The present experiments focused on the morphological interaction between serotonergic (5-HT) and noradrenergic (NA) axons during regeneration following partial axonal denervation in the cerebral cortex in adult rats. The denervation paradigm used employed two neurotoxins, one for 5-HT and one for NA axons, infused together at one cortical site while a single neurotoxin to either 5-HT or NA was infused at the symmetrical cortical site in the other hemisphere. This treatment enabled us to assess the role of 5-HT or NA axons in the regeneration of the other monoaminergic axon. 5-HT axon regeneration became apparent as early as 28 days after the toxin injection, whereas the regeneration of NA axons was not evident even at 60 days after the toxin injection. Since NA axons revealed marked regeneration in the cortical site with denervation of 5-HT axons, intact 5-HT axons may be inhibitory on the regeneration of NA axons. In contrast, since the regeneration of 5-HT axons was suppressed in the absence of NA axons, NA axons appear to exert a facilitatory effect on 5-HT axon regeneration. These results suggest that the role of 5-HT axons in the regeneration of NA axons is opposite to that of NA axons in the regeneration of 5-HT axons. In addition, the regeneration of 5-HT axons occurred much faster than that of NA axons in response to axonal damage. The differential roles of 5-HT and NA axons in axonal regeneration may play a role in a variety of physiological functions related to these monoamines and possibly in the pathophysiology of clinical depression.

The human zinc finger protein 191 (ZNF191) is a member of the SCAN domain family of Krüppel-like zinc finger transcription factors. ZNF191 shows 94% identity to its mouse homologue zinc finger protein 191(Zfp191), which is the most highly conserved among the human-mouse SCAN family member orthologues pairs. Zfp191 is widely expressed during early embryogenesis and in adult organs. Moreover, Zfp191-/- embryos have been shown to be severely retarded in development and die approximately at embryonic day E7.5. ZNF191 can specifically interact with the widespread TCAT motif which constitutes the HUMTH01 microsatellite in the tyrosine hydroxylase (TH) gene. Allelic variations of HUMTH01 have been stated to have a quantitative silencing effect on TH gene expression and to correlate with quantitative and qualitative changes in the binding by ZNF191. In addition, ZNF191 displays a suppressive effect on the transcription; however, little downstream targets have identified.

In this paper we demonstrate that the anodic, bioelectrocatalytic performance of wastewater inoculum based, mixed culture microbial biofilms can be considerably improved by using a consecutive, purely electrochemical selection and biofilm acclimatization procedure. The procedure may represent an alternative to a repetitive mechanical biofilm removal, re-suspension and electrochemically facilitated biofilm formation. By using the proposed technique, the bioelectrocatalytic current density was increased from the primary to the secondary biofilm from 250 microAcm(-2) to about 500 microAcm(-2); and the power density of respective microbial fuel cells could be increased from 686 mWm(-2) to 1487 mWm(-2). The electrochemical characterization of the biofilms reveals a strong similarity to Geobacter sulfurreducens biofilms, which may indicate a dominating role of this bacterium in the biofilms.

Global comparisons of gene expression profiles between species provide significant insight into gene regulation, evolutionary processes and disease mechanisms. In this work, we describe a flexible and intuitive approach for global expression profiling of closely related species, using high-density exon arrays designed for a single reference genome. The high-density probe coverage of exon arrays allows us to select identical sets of perfect-match probes to measure expression levels of orthologous genes. This eliminates a serious confounding factor in probe affinity effects of species-specific microarray probes, and enables direct comparisons of estimated expression indexes across species. Using a newly designed Affymetrix exon array, with eight probes per exon for approximately 315,000 exons in the human genome, we conducted expression profiling in corresponding tissues from humans, chimpanzees and rhesus macaques. Quantitative real-time PCR analysis of differentially expressed candidate genes is highly concordant with microarray data, yielding a validation rate of 21/22 for human versus chimpanzee differences, and 11/11 for human versus rhesus differences. This method has the potential to greatly facilitate biomedical and evolutionary studies of gene expression in nonhuman primates and can be easily extended to expression array design and comparative analysis of other animals and plants.

Chromatin-associated histone H2AX is a key regulator of the cellular responses to DNA damage. However, non-nucleosomal functions of histone H2AX are poorly characterized. We have recently shown that soluble H2AX can trigger apoptosis but the mechanisms leading to non-chromatin-associated H2AX are unclear. Here, we tested whether stalling of DNA replication, a common event in cancer cells and the underlying mechanism of various chemotherapeutic agents, can trigger increased soluble H2AX.

MicroRNAs (miRNAs) are involved in the fine control of cell proliferation and differentiation during the development of the nervous system. MiR-124, a neural specific miRNA, is expressed from the beginning of eye development in Xenopus, and has been shown to repress cell proliferation in the optic cup, however, its role at earlier developmental stages is unclear. Here, we show that this miRNA exerts a different role in cell proliferation at the optic vesicle stage, the stage which precedes optic cup formation. We show that miR-124 is both necessary and sufficient to promote cell proliferation and repress neurogenesis at the optic vesicle stage, playing an anti-neural role. Loss of miR-124 upregulates expression of neural markers NCAM, N-tubulin while gain of miR-124 downregulates these genes. Furthermore, miR-124 interacts with a conserved miR-124 binding site in the 3'-UTR of NeuroD1 and negatively regulates expression of the proneural marker NeuroD1, a bHLH transcription factor for neuronal differentiation. The miR-124-induced effect on cell proliferation can be antagonized by NeuroD1. These results reveal a novel regulatory role of miR-124 in neural development and uncover a previously unknown interaction between NeuroD1 and miR-124.

In spite of decades-long studies, the mechanism of morphogenesis of plus-stranded RNA viruses belonging to the genus Enterovirus of Picornaviridae, including poliovirus (PV), is not understood. Numerous attempts to identify an RNA encapsidation signal have failed. Genetic studies, however, have implicated a role of the non-structural protein 2C(ATPase) in the formation of poliovirus particles. Here we report a novel mechanism in which protein-protein interaction is sufficient to explain the specificity in PV encapsidation. Making use of a novel "reporter virus", we show that a quasi-infectious chimera consisting of the capsid precursor of C-cluster coxsackie virus 20 (C-CAV20) and the nonstructural proteins of the closely related PV translated and replicated its genome with wild type kinetics, whereas encapsidation was blocked. On blind passages, encapsidation of the chimera was rescued by a single mutation either in capsid protein VP3 of CAV20 or in 2C(ATPase) of PV. Whereas each of the single-mutation variants expressed severe proliferation phenotypes, engineering both mutations into the chimera yielded a virus encapsidating with wild type kinetics. Biochemical analyses provided strong evidence for a direct interaction between 2C(ATPase) and VP3 of PV and CAV20. Chimeras of other C-CAVs (CAV20/CAV21 or CAV18/CAV20) were blocked in encapsidation (no virus after blind passages) but could be rescued if the capsid and 2C(ATPase) coding regions originated from the same virus. Our novel mechanism explains the specificity of encapsidation without apparent involvement of an RNA signal by considering that (i) genome replication is known to be stringently linked to translation, (ii) morphogenesis is known to be stringently linked to genome replication, (iii) newly synthesized 2C(ATPase) is an essential component of the replication complex, and (iv) 2C(ATPase) has specific affinity to capsid protein(s). These conditions lead to morphogenesis at the site where newly synthesized genomes emerge from the replication complex.

Type III galactosemia results from reduced activity of the enzyme UDP-galactose 4'-epimerase. Five disease-associated alleles (G90E, V94M, D103G, N34S and L183P) and three artificial alleles (Y105C, N268D, and M284K) were tested for their ability to alleviate galactose-induced growth arrest in a Saccharomyces cerevisiae strain which lacks endogenous UDP-galactose 4'-epimerase. For all of these alleles, except M284K, the ability to alleviate galactose sensitivity was correlated with the UDP-galactose 4'-epimerase activity detected in cell extracts. The M284K allele, however, was able to substantially alleviate galactose sensitivity, but demonstrated near-zero activity in cell extracts. Recombinant expression of the corresponding protein in Escherichia coli resulted in a protein with reduced enzymatic activity and reduced stability towards denaturants in vitro. This lack of stability may result from the introduction of an unpaired positive charge into a bundle of three α-helices near the surface of the protein. The disparities between the in vivo and in vitro data for M284K-hGALE further suggest that there are additional, stabilising factors present in the cell. Taken together, these results reinforce the need for care in the interpretation of in vitro, enzymatic diagnostic tests for type III galactosemia.

To report the usage of PGD for alpha-thalassaemia with the - -(SEA) genotype.

Da Chengqi Tang (DCT) is a common purgative formula in Chinese medicine. Flavanones are its major active compounds derived from Fructus Aurantii Immaturus. The present study developed an LC-MS/MS method to characterize two pairs of flavanone 7-glycoside isomers, i.e., hesperidin versus neohesperidin and naringin versus isonaringin.

Glycine receptors (GlyRs) play important roles in regulating hippocampal neural network activity and spinal nociception. Here we show that, in cultured rat hippocampal (HIP) and spinal dorsal horn (SDH) neurons, 17-beta-estradiol (E2) rapidly and reversibly reduced the peak amplitude of whole-cell glycine-activated currents (IGly). In outside-out membrane patches from HIP neurons devoid of nuclei, E2 similarly inhibited IGly, suggesting a non-genomic characteristic. Moreover, the E2 effect on IGly persisted in the presence of the calcium chelator BAPTA, the protein kinase inhibitor staurosporine, the classical ER (i.e. ERalpha and ERbeta) antagonist tamoxifen, or the G-protein modulators, favoring a direct action of E2 on GlyRs. In HEK293 cells expressing various combinations of GlyR subunits, E2 only affected the IGly in cells expressing alpha2, alpha2beta or alpha3beta subunits, suggesting that either alpha2-containing or alpha3beta-GlyRs mediate the E2 effect observed in neurons. Furthermore, E2 inhibited the GlyR-mediated tonic current in pyramidal neurons of HIP CA1 region, where abundant GlyR alpha2 subunit is expressed. We suggest that the neuronal GlyR is a novel molecular target of E2 which directly inhibits the function of GlyRs in the HIP and SDH regions. This finding may shed new light on premenstrual dysphoric disorder and the gender differences in pain sensation at the CNS level.

Killer-cell immunoglobulin-like receptor (KIR) genes are a polymorphic family expressed on NK cells, and "senescent" CD28- T cells implicated in cardiovascular disease. KIR promoters are highly homologous, and NK expression is regulated by DNA methylation. T cell KIR regulation is poorly understood. We asked if epigenetic mechanisms and/or transcription factor alterations determine T cell KIR expression. DNA methylation inhibition activated multiple KIR genes in normal T cells. KIR2DL2 and KIR2DL4 were selected for further study. Expression of both was associated with promoter demethylation, and methylation of the promoters in reporter constructs suppressed expression. KIR reporter construct expression also increased in demethylated T cells and required Ets1, Sp1 and AML sites, implying effects on transcription factors. This was confirmed for Sp1. These results indicate that KIR genes are suppressed by DNA methylation in most T cells, and DNA demethylation promotes their expression through effects on both chromatin structure and transcription factors.

We have generated gene expression databases for human glial precursors, neuronal precursors, astrocyte precursors and neural stem cells and focused on comparing the profile of glial precursors with that of other populations.

HIV-1 Tat protein is an important pathogenic factor in HIV-1-associated neurological diseases. One hallmark of HIV-1 infection of the central nervous system (CNS) is astrocytosis, which is characterized by elevated glial fibrillary acidic protein (GFAP) expression in astrocytes. We have shown that Tat activates GFAP expression in astrocytes [Zhou et al., (2004) Mol Cell Neurosci 27:296-305] and that GFAP is an important regulator of Tat neurotoxicity [Zou et al., (2007) Am J Pathol 171:1293-1935]. However, the underlying mechanisms for Tat-mediated GFAP up-regulation are not understood. In this study, we reported concurrent up-regulation of adenovirus E1a-associated 300 kDa protein p300 and GFAP in Tat-expressing human astrocytoma cells and primary astrocytes. We showed that p300 was indeed induced by Tat expression and HIV-1 infection and that the induction occurred at the transcriptional level through the cis-acting elements of early growth response 1 (egr-1) within its promoter. Using siRNA, we further showed that p300 regulated both constitutive and Tat-mediated GFAP expression. Moreover, we showed that ectopic expression of p300 potentiated Tat transactivation activity and increased proliferation of HIV-1-infected astrocytes, but had little effect on HIV-1 replication in these cells. Taken together, these results demonstrate for the first time that Tat is a positive regulator of p300 expression, which in turn regulates GFAP expression, and suggest that the Tat-Egr-1-p300-GFAP axis likely contributes to Tat neurotoxicity and predisposes astrocytes to be an HIV-1 sanctuary in the CNS.

Multiple myeloma (MM) is a disease of cell cycle dysregulation while cell cycle modulation can be a target for MM therapy. In this study we investigated the effects and mechanisms of action of a sesquiterpene lactone 6-O-angeloylplenolin (6-OAP) on MM cells.

Myeloid-derived suppressor cells (MDSCs) have been shown to inhibit T-cell responses in many diseases, but, in hepatitis C virus (HCV) infected patients, MDSCs are still poorly studied. In this assay, we investigated the phenotype and frequency of two new populations of MDSCs denoted as monocytic and granulocytic MDSCs (M-MDSCs and G-MDSCs) in HCV infected patients and analyzed their clinical significance in these patients respectively. We found that the frequency of CD14(+)HLA-DR(-/low) cells (M-MDSCs) from HCV infected patients (mean ± SE, 3.134% ± 0.340%) was significantly increased when compared to healthy controls (mean ± SE, 1.764% ± 0.461%) (Z = -2.438, P = 0.015), while there was no statistical difference between the frequency of HLA-DR(-/low)CD33(+)CD11b(+)CD15(+) (G-MDSCs) of HCV infected patients and healthy donors (0.201% ± 0.038% versus 0.096% ± 0.026%, P > 0.05), which suggested that HCV infection could cause the proliferation of M-MDSCs instead of G-MDSCs. Besides, we found that the frequency of M-MDSCs in HCV infected patients had certain relevance with age (r = 0.358, P = 0.003); patients older than 40 years old group (mean ± SE, 3.673% ± 0.456%) had a significantly higher frequency of M-MDSCs than that of age less than 40 years old group (mean ± SE, 2.363% ± 0.482%) (Z = -2.685, P = 0.007). The frequency of M-MDSCs, however, had no correlation with HCV RNA loads, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and the level of liver inflammation degree.

Peripheral nerve injury has been shown to result in ectopic spontaneous discharges on soma and injured sites of sensory neurons, thereby inducing neuropathic pain. With the increase of membrane proteins on soma and injured site neurons, the negatively charged sialic acids bind to the external domains of membrane proteins, resulting in an increase of this charge. We therefore speculate that the electrophoretic velocity of injured neurons may be faster than non-injured neurons. The present study established rat models of neuropathic pain via chronic constriction injury. Results of the cell electrophoresis test revealed that the electrophoretic velocity of injured neuronal cells was faster than that of non-injured (control) cells. We then treated cells with divalent cations of Ca(2+) and organic compounds with positive charges, polylysine to counteract the negatively charged sialic acids, or neuraminidase to specifically remove sialic acids from the membrane surface of injured neurons. All three treatments significantly reduced the electrophoretic velocity of injured neuronal cells. These findings suggest that enhanced sialic acids on injured neurons may accelerate the electrophoretic velocity of injured neurons.

Zinc plays an essential role in the regulation of pancreatic β cell function, affecting important processes including insulin biosynthesis, glucose-stimulated insulin secretion, and cell viability. Mutations in the zinc efflux transport protein ZnT8 have been linked with both type 1 and type 2 diabetes, further supporting an important role for zinc in glucose homeostasis. However, very little is known about how cytosolic zinc is controlled by zinc influx transporters (ZIPs). In this study, we examined the β cell and islet ZIP transcriptome and show consistent high expression of ZIP6 (Slc39a6) and ZIP7 (Slc39a7) genes across human and mouse islets and MIN6 β cells. Modulation of ZIP6 and ZIP7 expression significantly altered cytosolic zinc influx in pancreatic β cells, indicating an important role for ZIP6 and ZIP7 in regulating cellular zinc homeostasis. Functionally, this dysregulated cytosolic zinc homeostasis led to impaired insulin secretion. In parallel studies, we identified both ZIP6 and ZIP7 as potential interacting proteins with GLP-1R by a membrane yeast two-hybrid assay. Knock-down of ZIP6 but not ZIP7 in MIN6 β cells impaired the protective effects of GLP-1 on fatty acid-induced cell apoptosis, possibly via reduced activation of the p-ERK pathway. Therefore, our data suggest that ZIP6 and ZIP7 function as two important zinc influx transporters to regulate cytosolic zinc concentrations and insulin secretion in β cells. In particular, ZIP6 is also capable of directly interacting with GLP-1R to facilitate the protective effect of GLP-1 on β cell survival.

Novelty seeking (NS) is a personality trait reflecting excitement in response to novel stimuli. High NS is usually a predictor of risky behaviour such as drug abuse. However, the relationships between NS and risk-related cognitive processes, including individual risk preference and the brain activation associated with risk prediction, remain elusive. In this fMRI study, participants completed the Tridimensional Personality Questionnaire to measure NS and performed a probabilistic decision making task. Using a mathematical model, we estimated individual risk preference. Brain regions associated with risk prediction were determined via fMRI. The NS score showed a positive correlation with risk preference and a negative correlation with the activation elicited by risk prediction in the right posterior insula (r-PI), left anterior insula (l-AI), right striatum (r-striatum) and supplementary motor area (SMA). Within these brain regions, only the activation associated with risk prediction in the r-PI showed a correlation with NS after controlling for the effect of risk preference. Resting-state functional connectivity between the r-PI and r-striatum/l-AI was negatively correlated with NS. Our results suggest that high NS may be associated with less aversion to risk and that the r-PI plays an important role in relating risk prediction to NS.

Type 2 diabetes mellitus (T2DM) is regarded as one of the serious risk factors for age-related cognitive impairment; however, a causal link between these two diseases has so far not been established. It was recently discovered that, apart from high D-glucose levels, T2DM patients also display abnormally high concentrations of uric D-ribose. Here, we show for the first time that the administration of D-ribose, the most active glycator among monosaccharides, produces high levels of advanced glycation end products (AGEs) and, importantly, triggers hyperphosphorylation of Tau in the brain of C57BL/6 mouse and neuroblastoma N2a cells. However, the administration of D-glucose showed no significant changes in Tau phosphorylation under the same experimental conditions. Crucially, suppression of AGE formation using an AGEs inhibitor (aminoguanidine) effectively prevents hyperphosphorylation of Tau protein. Further study shows AGEs resulted from ribosylation activate calcium-/calmodulin-dependent protein kinase type II (CaMKII), a key kinase responsible for Tau hyperphosphorylation. These data suggest that there is indeed a mechanistic link between ribosylation and Tau hyperphosphorylation. Targeting ribosylation by inhibiting AGE formation may be a promising therapeutic strategy to prevent Alzheimer's disease-like Tau hyperphosphorylation and diabetic encephalopathies.