The aim of this study was to explore the effects of 22(S)-hydroxycholesterol (22(S)-HC) on lipid and glucose metabolism in human-derived cells from metabolic active tissues. Docking of T0901317 and 22(S)-HC showed that both substances fitted into the ligand binding domain of liver X receptors (LXR). Results show that while several lipogenic genes were induced by T0901317 in myotubes, HepG2 cells and SGBS cells, effect of 22(S)-HC varied more between cell types. In myotubes, most lipogenic genes were downregulated or unchanged by 22(S)-HC, whereas a more diverse pattern was found in HepG2 and SGBS cells. Treatment with 22(S)-HC induced sterol regulatory element binding transcription factor 1 in SGBS and HepG2 cells, but not in myotubes. Fatty acid synthase was downregulated by 22(S)-HC in myotubes, upregulated in SGBS and unchanged in HepG2 cells. De novo lipogenesis was increased by T0901317 in all cell models, whereas differently affected by 22(S)-HC depending on the cell type; decreased in myotubes and HepG2 cells, whereas increased in SGBS cells. Oxidation of linoleic acid was reduced by 22(S)-HC in all cell models while glucose uptake increased and tended to increase in myotubes and SGBS cells, respectively. Cholesterol efflux was unaffected by 22(S)-HC treatment. These results show that 22(S)-HC affects LXR-regulated processes differently in various cell types. Ability of 22(S)-HC to reduce lipogenesis and lipid accumulation in myotubes and hepatocytes indicate that 22(S)-HC might reduce lipid accumulation in non-adipose tissues, suggesting a potential role for 22(S)-HC or a similar LXR modulator in the treatment of type 2 diabetes.

Parkinson's disease (PD) is characterized by accumulation of α-synuclein aggregates and degeneration of melanized, catecholaminergic neurons. The tissue transglutaminase (tTG) enzyme catalyzes molecular protein cross-linking. In PD, tTG levels are increased and cross-linking has been identified as an important factor in α-synuclein aggregation. In our quest to link tTGs distribution in the human brain to the hallmarks of PD pathology, we recently reported that catecholaminergic neurons in PD disease-affected brain areas display typical endoplasmic reticulum (ER) granules showing tTG immunoreactivity. In the present study, we set out to elucidate the nature of the interaction between tTG and the ER in PD pathogenesis, using retinoic-acid differentiated SH-SY5Y cells exposed to the PD-mimetic 1-methyl-4-phenylpyridinium (MPP(+)). Alike our observations in PD brain, MPP(+)-treated cells displayed typical TG-positive granules, that were also induced by other PD mimetics and by ER-stress inducing toxins. Additional immunocytochemical and biochemical investigation revealed that tTG is indeed associated to the ER, in particular at the cytoplasmic face of the ER. Upon MPP(+) exposure, additional recruitment of tTG toward the ER was found. In addition, we observed that MPP(+)-induced tTG activity results in transamidation of ER membrane proteins, like calnexin. Our data provide strong evidence for a, so far unrecognized, localization of tTG at the ER, at least in catecholaminergic neurons, and suggests that in PD activation of tTG may have a direct impact on ER function, in particular via post-translational modification of ER membrane proteins.

The extremely radioresistant bacterium Deinococcus radiodurans possesses a rapid and efficient but poorly known DNA damage response mechanism that mobilizes one-third of its genome to survive lethal radiation damage. Deinococcal PprI serves as a general switch to regulate the expression of dozens of proteins from different pathways after radiation, including the DNA repair proteins RecA, PprA and SSB. However, the underlying mechanism is poorly understood. In this study, we analyzed the dynamic alteration in global transcriptional profiles in wildtype and pprI mutant strains by combining microarrays and time-course sampling. We found that PprI up-regulated transcription of at least 210 genes after radiation, including 21 DNA repair and replication-related genes. We purified PprI and a helix-turn-helix (HTH) domain mutant and found that PprI specifically bound to the promoters of recA and pprA in vitro but did not bind nonspecific double-strand DNA. Chromatin immunoprecipitation (ChIP) assays confirmed that PprI specifically interacted with the promoter DNA of recA and pprA after radiation. Finally, we showed that a DNA-binding activity-deficient pprI mutant only partially restored resistance of the pprI mutant strain to γ radiation, UV radiation, and mitomycin C. Taken together, these results indicate that DNA-binding activity is essential for PprI to program the DNA repair process and cellular survival of D. radiodurans in response to radiation damage.

Ultraviolet-B (UV-B) radiation affects the genome stability of aquatic organisms by absorption of certain wavelength at the molecular level. Recently, extensive gene information has been identified from the intertidal copepod, Tigriopus japonicus. Here, we developed a 55K (54,254 genes) oligomicroarray and tested its usefulness to identify the effect of single dose of UV-B irradiation (12 kJ/m(2)) on transcriptomes of the copepod T. japonicus. A total of 35,361 spots were identified to be significantly modulated on the 55K oligomicroarray by hierarchical clustering after exposure to UV-B irradiation over 48 h (6, 12, 24, and 48 h). Of them, 1300 and 588 genes were observed to be up-regulated and down-regulated at all time points, respectively. Particularly, it was observed that several genes involved in DNA repair mechanism were significantly modulated in the UV-B-exposed T. japonicus by microarray and quantitative real-time RT-PCR analysis. In detail, UV-B irradiation specifically up-regulated some genes in non-homologous end-joining (NHEJ), homologous recombination (HR), base excision repair (BER), and mismatch repair (MMR) pathways. On the other hand, a majority of down-regulated genes were representatives for the nucleotide excision repair (NER) mechanism. These results demonstrated that DNA damage would be induced by UV-B irradiation in this species, resulting in reliable induction or repression of various DNA repair mechanism on UV-B-induced DNA damage. In this report, we suggest that a high density microarray-based approach for risk assessment of UV-B irradiation would be useful to elucidate the mechanistic analysis in a non-model organism. This study could also provide a better understanding of molecular mechanisms of cellular protection against UV-B-induced stress.

Stable severe chronic obstructive pulmonary disease (COPD) patients with chronic hypercapnic respiratory failure treated by nocturnal bi-level positive pressure non-invasive ventilation (NIV) may experience severe morning deventilation dyspnea. We hypothesised that in these patients, progressive hyperinflation, resulting from inappropriate ventilator settings, leads to patient-ventilator asynchrony (PVA) with a high rate of unrewarded inspiratory efforts and morning discomfort.

These guidelines update previous guidance published in 2005. They have been revised by a group who are members of the UK and Ireland Neuroendocrine Tumour Society with endorsement from the clinical committees of the British Society of Gastroenterology, the Society for Endocrinology, the Association of Surgeons of Great Britain and Ireland (and its Surgical Specialty Associations), the British Society of Gastrointestinal and Abdominal Radiology and others. The authorship represents leaders of the various groups in the UK and Ireland Neuroendocrine Tumour Society, but a large amount of work has been carried out by other specialists, many of whom attended a guidelines conference in May 2009. We have attempted to represent this work in the acknowledgements section. Over the past few years, there have been advances in the management of neuroendocrine tumours, which have included clearer characterisation, more specific and therapeutically relevant diagnosis, and improved treatments. However, there remain few randomised trials in the field and the disease is uncommon, hence all evidence must be considered weak in comparison with other more common cancers.

Evaluation of the association of 31 common single nucleotide polymorphisms (SNPs) with fasting glucose, fasting insulin, HOMA-beta cell function (HOMA-β), HOMA-insulin resistance (HOMA-IR) and type 2 diabetes in the Indian population.

Voltage clamping is an important tool for measuring individual currents from an electrically active cell. However, it is difficult to isolate individual currents without pharmacological or voltage inhibition. Herein, we present a technique that involves inserting a noise function into a standard voltage step protocol, which allows one to characterize the unique frequency response of an ion channel at different step potentials. Specifically, we compute the fast Fourier transform for a family of current traces at different step potentials for the inward rectifying potassium channel, K(ir)2.1, and the channel encoding the cardiac fast sodium current, Na(v)1.5. Each individual frequency magnitude, as a function of voltage step, is correlated to the peak current produced by each channel. The correlation coefficient vs. frequency relationship reveals that these two channels are associated with some unique frequencies with high absolute correlation. The individual IV relationship can then be recreated using only the unique frequencies with magnitudes of high absolute correlation. Thus, this study demonstrates that ion channels may exhibit unique frequency responses.

Epstein-Barr virus (EBV) infection leads to Hodgkin's disease (HD) in some immunocompetent hosts. The malignant Reed-Sternberg cells of HD only express a limited array of subdominant EBV antigens to evade pre-existing immune responses to EBV. The EBV-encoded latent membrane proteins (LMP1 and LMP2), which are expressed by HD and various EBV-associated malignancies, have been proposed as a potential target for cytotoxic T lymphocyte (CTL)-based therapy. However, the precursor frequency for LMP-specific CTL is generally low in healthy EBV-infected hosts, and immunotherapy based on these antigens is often compromised by the poor immunogenicity and the oncogenic potential. In the present study, we report that transiently expressing an inhibitor of A20, a key negative regulator of inflammatory signaling pathways, together with the LMP antigens (truncated LMP1 and full-length LMP2) greatly enhances maturation and cytokine production of human (h) monocyte-derived dendritic cells (DCs). As a consequence, LMP1/2-expressed, A20-silenced hDCs have an enhanced potency to prime LMP-specific T-cell response. When the in vitro primed T cells are adoptively transferred into tumor-xenografted, severe-combined immunodeficient mice, some of the xenografted tumors approach complete regression. Thus, the study may provide an available resource of LMP-specific T cells for T-cell immunotherapy.

SINE-VNTR-Alu (SVA) elements are non-autonomous, hominid-specific non-LTR retrotransposons and distinguished by their organization as composite mobile elements. They represent the evolutionarily youngest, currently active family of human non-LTR retrotransposons, and sporadically generate disease-causing insertions. Since preexisting, genomic SVA sequences are characterized by structural hallmarks of Long Interspersed Elements 1 (LINE-1, L1)-mediated retrotransposition, it has been hypothesized for several years that SVA elements are mobilized by the L1 protein machinery in trans. To test this hypothesis, we developed an SVA retrotransposition reporter assay in cell culture using three different human-specific SVA reporter elements. We demonstrate that SVA elements are mobilized in HeLa cells only in the presence of both L1-encoded proteins, ORF1p and ORF2p. SVA trans-mobilization rates exceeded pseudogene formation frequencies by 12- to 300-fold in HeLa-HA cells, indicating that SVA elements represent a preferred substrate for L1 proteins. Acquisition of an AluSp element increased the trans-mobilization frequency of the SVA reporter element by ~25-fold. Deletion of (CCCTCT)(n) repeats and Alu-like region of a canonical SVA reporter element caused significant attenuation of the SVA trans-mobilization rate. SVA de novo insertions were predominantly full-length, occurred preferentially in G+C-rich regions, and displayed all features of L1-mediated retrotransposition which are also observed in preexisting genomic SVA insertions.

Voltage-gated calcium (Ca(V)) channels are transmembrane proteins that form Ca(2+)-selective pores gated by depolarization and are essential regulators of the intracellular Ca(2+) concentration. By providing a pathway for rapid Ca(2+) influx, Ca(V) channels couple membrane depolarization to a wide array of cellular responses including neurotransmission, muscle contraction and gene expression. Ca(V) channels fall into two major classes, low voltage-activated (LVA) and high voltage-activated (HVA). The ion-conducting pathway of HVA channels is the α(1) subunit, which typically contains associated β and α(2)δ ancillary subunits that regulate the properties of the channel. Although it is widely acknowledged that α(2)δ-1 is post-translationally cleaved into an extracellular α(2) polypeptide and a membrane-anchored δ protein that remain covalently linked by disulfide bonds, to date the contribution of different cysteine (Cys) residues to the formation of disulfide bridges between these proteins has not been investigated. In the present report, by predicting disulfide connectivity with bioinformatics, molecular modeling and protein biochemistry experiments we have identified two Cys residues involved in the formation of an intermolecular disulfide bond of critical importance for the structure and function of the α(2)δ-1 subunit. Site directed-mutagenesis of Cys404 (located in the von Willebrand factor-A region of α(2)) and Cys1047 (in the extracellular domain of δ) prevented the association of the α(2) and δ peptides upon proteolysis, suggesting that the mature protein is linked by a single intermolecular disulfide bridge. Furthermore, co-expression of mutant forms of α(2)δ-1 Cys404Ser and Cys1047Ser with recombinant neuronal N-type (Ca(V)2.2α(1)/β(3)) channels, showed decreased whole-cell patch-clamp currents indicating that the disulfide bond between these residues is required for α(2)δ-1 function.

Probiotics have been proven effective for preventing caries. In contrast, the effect of probiotics on improving oral diseases such as gingivitis, periodontitis and halitosis has been less explored.

Exendin-4 is a GLP-1 agonist that is clinically used for the treatment of diabetes mellitus and may also have neuroprotective effect. We explored the effect of repeated administration of exendin-4 (0.5 μg/kg, intraperitoneal twice a day for 7 days) on infarct volume, neurological deficit (neurological score, grip test, foot fault and rota rod tests), oxidative stress parameters (malondialdehyde, reduced glutathione, and superoxide dismutase) and expression of endothelin (ET) ET(A) and ET(B) receptors following cerebral ischemia produced in rats by permanent middle cerebral artery occlusion (MCAO). Since ET(A) receptors in the central nervous system (CNS) are involved in cerebral ischemia, we determined the effect of a specific ET(A) receptor antagonist, BQ123 (1mg/kg, intravenously administered thrice: 30 min, 2h and 4h after MCAO for a total dose of 3 mg/kg) on cerebral ischemia in control and exendin-4 treated rats. Results indicate that exendin-4 treated rats had significant protection following MCAO induced cerebral ischemia. The infarct volume was 27% less compared to vehicle treated rats. The neurological deficit following MCAO was lower and oxidative stress parameters were improved in exendin-4 treated rats compared to control. BQ123 significantly improved infarct volume, oxidative stress parameters and neurological deficit in ischemic rats treated with vehicle or exendin-4. BQ123 induced protection from cerebral ischemia was similar in vehicle or exendin-4 treated rats. Expression of ET(A) receptors was significantly increased following cerebral ischemia which was not affected by exendin-4 treatment or by BQ123 administration. No change in expression of ET(B) receptors was observed following cerebral ischemia or any treatment. It is concluded that exendin-4 protects the CNS from damage due to cerebral ischemia by reducing oxidative stress and is independent of ET receptor involvement.

Diverse experimental evidence exists implicating the activation of various different cell surface receptors and intracellular pathways by antiphospholipid antibodies (aPL). This evidence has been generated using a number of different cell types with varying numbers of aPL from different sources and disease subtypes. This experimental variability complicates the comparison of results from different studies. We therefore undertook a systematic review of the literature to provide a critical analysis of the strength of the evidence that specific receptors and signaling pathways are important in the pathogenesis of antiphospholipid syndrome.

Cortical heterotopia, a malformation of the developing cortex, are a major cause of epilepsy and mental retardation in humans. Hemorrhagic hydrocephalus (hhy) mutation on mouse chromosome 12 results in subcortical heterotopia and nonobstructive hydrocephalus with frequent brain hemorrhage. Here, we show that coiled-coil domain-containing 85C (Ccdc85c), consisting of 6 exons that encode a 420 amino acid protein, is disrupted by replacement of a 3.2-kb sequence, including exon 2 in Ccdc85c by a 1.5-kb retrotransposon-like repeat sequence in the hhy mutant. Immunoreactivity to Ccdc85C was detected predominantly at the apical junctions of radial glia in the wall of lateral ventricles of the developing brain. In the hhy brain at embryonic (E) day 18 (E18), radial glial demise followed by agenesis of the ependymal layer lining the neonatal cortex and accumulation of neuronal specific nuclear protein (NeuN)-positive postmigratory neurons in the subcortical area occurred. Accumulation of E15-born, but not of E13-born, 5-bromo-2'-deoxyuridine labeled neurons expressing special AT-rich sequence binding protein 2 was detected in both heterotopia and the superficial layers of the hhy neocortex at postnatal day 7. Ccdc85c deficiency permitted radial scattering of paired box gene 6-positive neural progenitors in the ventricular zone, likely resulting in reduced self-renewal of the progenitors in the developing hhy cortex. These findings indicate an important role of Ccdc85C in cortical development and provide a mouse model to study pathogenesis of subcortical heterotopia and hydrocephalus.

Matrix metalloproteinases (MMPs) have been implicated in the modulation of synaptic plasticity, glial activation, and long-term potentiation in the CNS. Here we demonstrate for the first time a mechanism for the regulation of nociceptive processing by spinal MMP-3 during peripheral inflammation. We first determined by western blotting that the catalytic (active) form of MMP-3 (cMMP-3) is increased in lumbar spinal cord following peripheral inflammation in rats. The peripheral inflammation-induced thermal hyperalgesia and tactile hypersensitivity was transiently (2-3 h) attenuated by intrathecal (IT) pretreatment with either an MMP-3 inhibitor (NNGH), or a broad spectrum MMP inhibitor (GM6001). In addition, IT delivery of cMMP-3 evoked hypersensitivity, whereas the pro (enzymatically inactive) form of MMP-3 did not. This suggests a pro-algesic effect of spinal MMP-3 mediated by an enzymatic mechanism. This cMMP-3-induced hypersensitivity is concurrent with increased tumor necrosis factor (TNF) in the spinal cord. The hypersensitivity behavior is prevented by intrathecal etanercept (TNF blockade). Treatment with cMMP-3 resulted in an increase in TNF release from spinal primary microglial, but not astrocyte cultures. These findings thus present direct evidence implicating MMP-3 in the coordination of spinal nociceptive processing via a spinal TNF-dependent mechanism.

During mouse pancreas development, the transient expression of Neurogenin3 (Neurog3) in uncommitted pancreas progenitors is required to determine endocrine destiny. However it has been reported that Neurog3-expressing cells can eventually adopt acinar or ductal fates and that Neurog3 levels were important to secure the islet destiny. It is not known whether the competence of Neurog3-induced cells to give rise to non-endocrine lineages is an intrinsic property of these progenitors or depends on pancreas developmental stage. Using temporal genetic labeling approaches we examined the dynamic of endocrine progenitor differentiation and explored the plasticity of Neurog3-induced cells throughout development. We found that Neurog3(+) progenitors develop into hormone-expressing cells in a fast process taking less then 10h. Furthermore, fate-mapping studies in heterozygote (Neurog3(CreERT/+)) and Neurog3-deficient (Neurog3(CreERT/CreERT)) embryos revealed that Neurog3-induced cells have different potential over time. At the early bud stage, failed endocrine progenitors can adopt acinar or ductal fate, whereas later in the branching pancreas they do not contribute to the acinar lineage but Neurog3-deficient cells eventually differentiate into duct cells. Thus these results provide evidence that the plasticity of Neurog3-induced cells becomes restricted during development. Furthermore these data suggest that during the secondary transition, endocrine progenitor cells arise from bipotent precursors already committed to the duct/endocrine lineages and not from domain of cells having distinct potentialities.

Chitinase 3-like 1 (CHI3L1), one of the mammalian members of the chitinase family, is expressed in several types of human cancer, and elevated serum level of CHI3L1 is suggested to be a biomarker of poor prognosis in advanced cancer patients. However, the overall biological function of CHI3L1 in human cancers still remains unknown. Studies were performed to characterize the role of CHI3L1 in cancer pathophysiology utilizing human colorectal cancer samples and human cell lines. Plasma protein and tissue mRNA expression levels of CHI3L1 in colorectal cancer were strongly upregulated. Immunohistochemical analysis showed that CHI3L1 was expressed in cancer cells, and CHI3L1 expression had a significant association with the number of infiltrated macrophages and microvessel density (MVD). By utilizing transwell migration and tube-formation assays, overexpression of CHI3L1 in SW480 cells (human colon cancer cells) enhanced the migration of THP-1 cells (human macrophage cells) and HUVECs (human endothelial cells), and the tube formation of HUVECs. The knockdown of CHI3L1 by RNA interference or the neutralization of CHI3L1 by anti-CHI3L1 antibody displayed strong suppression of CHI3L1-induced migration and tube formation. Cell proliferation assay showed that CHI3L1 overexpression significantly enhanced the proliferation of SW480 cells. Enzyme-linked immunosorbent assay (ELISA) analysis showed that CHI3L1 increased the secretion of inflammatory chemokines, IL-8 and monocyte chemoattractant protein-1 (MCP-1), from SW480 cells through mitogen-activated protein kinase (MAPK) signaling pathway. Both neutralization of IL-8 or MCP-1 and inhibition or knockdown of MAPK in SW480 cells significantly inhibited CHI3L1-induced migration and tube formation. In a xenograft mouse model, overexpression of CHI3L1 in HCT116 cells (human colon cancer cells) enhanced the tumor growth as well as macrophage infiltration and MVD. In conclusion, CHI3L1 expressed in colon cancer cells promotes cancer cell proliferation, macrophage recruitment and angiogenesis. Thus, the inhibition of CHI3L1 activity may be a novel therapeutic strategy for human colorectal cancer.

Overexposure to waterborne manganese (Mn) is linked with cognitive impairment in children and neurochemical abnormalities in other experimental models. In order to characterize the threshold between Mn-exposure and altered neurochemistry, it is important to identify biomarkers that positively correspond with brain Mn-accumulation. The objective of this study was to identify Mn-induced alterations in plasma, liver, and brain metabolites using liquid/gas chromatography-time of flight-mass spectrometry metabolomic analyses; and to monitor corresponding Mn-induced behavior changes. Weanling Sprague-Dawley rats had access to deionized drinking water either Mn-free or containing 1g Mn/L for 6 weeks. Behaviors were monitored during the sixth week for a continuous 24h period while in a home cage environment using video surveillance. Mn-exposure significantly increased liver, plasma, and brain Mn concentrations compared to control, specifically targeting the globus pallidus (GP). Mn significantly altered 98 metabolites in the brain, liver, and plasma; notably shifting cholesterol and fatty acid metabolism in the brain (increased oleic and palmitic acid; 12.57 and 15.48 fold change (FC), respectively), and liver (increased oleic acid, 14.51 FC; decreased hydroxybutyric acid, -14.29 FC). Additionally, Mn-altered plasma metabolites homogentisic acid, chenodeoxycholic acid, and aspartic acid correlated significantly with GP and striatal Mn. Total distance traveled was significantly increased and positively correlated with Mn-exposure, while nocturnal stereotypic and exploratory behaviors were reduced with Mn-exposure and performed largely during the light cycle compared to unexposed rats. These data provide putative biomarkers for Mn-neurotoxicity and suggest that Mn disrupts the circadian cycle in rats.

The apicoplast is a highly specialized organelle that mediates required functions in the growth and replication of apicomplexan parasites. Despite structural conservation of the apicoplast among different parasite genera and species, there are also critical differences in the metabolic requirements of different parasites and at different stages of the life cycle. To specifically compare apicoplast pathways between parasites that have both common and unique stages, we characterized the apicoplast in Babesia bovis, which has only intraerythrocytic asexual stages in the mammalian host, and compared it to that of Plasmodium falciparum, which has both asexual intraerythrocytic and hepatic stages. Specifically focusing on the type II fatty acid (FASII) and isoprenoid (MEP) biosynthesis pathways, we searched for pathway components and retention of active sites within the genome, localized key components [acyl carrier protein (ACP) and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB)] to the apicoplast, and demonstrated that the N-terminal bipartite signals of both proteins are required and sufficient for trafficking to the apicoplast lumen. Using specific pharmacologic inhibition, we demonstrated that MEP biosynthesis may be disrupted and its presence is required for intraerythrocytic growth of B. bovis asexual stages, consistent with the genomic pathway analysis and with its requirement in the asexual erythrocytic stages of P. falciparum. In contrast, FASII biosynthesis may or may not be present and specific drug targets did not have any inhibitory effect to B. bovis intraerythrocytic growth, which is consistent with the lack of requirement for P. falciparum intraerythrocytic growth. However, genomic analysis revealed the loss of FASII pathway components in B. bovis whereas the pathway is intact for P. falciparum but regulated to be expressed when needed (hepatic stages) and silent when not (intraerythrocytic stages). The results indicate specialized molding of apicoplast biosynthetic pathways to meet the requirements of individual apicomplexan parasites and their unique intracellular niches.