes correlated with perifosine cytotoxicity are connected by certain known pathways that lead to the mitogen-activated protein kinase signalling pathway and apoptosis. Biological processes such as 'response to stress', 'inflammatory response' and 'ubiquitin cycle' were enriched among these genes. Three single nucleotide polymorphisms (SNPs) located in CACNA2D1 and EXOC4 were found to be correlated with perifosine cytotoxicity. Our results provided a manageable list of genes whose expression levels or genotypic variation were strongly correlated with the cytotoxcity of perifosine. These genes could be targets for further studies using candidate-gene approaches. The results also provided insights into the pharmacodynamics of perifosine.

Interleukin-1 receptor-associated kinase-4 (IRAK-4) encodes a kinase that is essential for NF-kB activation in Toll-like receptor and T-cell receptor signaling pathways, indicating a possible crosstalk between innate and acquired immunities. We attempted to determine whether the polymorphisms in the Interleukin-1 receptor-associated kinase-4 (IRAK-4) gene are associated with allergic rhinitis (AR) in the Han Chinese population.

Microglial activation plays an important role in neurodegenerative diseases by producing several proinflammatory enzymes and proinflammatory cytokines. The phenolic glucoside gastrodin, a main constituent of a Chinese herbal medicine, has been known to display anti-inflammatory properties. The current study investigates the potential mechanisms whereby gastrodin affects the expression of potentially pro-inflammatory proteins by cultured murine microglial BV-2 cells stimulated with lipopolysaccharide (LPS).

Influenza A viruses are a severe threat worldwide, causing large epidemics that kill thousands every year. Prevention of influenza infection is complicated by continuous viral antigenic changes. Newer anti-influenza agents include MEK/ERK and protein kinase C inhibitors; however, the downstream effectors of these pathways have not been determined. In this study, we identified a common mechanism for the inhibitory effects of a significant group of anti-influenza agents. Our studies showed that influenza infection activates a series of signaling pathways that converge to induce myosin light chain (MLC) phosphorylation and remodeling of the actin cytoskeleton. Inhibiting MLC phosphorylation by blocking RhoA/Rho kinase, phospholipase C/protein kinase C, and HRas/Raf/MEK/ERK pathways with the use of genetic or chemical manipulation leads to the inhibition of influenza proliferation. In contrast, the induction of MLC phosphorylation enhances influenza proliferation, as does activation of the HRas/Raf/MEK/ERK signaling pathway. This effect is attenuated by inhibiting MLC phosphorylation. Additionally, in intracellular trafficking studies, we found that the nuclear export of influenza ribonucleoprotein depends on MLC phosphorylation. Our studies provide evidence that modulation of MLC phosphorylation is an underlying mechanism for the inhibitory effects of many anti-influenza compounds.

Expression of atheroprotective genes in the blood vessel wall is potentially an effective means of preventing or reversing atherosclerosis. Development of this approach has been hampered by lack of a suitable gene-transfer vector. We used a helper-dependent adenoviral (HDAd) vector to test whether expression of apolipoprotein A-I (apoA-I) in the artery wall could retard the development of atherosclerosis in hyperlipidemic rabbits. Carotid arteries were infused with an HDAd expressing rabbit apoA-I or a "null" HDAd and harvested 2 and 4 weeks later. ApoA-I mRNA and protein were detected only in HDAdApoAI arteries. Lesion size, lipid and macrophage content, and adhesion molecule expression were similar in both groups at 2 weeks. Between 2 and 4 weeks, most of these measures of atherosclerosis increased in HDAdNull arteries, but were stable or decreased in HDAdApoAI arteries (P ≤ 0.04 for all end points in 4-week HDAdApoAI versus HDAdNull arteries). A longer-term study in chow-fed rabbits revealed persistence of HDAd vector DNA and apoA-I expression for ≥48 weeks, with stable vector DNA content and apoA-I expression from 4 to 48 weeks. Expression of apoA-I in arterial endothelium significantly retards atherosclerosis. HDAd provides prolonged, stable expression of a therapeutic transgene in the artery wall.