To investigate the effect of valproate treatment on the K562 cell line, a model for chronic myelogenous leukaemia, the growth and survival of the K562 cell line were investigated using the Annexin-V/PI dual staining method, and global profiles of gene expression and alternative splicing in K562 cells were assessed using exon microarrays. A significant increase in cell apoptosis was observed in valproate-exposed K562 cells using flow cytometry. A total of 628 transcripts were identified as being significantly differentially expressed. The number of genes demonstrating increased expression levels was greater than the number of genes demonstrating decreased expression levels (445 genes vs. 183 genes, respectively). The significant enrichment analysis of GO terms for the differentially expressed genes revealed that these genes are involved in many important biological processes such as apoptosis. Six of the genes observed to be differentially expressed that might be involved in apoptosis were selected to undergo qRT-PCR validation. In total, 198 candidates of alternative splicing variants were identified. Among them, three alternative splicing events were selected for validation, and CBLC and TBX1 were confirmed to be alternatively spliced by semi-nested PCR. In conclusion, valproate exposure facilitated cell apoptosis, altered mRNA expression and alternative splicing events in the K562 cell line.

Interleukin 7 (IL-7) promotes pre-B cell survival and proliferation by activating the Pim1 and Akt kinases. These signals must be attenuated to induce G1 cell cycle arrest and expression of the RAG endonuclease, which are both required for IgL chain gene rearrangement. As lost IL-7 signals would limit pre-B cell survival, how cells survive during IgL chain gene rearrangement remains unclear. We show that RAG-induced DNA double-strand breaks (DSBs) generated during IgL chain gene assembly paradoxically promote pre-B cell survival. This occurs through the ATM-dependent induction of Pim2 kinase expression. Similar to Pim1, Pim2 phosphorylates BAD, which antagonizes the pro-apoptotic function of BAX. However, unlike IL-7 induction of Pim1, RAG DSB-mediated induction of Pim2 does not drive proliferation. Rather, Pim2 has antiproliferative functions that prevent the transit of pre-B cells harboring RAG DSBs from G1 into S phase, where these DNA breaks could be aberrantly repaired. Thus, signals from IL-7 and RAG DSBs activate distinct Pim kinase family members that have context-dependent activities in regulating pre-B cell proliferation and survival.

Endophytic fungi from the Chinese medicinal plant Actinidia macrosperma were isolated and identified for the first time. This was the first study to evaluate their cytotoxic and antitumour activities against brine shrimp and five types of tumour cells, respectively. In total, 17 fungal isolates were obtained. Five different taxa were represented by 11 isolates, and six isolates were grouped into the species of Ascomycete Incertae sedis with limited morphological and molecular data. Cytotoxic activity has been found in most isolates except AM05, AM06, and AM10. The isolates AM07 (4.86 μg/mL), AM11 (7.71 μg/mL), and AM17 (14.88 μg/mL) exhibited significant toxicity against brine shrimp. The results of the MTT assay to assess antitumour activity revealed that 82.4% of isolate fermentation broths displayed growth inhibition (50% inhibitory concentration IC(50)< 100 μg/mL). Moreover, AM07, AM11, and AM17 showed strong antitumour activity in all the cell lines examined. These results suggest that endophytic fungi in A. macrosperma are valuable for the isolation and identification of novel cytotoxic and antitumour bioactive agents.

This study was conducted to determine if oral administration of the novel herbal medicine, MA, and its Lactobacillus acidophilus fermented product, MA128, have therapeutic properties for the treatment of asthma. Asthma was induced in BALB/c mice by systemic sensitization to ovalbumin (OVA) followed by intratracheal, intraperitoneal, and aerosol allergen challenges. MA and MA128 were orally administered 6 times a week for 4 weeks. At 1 day after the last ovalbumin exposure, airway hyperresponsiveness was assessed and samples of bronchoalveolar lavage fluid, lung cells, and serum were collected for further analysis. We investigated the effect of MA and MA128 on airway hyperresponsiveness, pulmonary eosinophilic infiltration, various immune cell phenotypes, Th2 cytokine production, OVA-specific IgE production, and Th1/Th2 cytokine production in this mouse model of asthma. In BALB/c mice, we found that MA and MA128 treatment suppressed eosinophil infiltration into airways and blood, allergic airway inflammation and AHR by suppressing the production of IL-5, IL-13, IL-17, Eotaxin, and OVA-specific IgE, by upregulating the production of OVA-specific Th1 cytokine (IFN-γ), and by downregulating OVA-specific Th2 cytokine (IL-4) in the culture supernatant of spleen cells. The effectiveness of MA was increased by fermentation with Lactobacillus acidophilus.

The aim of the present investigation was to elucidate the cellular mechanisms whereby Tanshinone IIA (Tan IIA) leads to cell cycle arrest and apoptosis in vitro in keratinocytes, the target cells in psoriasis. Tan IIA inhibited proliferation of mouse keratinocytes in a dose- and time-dependent manner and induced apoptosis, resulting in S phase arrest accompanied by down-regulation of pCdk2 and cyclin A protein expression. Furthermore, Tan IIA-induced apoptosis and mitochondrial membrane potential changes were also further demonstrated by DNA fragmentation, single-cell gel electrophoresis assay (SCGE), and flow cytometry methods. Apoptosis was partially blocked by the caspase-3 inhibitor Ac-DEVD-CHO. Mitochondrial regulation of apoptosis further downstream was investigated, showing changes in the mitochondrial membrane potential, cytochrome c release into the cytoplasm, and enhanced activation of cleaved caspase-3 and Poly ADP-ribose polymerase (PARP). There was also no translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus in apoptotic keratinocytes, indicating Tan IIA-induced apoptosis occurs mainly through the caspase pathway. Our findings provide the molecular mechanisms by which Tan IIA can be used to treat psoriasis and support the traditional use of Salvia miltiorrhiza Bungee (Labiatae) for psoriasis and related skin diseases.

Background. Despite controversy, bipolar disorder (BD) is being increasingly diagnosed in under 18s. There is scant information regarding its treatment and uncertainty regarding the status of "severe mood dysregulation (SMD)" and how it overlaps with BD. This article collates available research on treatment of BD in under 18s and explores the status of SMD. Methods. Literature on treatment of BD in under 18s and on SMD were identified using major search engines; these were then collated and reviewed. Results. Some markers have been proposed to differentiate BD from disruptive behaviour disorders (DBD) in children. Pharmacotherapy restricted to short-term trials of mood-stabilizers and atypical-antipsychotics show mixed results. Data on maintenance treatment and non-pharmacological interventions are scant. It is unclear whether SMD is an independent disorder or an early manifestation of another disorder. Conclusions. Valproate, lithium, risperidone, olanzapine, aripiprazole and quetiapine remain first line treatments for acute episodes in the under 18s with BD. Their efficacy in maintenance treatment remains unclear. There is no validated treatment for SMD. It is likely that some children who are currently diagnosed with BD and DBD and possibly most children currently diagnosed with SMD will be subsumed under the proposed category in the DSM V of disruptive mood dysregulation disorder with dysphoria.

Assembly and disassembly of adhesion structures such as focal adhesions (FAs) and podosomes regulate cell adhesion and differentiation. On antigen-presenting dendritic cells (DCs), acquisition of a migratory and immunostimulatory phenotype depends on podosome dissolution by prostaglandin E(2) (PGE(2)). Whereas the effects of physico-chemical and topographical cues have been extensively studied on FAs, little is known about how podosomes respond to these signals. Here, we show that, unlike for FAs, podosome formation is not controlled by substrate physico-chemical properties. We demonstrate that cell adhesion is the only prerequisite for podosome formation and that substrate availability dictates podosome density. Interestingly, we show that DCs sense 3-dimensional (3-D) geometry by aligning podosomes along the edges of 3-D micropatterned surfaces. Finally, whereas on a 2-dimensional (2-D) surface PGE(2) causes a rapid increase in activated RhoA levels leading to fast podosome dissolution, 3-D geometric cues prevent PGE(2)-mediated RhoA activation resulting in impaired podosome dissolution even after prolonged stimulation. Our findings indicate that 2-D and 3-D geometric cues control the spatial organization of podosomes. More importantly, our studies demonstrate the importance of substrate dimensionality in regulating podosome dissolution and suggest that substrate dimensionality plays an important role in controlling DC activation, a key process in initiating immune responses.

Monkeypox virus (MPXV) is comprised of two clades: Congo Basin MPXV, with an associated case fatality rate of 10%, and Western African MPXV, which is associated with less severe infection and minimal lethality. We thus postulated that Congo Basin and West African MPXV would differentially modulate host cell responses and, as many host responses are regulated through phosphorylation independent of transcription or translation, we employed systems kinomics with peptide arrays to investigate these functional host responses. Using this approach we have demonstrated that Congo Basin MPXV infection selectively down-regulates host responses as compared with West African MPXV, including growth factor- and apoptosis-related responses. These results were confirmed using fluorescence-activated cell sorting analysis demonstrating that West African MPXV infection resulted in a significant increase in apoptosis in human monocytes as compared with Congo Basin MPXV. Further, differentially phosphorylated kinases were identified through comparison of our MPXV data sets and validated as potential targets for pharmacological inhibition of Congo Basin MPXV infection, including increased Akt S473 phosphorylation and decreased p53 S15 phosphorylation. Inhibition of Akt S473 phosphorylation resulted in a significant decrease in Congo Basin MPXV virus yield (261-fold) but did not affect West African MPXV. In addition, treatment with staurosporine, an apoptosis activator resulted in a 49-fold greater decrease in Congo Basin MPXV yields as compared with West African MPXV. Thus, using a systems kinomics approach, our investigation demonstrates that West African and Congo Basin MPXV differentially modulate host cell responses and has identified potential host targets of therapeutic interest.

Members of the Nogo66 receptor family (NgR) are closely associated with nerve growth inhibition and plasticity in the CNS. All three members, NgR1, NgR2 and NgR3, are GPI anchored and highly glycosylated proteins. The binding and signaling properties of NgR1 are well described, but largely unknown for NgR2. At present the only known ligands are myelin associated glycoprotein (MAG) and amyloid beta precursor protein (APP). Despite the requirement of co-receptors for signaling no other binding partner has been uncovered. To learn more about the interactome of NgR2 we performed pull down experiments and were able to identify F-box protein that recognizes sugar chain 1 (Fbs1) as binding partner. We confirmed this finding with co-immunoprecipitations and in vitro binding assays and showed that the binding is mediated by the substrate recognition domain of Fbs1. As a substrate recognition protein of the SCF complex, Fbs1 binding leads to polyubiquitination and finally degradation of its substrates. This is the first time a member of the Nogo receptor family has been connected with an intracellular degradation pathway, which has not only implications for its production, but also for amyloid deposition in Alzheimer's disease.

Many Gram-negative bacteria use N-acyl-homoserine lactones (AHLs) as quorum sensing (QS) signaling molecules to coordinate their group behavior. Recently, it was shown that plants can perceive and respond to these bacterial AHLs. However, little is known about the molecular mechanism underlying the response of plants to bacterial QS signals. In this study, we show that the promotion of root elongation in wild type Arabidopsis thaliana induced by the AHLs N-3-oxo-hexanoyl-homoserine lactone (3OC6-HSL) or N-3-oxo-octanoyl-homoserine lactone (3OC8-HSL) was completely abolished in plants with loss-of-function mutations in two candidate G-protein Coupled Receptors (GPCRs), Cand2 and Cand7. Furthermore, real-time PCR analysis revealed that the expression levels of Cand2 and Cand7 were elevated in plants treated with 3OC6-HSL or 3OC8-HSL. These results suggest that Cand2 and Cand7 are involved in the regulation of root growth by bacterial AHLs and that GPCRs play a role in mediating interactions between plants and microbes.

Dynamic cell interaction with ECM components has profound influence in cancer progression. SPARC is a component of the ECM, impairs the proliferation of different cell types and modulates tumor cell aggressive features. We previously reported that SPARC expression significantly impairs medulloblastoma tumor growth in vivo. In this study, we demonstrate that expression of SPARC inhibits medulloblastoma cell proliferation. MTT assay indicated a dose-dependent reduction in tumor cell proliferation in adenoviral mediated expression of SPARC full length cDNA (Ad-DsRed-SP) in D425 and UW228 cells. Flow cytometric analysis showed that Ad-DsRed-SP-infected cells accumulate in the G2/M phase of cell cycle. Further, immunoblot and immunoprecipitation analyses revealed that SPARC induced G2/M cell cycle arrest was mediated through inhibition of the Cyclin-B-regulated signaling pathway involving p21 and Cdc2 expression. Additionally, expression of SPARC decreased STAT3 phosphorylation at Tyr-705; constitutively active STAT3 expression reversed SPARC induced G2/M arrest. Ad-DsRed-SP significantly inhibited the pre-established orthotopic tumor growth and tumor volume in nude-mice. Immunohistochemical analysis of tumor sections from mice treated with Ad-DsRed-SP showed decreased immunoreactivity for pSTAT3 and increased immunoreactivity for p21 compared to tumor section from mice treated with mock and Ad-DsRed. Taken together our studies further reveal that STAT3 plays a key role in SPARC induced G2/M arrest in medulloblastoma cells. These new findings provide a molecular basis for the mechanistic understanding of the effects of SPARC on medulloblastoma tumor cell proliferation.

Molecular mechanisms by which fertilization competent acrosome-reacted sperm bind to the oolemma remain uncharacterized. To identify oolemmal binding partner(s) for sperm acrosomal ligands, affinity panning was performed with mouse oocyte lysates using sperm acrosomal protein, SLLP1 as a target. An oocyte specific membrane metalloproteinase, SAS1B (Sperm Acrosomal SLLP1 Binding), was identified as a SLLP1 binding partner. cDNA cloning revealed six SAS1B splice variants, each containing a zinc binding active site and a putative transmembrane domain, with signal peptides in three variants. SAS1B transcripts were ovary specific. SAS1B protein was first detected in early secondary follicles in day 3 ovaries. Immunofluorescence localized SAS1B to the microvillar oolemma of M2 oocytes. After fertilization, SAS1B decreased on the oolemma and became virtually undetectable in blastocysts. In transfected CHO-K1 cells SAS1B localized to the surface of unpermeabilized cells. Recombinant and native SLLP1 co-localized with SAS1B to the microvillar domain of ovulated M2 oocytes. Molecular interactions between mouse SLLP1 and SAS1B were demonstrated by surface plasmon resonance, far-western, yeast two-hybrid, recombinant- and native-co-IP analyses. SAS1B bound to SLLP1 with high affinity. SAS1B had protease activity, and SAS1B protein or antibody significantly inhibited fertilization. SAS1B knockout female mice showed a 34% reduction in fertility. The study identified SAS1B-SLLP1 as a pair of novel sperm-egg binding partners involving the oolemma and intra-acrosomal compartment during fertilization.

Animal models and behavioral paradigms are critical for elucidating the neural mechanism involved in complex behaviors, including social cognition. Both genotype and phenotype based models have implicated the neuropeptide oxytocin (OT) in the regulation of social behavior. Based on the findings in animal models, alteration of the OT system has been hypothesized to play a role in the social deficits associated with autism and other neuropsychiatric disorders. While the evidence linking the peptide to the etiology of the disorder is not yet conclusive, evidence from multiple animal models suggest modulation of the OT system may be a viable strategy for the pharmacological treatment of social deficits. In this review, we will discuss how animal models have been utilized to understand the role of OT in social cognition and how those findings can be applied to the conceptualization and treatment of the social impairments in ASD. Animal models with genetic alterations of the OT system, like the OT, OT receptor and CD38 knock-out mice, and those with phenotypic variation in social behavior, like BTBR inbred mice and prairie voles, coupled with behavioral paradigms with face and construct validity may prove to have predictive validity for identifying the most efficacious methods of stimulating the OT system to enhance social cognition in humans. The widespread use of strong animal models of social cognition has the potential yield pharmacological, interventions for the treatment social impairments psychiatric disorders. This article is part of a Special Issue entitled Oxytocin, Vasopressin, and Social Behavior.

Dysfunction in central glucocorticoid signaling is implicated in hypothalamic-pituitary-adrenocortical (HPA) axis dysregulation and major depression. In comparison with men, women are twice as likely to suffer from depression and have heightened HPA axis responses to stress. We hypothesized that this striking increase in stress vulnerability in females may be because of sex differences in central glucocorticoid signaling. The current study tests the role of the forebrain type II glucocorticoid receptor (GR) on HPA axis function in female mice and depression-like behavior in both female and male mice. This was accomplished by using mice with selective deletion of GR in forebrain cortico-limbic sites including the prefrontal cortex, hippocampus, and basolateral amygdala (forebrain glucocorticoid receptor knockout mouse (FBGRKO)). In order to examine HPA axis function in female FBGRKO, we measured nadir, peak circadian and restraint-induced corticosterone concentrations in female FBGRKO. The data indicate that unlike male FBGRKO, basal and stress-induced corticosterone concentrations are not increased in female FBGRKO. Given the pronounced effect of central glucocorticoid signaling on mood, we also examined the necessity of corticolimbic GR on depression-like behavior with the sucrose preference and forced swim tests (FST) in male and female FBGRKO mice. Consistent with previous studies, male FBGRKO displayed increased depression-like behavior as indicated by greater immobility in the FST and decreased sucrose preference compared with littermate controls, effects that were not observed in females. Overall the findings indicate a marked sex difference in the function of forebrain GR on HPA axis regulation and depression-like behaviors, and may have implications for therapeutic approaches using GR-modulating drugs.

The anorexigenic gastrointestinal hormone Peptide YY plays an important role in the communication between the gastrointestinal tract and the central nervous system. PYY has been shown to modulate brain activity in regions implicated in reward and food related behavior. Its effects on brain structure however, remain unknown. Voxel-based morphometry was used to investigate the relationship between fasting and postprandial plasma PYY concentrations and regional gray matter volume (GMV). For this analysis twenty adult, non diabetic Caucasians were included (18F/2M, age 31±9 y, percentage of body fat [PFAT] 32±8%) who had volumetric brain magnetic resonance images and underwent H(2)(15)O positron emission tomographic (PET) measurements of regional cerebral blood flow (rCBF), a marker of local neuronal activity, and measurements of plasma total PYY, prior to (fasting) and following a satiating liquid meal. Voxel-wise analysis revealed a regional positive association between postprandial PYY and gray matter volume bilaterally in the caudate nuclei. These associations remained significant (p<0.05) after small volume correction for multiple comparisons. Based on these findings we investigated whether postprandial PYY is associated with PET measured rCBF of the caudate nucleus. We found a significant negative association between average postprandial caudate rCBF and postprandial plasma PYY concentrations (r=-0.60, p<0.02, age, sex and PFAT adjusted). Average postprandial caudate rCBF was also negatively associated with rCBF in the right medial orbitofrontal cortex and the right hippocampal formation (p<0.05, corrected for multiple comparisons). Total PYY is positively associated with gray matter but negatively with postprandial activity in the caudate nuclei while caudate activity is negatively associated with rCBF in prefrontal and paralimbic regions implicated in reward behavior. Thus, PYY may act centrally to modulate eating behavior via striatal networks.

Homeobox proteins are critical regulators of developmental gene transcription and cell specification. Many insights into transcriptional regulation have been gained from studies in the nematode Caenorhabditis elegans. We investigated the expression and regulation of the C. elegans homeobox gene ceh-63, which encodes a single-homeodomain transcription factor of 152 amino acids. ceh-63 is expressed in the interneuron DVC in both sexes, from late embryogenesis through adulthood, and two pairs of uterine cells in reproductive hermaphrodites only. A reporter gene fusion, encoding GFP fused to the full-length CEH-63, also drove weak inconsistent expression in additional unidentified cells in the head and tail. A potential ceh-63 null mutant had no obvious abnormalities, except for a possible increase in subtle defects of the DVC axon projection. No behavioural responses were observed upon either laser ablation of DVC or activation of DVC through light stimulation of channelrhodopsin-2 specifically expressed in this neuron. The function of DVC therefore remains enigmatic. A transcriptional regulatory cascade operating in DVC was defined from the LIM-homeodomain protein CEH-14 through CEH-63 to the helix-turn-helix transcription factor MBR-1. Both CEH-14 and CEH-63 individually bound the mbr-1 promoter in a yeast one-hybrid assay. A model is proposed suggesting that CEH-14 activates ceh-63 and then along with CEH-63 co-ordinately activates mbr-1.

Growth hormone 1 (GH1), a pituitary hormone, plays a key role in the regulation of growth. Both excess GH1 treatment and overexpression of a GH1 transgene promote growth of salmon, but these animals exhibit physiological abnormalities in viability, fertility and metabolism, which might be related to pituitary function. However, the molecular dynamics induced in the pituitary by excess GH1 remain unknown. In this study, we performed iTRAQ proteome analysis of the amago salmon pituitary, with and without excess GH1 treatment, and found that the expression levels of proteins related to endocrine systems, metabolism, cell growth and proliferation were altered in the GH1-treated pituitary. Specifically, pituitary hormone prolactin (2.29 fold), and somatolactin α (0.14 fold) changed significantly. This result was confirmed by proteome and transcriptome analyses of pituitary from the GH1-transgenic (GH1-Tg) amago salmon. The dynamics of protein and gene expression in the pituitary of GH1-Tg amago salmon were similar to those of pituitary treated with excess GH1. Our findings suggest that not only excess GH1 hormone, but also the quantitative changes in other pituitary hormones, might be essential for the abnormal growth of amago salmon. These data will be useful in future attempts to increase the productivity of fish farming.

Cannabinoid CB1 receptors (CB1Rs) are known to be downregulated in patients and in animal models of Huntington's disease (HD). However, the functional meaning of this reduction, if any, is still unclear. Here, the effects of the cannabinoid receptor agonist WIN 55,212-2 (WIN) were investigated on striatal synaptic transmission and on glutamate and GABA release in symptomatic R6/2 mice, a genetic model of HD. The expression levels of CB1Rs in glutamatergic and GABAergic synapses were also evaluated. We found that in R6/2 mice, WIN effects on synaptic transmission and glutamate release were significantly increased with respect to wild type mice. On the contrary, a decrease in WIN-induced reduction of GABA release was found in R6/2 versus WT mice. The expression of CB1Rs in GABAergic neurons was drastically reduced, while CB1Rs levels in glutamatergic neurons were unchanged. These results demonstrate that the expression and functionality of CB1Rs are differentially affected in GABAergic and glutamatergic neurons in R6/2 mice. As a result, the balance between CB1Rs expressed by the two neuronal populations and, thus, the net effect of CB1R stimulation, is profoundly altered in HD mice.

Wan-Sheng-Hua-Feng-Dan (WSHFD) is a traditional Chinese medicine used for the treatment of neurological disorders. Cinnabar (HgS) and realgar (As(4)S(4)) are included in WSHFD. Are they remedies or poisons?

p97/VCP is a hexameric ATPase that is coupled to diverse cellular processes, such as membrane fusion and proteolysis. How p97 activity is regulated is not fully understood. Here we studied the potential role of TUG, a widely expressed protein containing a UBX domain, to control mammalian p97. In HEK293 cells, the vast majority of TUG was bound to p97. Surprisingly, the TUG UBX domain was neither necessary nor sufficient for this interaction. Rather, an extended sequence, comprising three regions of TUG, bound to the p97 N-terminal domain. The TUG C terminus resembled the Arabidopsis protein PUX1. Similar to the previously described action of PUX1 on AtCDC48, TUG caused the conversion of p97 hexamers into monomers. Hexamer disassembly was stoichiometric rather than catalytic and was not greatly affected by the p97 ATP-binding state or by TUG N-terminal regions in vitro. In HeLa cells, TUG localized to the endoplasmic reticulum-to-Golgi intermediate compartment and endoplasmic reticulum exit sites. Although siRNA-mediated TUG depletion had no marked effect on total ubiquitylated proteins or p97 localization, TUG overexpression caused an accumulation of ubiquitylated substrates and targeted both TUG and p97 to the nucleus. A physiologic role of TUG was revealed by siRNA-mediated depletion, which showed that TUG is required for efficient reassembly of the Golgi complex after brefeldin A removal. Together, these data support a model in which TUG controls p97 oligomeric status at a particular location in the early secretory pathway and in which this process regulates membrane trafficking in various cell types.