Insulin resistance (IR) is a complex disorder caused by an interplay of both genetic and environmental factors. Recent studies identified a significant interaction between body mass index (BMI) and the rs1800795 polymorphism of the interleukin-6 gene that influences both IR and onset of type 2 diabetes mellitus, with obese individuals homozygous for the C allele demonstrating the highest level of IR and greatest risk for type 2 diabetes mellitus. Replication of a gene-environment interaction is important to confirm the validity of the initial finding and extend the generalizability of the results to other populations. Thus, the objective of this study was to replicate this gene-environment interaction on IR in a hypertensive population and perform a meta-analysis with prior published results. The replication analysis was performed using white individuals with hypertension from the Hypertensive Pathotype cohort (N = 311), genotyped for rs1800795. Phenotype studies were conducted after participants consumed 2 diets--high sodium (200 mmol/d) and low sodium (10 mmol/d)--for 7 days each. Measurements for plasma glucose, insulin, and interleukin-6 were obtained after 8 hours of fasting. Insulin resistance was characterized by the homeostatic model assessment (HOMA-IR). In Hypertensive Pathotype, BMI was a significant effect modifier of the relationship between rs1800795 and HOMA-IR; higher BMI was associated with higher HOMA-IR among homozygote CC individuals when compared with major allele G carriers (P = .003). Furthermore, the meta-analysis in 1028 individuals confirmed the result, demonstrating the same significant interaction between rs1800795 and BMI on HOMA-IR (P = 1.05 × 10(-6)). This rare replication of a gene-environment interaction extends the generalizability of the results to hypertension while highlighting this polymorphism as a marker of IR in obese individuals.

The signaling pathways utilized by naïve and experienced effector CD4 T cells during activation and proliferation were evaluated. While inhibition of either mTOR or MAPK alone was able to inhibit naïve T cell proliferation, both mTOR and MAPK (ERK) pathway inhibition was required to efficiently block experienced, effector CD4 T cell proliferation. This was demonstrated both in vitro, and in vivo by treating mice with collagen-induced arthritis using mTOR and/or ERK inhibitors. The combination of mTOR and ERK inhibition prevented or treated disease more efficiently than either agent alone. These data illustrate the different requirements of naïve and experienced effector CD4 T cells in the use of the mTOR and MAPK pathways in proliferation, and suggest that therapies targeting both the mTOR and MAPK pathways may be more effective than targeting either pathway alone in the treatment of CD4 T cell-mediated autoimmunity.

Microbial colonization on new terrestrial substrates represents the initiation of new soil ecosystem formation. In this study, we analyzed early bacterial communities growing on volcanic ash deposits derived from the 2000 Mount Oyama eruption on the island of Miyake (Miyake-jima), Japan. A site was established in an unvegetated area near the summit and investigated over a 6-year period from 2003 to 2009. Collected samples were acidic (pH 3.0-3.6), did not utilize any organic substrates in ECO microplate assays (Biolog), and harbored around 106 cells (g dry weight)(-1) of autotrophic Fe(II) oxidizers by most-probable-number (MPN) counts. Acidithiobacillus ferrooxidans, Acidithiobacillus ferrivorans, and the Leptospirillum groups I, II and III were found to be abundant in the deposits by clone library analysis of bacterial 16S rRNA genes. The numerical dominance of Acidithiobacillus ferrooxidans was also supported by analysis of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Comparing the 16S rRNA gene clone libraries from samples differing in age, shifts in Fe(II)-oxidizing populations seemed to occur with deposit aging. The detection of known 16S rRNA gene sequences from Fe(III)-reducing acidophiles promoted us to propose the acidity-driven iron cycle for the early microbial ecosystem on the deposit.

Ammonia monooxygenase subunit A gene (amoA) is frequently used as a functional gene marker for diversity analysis of ammonia-oxidizing bacteria (AOB). To select a suitable amoA primer for real-time PCR and PCR-denaturing gradient gel electrophoresis (DGGE), three reverse primers (degenerate primer amoA-2R; non-degenerate primers amoA-2R-GG and amoA-2IR) were examined. No significant differences were observed among the three primers in terms of quantitative values of amoA from environmental samples using real-time PCR. We found that PCR-DGGE analysis with the amoA-2IR primer gave the best results in this studied soil. These results indicate that amoA-2IR is a suitable primer for community analysis of AOB in the environment.

HERG (human ether-à-go-go-related gene) K(+) currents fulfill important ionic functions in cardiac and other excitable cells. In addition, HERG channels influence cell growth and migration in various types of tumor cells. The mechanisms underlying these functions are still not resolved. Here, we investigated the role of HERG channels for cell growth in a cell line (SW2) derived from small cell lung cancer (SCLC), a malignant variant of lung cancer. The two HERG1 isoforms (HERG1a, HERG1b) as well as HERG2 and HERG3 are expressed in SW2 cells. Inhibition of HERG currents by acute or sustained application of E-4031, a specific ERG channel blocker, depolarized SW2 cells by 10-15 mV. This result indicated that HERG K(+) conductance contributes considerably to the maintenance of the resting potential of about -45 mV. Blockage of HERG channels by E-4031 for up to 72 h did not affect cell proliferation. In contrast, siRNA-induced inhibition of HERG1 protein expression decreased cell proliferation by about 50%. Reduction of HERG1 protein expression was confirmed by Western blots. HERG current was almost absent in SW2 cells transfected with siRNA against HERG1. Qualitatively similar results were obtained in three other SCLC cell lines (OH1, OH3, H82), suggesting that the HERG1 channel protein is involved in SCLC cell growth, whereas the ion-conducting function of HERG1 seems not to be important for cell growth.

Retinoic acid, the active form of the nutrient vitamin A, regulates several facets of neuronal plasticity in the hippocampus, including neurogenesis and synaptic strength, acting via specific retinoic acid receptors (RARs). Essential for conversion of vitamin A to retinoic acid is the enzyme retinaldehyde dehydrogenase (RALDH) and in the rodent hippocampus this is only present in the adjacent meninges where it must act as a locally released paracrine hormone. Little is known though about the expression of RALDHs and RARs in the human hippocampus. This study confirms that RALDH levels are very low in mouse neurons but, surprisingly, strong expression of RALDH protein is detected by immunohistochemistry in hippocampal neurons. The receptors RARα, β and γ were also detected, each receptor exhibiting differing subcellular locations implying their potential regulation of both transcription and non-genomic actions. These results imply an essential function of retinoic acid in the human hippocampus likely to include regulation of neuronal plasticity.

The influenza A virus genome consists of eight viral RNAs (vRNAs) that form viral ribonucleoproteins (vRNPs). Even though evidence supporting segment-specific packaging of vRNAs is accumulating, the mechanism ensuring selective packaging of one copy of each vRNA into the viral particles remains largely unknown. We used electron tomography to show that the eight vRNPs emerge from a common 'transition zone' located underneath the matrix layer at the budding tip of the virions, where they appear to be interconnected and often form a star-like structure. This zone appears as a platform in 3D surface rendering and is thick enough to contain all known packaging signals. In vitro, all vRNA segments are involved in a single network of intermolecular interactions. The regions involved in the strongest interactions were identified and correspond to known packaging signals. A limited set of nucleotides in the 5' region of vRNA 7 was shown to interact with vRNA 6 and to be crucial for packaging of the former vRNA. Collectively, our findings support a model in which the eight genomic RNA segments are selected and packaged as an organized supramolecular complex held together by direct base pairing of the packaging signals.

The human prolyl-4-hydroxylase domain (PHD) proteins 1-3 are known as cellular oxygen sensors, acting via the degradation of hypoxia-inducible factor (HIF) α-subunits. PHD2 and PHD3 genes are inducible by HIFs themselves, suggesting a negative feedback loop that involves PHD abundance. To identify novel regulators of the PHD2 gene, an expression array of 704 transcription factors was screened by a method that allows distinguishing between HIF-dependent and HIF-independent promoter regulation. Among others, the E-twenty six transcription factor ETS translocation variant 4 (ETV4) was found to contribute to PHD2 gene expression particularly under hypoxic conditions. Mechanistically, complex formation between ETV4 and HIF-1/2α was observed by mammalian two-hybrid and fluorescence resonance energy transfer analysis. HIF-1α domain mapping, CITED2 overexpression and factor inhibiting HIF depletion experiments provided evidence for cooperation between HIF-1α and p300/CBP in ETV4 binding. Chromatin immunoprecipitation confirmed ETV4 and HIF-1α corecruitment to the PHD2 promoter. Of 608 hypoxically induced transcripts found by genome-wide expression profiling, 7.7% required ETV4 for efficient hypoxic induction, suggesting a broad role of ETV4 in hypoxic gene regulation. Endogenous ETV4 highly correlated with PHD2, HIF-1/2α and several established markers of tissue hypoxia in 282 human breast cancer tissue samples, corroborating a functional interplay between the ETV4 and HIF pathways.

The RNA binding protein HuD plays essential roles in neuronal development and plasticity. We have previously shown that HuD stimulates translation. Key for this enhancer function is the linker region and the poly(A) binding domain of HuD that are also critical for its function in neurite outgrowth. Here, we further explored the underlying molecular interactions and found that HuD but not the ubiquitously expressed HuR interacts directly with active Akt1. We identify that the linker region of HuD is required for this interaction. We also show by using chimeric mutants of HuD and HuR, which contain the reciprocal linker between RNA-binding domain 2 (RBD2) and RBD3, respectively, and by overexpressing a dominant negative mutant of Akt1 that the HuD-Akt1 interaction is functionally important, as it is required for the induction of neurite outgrowth in PC12 cells. These results suggest the model whereby RNA-bound HuD functions as an adapter to recruit Akt1 to trigger neurite outgrowth. These data might also help to explain how HuD enhances translation of mRNAs that encode proteins involved in neuronal development.

Adenoviruses have been identified in humans and a wide range of vertebrate animals, but not previously from the polar region. Here, we report the entire 26,340-bp genome of a novel adenovirus, detected by PCR, in tissues of six of nine South Polar skuas (Catharacta maccormicki), collected in Lake King Sejong, King George Island, Antarctica, from 2007 to 2009. The DNA polymerase, penton base, hexon and fiber genes of the South Polar skua adenovirus (SPSAdV) exhibited 68.3%, 75.4%, 74.9% and 48.0% nucleotide sequence similarity with their counterparts in turkey hemorrhagic enteritis virus. Phylogenetic analysis based on the entire genome revealed that SPSAdV belonged to the genus Siadenovirus, family Adenoviridae. This is the first evidence of a novel adenovirus, SPSAdV, from a large polar seabird (family Stercorariidae) in Antarctica.

Mutations in the valosin-containing protein (VCP) gene were recently reported to be the cause of 1%-2% of familial amyotrophic lateral sclerosis (ALS) cases. VCP mutations are known to cause inclusion body myopathy (IBM) with Paget's disease (PDB) and frontotemporal dementia (FTD). The presence of VCP mutations in patients with sporadic ALS, sporadic ALS-FTD, and progressive muscular atrophy (PMA), a known clinical mimic of inclusion body myopathy, is not known. To determine the identity and frequency of VCP mutations we screened a cohort of 93 familial ALS, 754 sporadic ALS, 58 sporadic ALS-FTD, and 264 progressive muscular atrophy patients for mutations in the VCP gene. Two nonsynonymous mutations were detected; 1 known mutation (p.R159H) in a patient with familial ALS with several family members suffering from FTD, and 1 mutation (p.I114V) in a patient with sporadic ALS. Conservation analysis and protein prediction software indicate the p.I114V mutation to be a rare benign polymorphism. VCP mutations are a rare cause of familial ALS. The role of VCP mutations in sporadic ALS, if present, appears limited.

Task effects in semantic processing were investigated by contrasting the neural activation associated with two semantic categorization tasks (SCT) using event-related fMRI. The two SCTs involved different decision categories: is it an animal? vs. is it a concrete thing? Participants completed both tasks and, across participants, the same core set of items were presented in both tasks. Results showed task differences in the neural activation associated with these items: in the animal SCT there was greater activation in a number of frontal and temporal regions, including left superior and middle temporal gyri, while in the concrete SCT there was greater activation in left medial frontal gyrus and bilaterally in the precentral gyri. These results are interpreted as evidence of top-down modulation of semantic processing; participants make adjustments to optimize performance in a given task and these adjustments have consequences for the activation observed.

Oncomodulin, an ~12 kDa Ca(2+)-binding protein secreted from activated macrophages, has been shown to promote axonal regeneration from retinal ganglion cells (RGCs) following optic nerve injury. However, to date, the axonal growth-promoting capacity of oncomodulin in other models of 'regenerative failure' has not been evaluated. We assessed the capability of preconditioning treatment with oncomodulin to promote sensory axonal regeneration in an in vitro spot model of regenerative failure, and across the dorsal root zone (DREZ) after root crush injury. Neither the direct exposure of adult rat DRGs to oncomodulin, nor preconditioning of DRGs by intraganglionic injection of oncomodulin, stimulated axonal outgrowth in the in vitro proteoglycan spot gradient assay. However, direct exposure of unconditioned DRGs to both oncomodulin and db-cAMP in vitro, as well as preconditioning of DRGs with the combined treatment in vivo, resulted in significant, albeit modest, neurite extension across the inhibitory proteoglycan barrier. We next quantified axon regeneration through the C8 DREZ in adult rats after oncomodulin and/or db-cAMP preconditioning and chondroitinase (ChABC) injection into the DREZ immediately following a root crush injury. Axonal regeneration across the DREZ was not observed in control animals, or after injection of ChABC-alone. Treatment with oncomodulin- or db-cAMP-alone resulted in extremely sparse regeneration. However, significant, but meager, sensory axon regeneration across the DREZ was observed using the oncomodulin/ db-cAMP combination (p<0.001), supporting findings from previous studies suggesting that cAMP is necessary for the growth-promoting effects of oncomodulin. Although our results support a role for oncomodulin in macrophage-induced axonal regeneration, the effects of oncomodulin/db-cAMP on sensory regeneration were extremely limited in comparison to previous studies in the same injury model using zymosan.

These experiments were completed as part of an NIH "Facilities of Research Excellence in Spinal Cord Injury" contract to support independent replication of published studies that could be considered for eventual clinical testing. Recent studies have reported that selective inhibition of the P2X7 receptor improves both the functional and histopathological consequences of a contusive spinal cord injury (SCI) in rats. We repeated two published studies reporting the beneficial effects of pyridoxal-5'-phosphate-6-azophenyl-2'-4'-disulphonic acid (PPADS) or Brilliant blue G (BBG) treatment after SCI (Wang et al., 2004 and Peng et al., 2009). Mild thoracic SCI was first produced in Experiment 1 by means of the MASCIS impactor at T10 (height 6.25 mm, weight 10 g) followed by intraspinal administration of a P2X7 antagonist (2 μl/10 mM) after injury. Treatment with PPADS or another highly selective P2X7R antagonist Brilliant Blue G (BBG) (2 μl/02 mM) did not improve locomotive (BBB rating scale) over a 7 week period compared to vehicle treated rats. Also, secondary histopathological changes in terms of overall lesion and cavity volume were not significantly different between the PPADS, BBG, and vehicle treated animals. In the second experiment, the systemic administration of BBG (10 or 50 mg/kg, iv) 15 min, 24 and 72 h after moderate (12.5 mm) SCI failed to significantly improve motor recovery or histopathological outcome over the 6 week observational period. Although we cannot conclude that there will be no long-term beneficial effects in other spinal cord injury models using selective P2X7 receptor antagonists at different doses or treatment durations, we caution researchers that this potentially exciting therapy requires further preclinical investigations before the implementation of clinical trials targeting severe SCI patients.

(1) To investigate the association between diabetes and marrow changes in the cuboid; and (2) to evaluate the influence of age, gender, body mass index (BMI) and use of insulin in the occurrence of marrow changes in the cuboid.

We herein studied the role of glutamate dehydrogenase (GDH), in response to leucine (LEU) supplementation, upon insulin secretion of malnourished rats. Weaned male Wistar rats were fed normal-protein (17%) or low-protein diet (6%, LP) for 8 weeks. Half of the rats of each group were supplemented with LEU (1.5%) in the drinking water for the following 4 weeks. Gene and protein expressions, static insulin secretion, and cytoplasmic Ca(2+) oscillations were measured. Glutamate dehydrogenase messenger RNA was 58% lower in LP islets, and LEU supplementation augmented it in 28%. The LP islets secreted less insulin when exposed to 20 mmol/L LEU, 20 mmol/L LEU + 2 mmol/L glutamine (with or without 5 mmol/L aminooxyacetic acid, a branched chain aminotransferase inhibitor, or 20 μmol/L epigallocatechin gallate, a GDH inhibitor), 20 mmol/L α-ketoisocaproate, glutamine + 20 mmol/L β-2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (a GDH activator), and 22.2 mmol/L glucose. Leucine supplementation augmented insulin secretion to levels found in normal-protein islets in all the above conditions, an effect that was blunted when islets were incubated with epigallocatechin gallate. The glutamine + β-2-aminobicyclo[2.2.1]heptane-2-carboxylic acid-induced increased [Ca(2+)](i) and oscillations were higher than those for LP islets. Leucine supplementation normalized these parameters in LP islets. Impaired GDH function was associated with lower insulin release in LP islets, and LEU supplementation normalized insulin secretion via restoration of GDH function. In addition, GDH may contribute to insulin secretion through ameliorations of Ca(2+) handling in LP islets.

The overexpression of epidermal growth factor receptor (EGFR) in tumors underlines the recent interest in EGFR as attractive target for the development of new cancer imaging agents. EGFR-tyrosine kinase inhibitors (EGFR-TKIs) based on the anilinoquinazoline scaffold have been explored as potential probes for EGFR imaging. However, up to now, no optimal radiotracer is available. Herein, we report the synthesis and biological evaluation of three novel halogenated 6-substituted 4-anilinoquinazoline based EGFR-TKIs. Radiosynthesis ((125)I and (18)F) of the corresponding analogues was also performed.

The protozoan parasite Trypanosoma brucei switches its variant surface glycoprotein (VSG) to subvert its mammalian hosts' immune responses. The T. brucei genome contains as many as 1600 VSG genes (VSGs), but most are silent noncoding pseudogenes. Only one functional VSG, located in a telomere-linked expression site, is transcribed at a time. Silent VSGs are copied into a VSG expression site through gene conversion. Truncated gene conversion events can generate new mosaic VSGs with segments of sequence identity to other VSGs. To examine the VSG family sub-structure within which these events occur, we combined the available VSG sequences and annotations with scripted BLAST searches to map the relationships among VSGs in the T. brucei genome. Clusters of related VSGs were visualized in 2- and 3-dimensions for different N- and C-terminal regions. Five types of N-termini (N1-N5) were observed, within which gene recombinational events are likely to occur, often with fully-coding 'functional' or 'atypical'VSGs centrally located between more dissimilar VSGs. Members of types N1, N3 and N4 are most closely related in the middle of the N-terminal region, whereas type N2 members are more similar near the N-terminus. Some preference occurs in pairing between specific N- and C-terminal types. Statistical analyses indicated no overall tendency for more related VSGs to be located closer in the genome than less related VSGs, although exceptions were noted. Many potential mosaic gene formation events within each N-terminal type were identified, contrasted by only one possible mosaic gene formation between N-terminal types (N1 and N2). These data suggest that mosaic gene formation is a major contributor to the overall VSG diversity, even though gene recombinational events between members of different N-terminal types occur only rarely.

Vasoactive intestinal peptide receptor-1 signaling in lymphocytes has been shown to regulate chemotaxis, proliferation, apoptosis and differentiation. During T cell activation, VPAC1 mRNA is downregulated, but the effect on its protein levels is less clear. A small number of studies have reported measurement of human VPAC1 by flow cytometry, but murine VPAC1 reagents are unavailable. Therefore, we set out to generate a reliable and highly specific α-mouse VPAC1 polyclonal antibody for use with flow cytometry. After successfully generating a rabbit α-VPAC1 polyclonal antibody (α-mVPAC1 pAb), we characterized its cross-reactivity and showed that it does not recognize other family receptors (mouse VPAC2 and PAC1, and human VPAC1, VPAC2 and PAC1) by flow cytometry. Partial purification of the rabbit α-VPAC1 sera increased the specific-activity of the α-mVPAC1 pAb by 20-fold, and immunofluorescence microscopy (IF) confirmed a plasma membrane subcellular localization for mouse VPAC1 protein. To test the usefulness of this specific α-mVPAC1 pAb, we showed that primary, resting mouse T cells express detectable levels of VPAC1 protein, with little detectable signal from activated T cells, or CD19 B cells. These data support our previously published data showing a downregulation of VPAC1 mRNA during T cell activation. Collectively, we have established a well-characterized, and highly species specific α-mVPAC1 pAb for VPAC1 surface measurement by IF and flow cytometry.

MicroRNAs (miR) are single-stranded short RNA molecules that regulate gene expression by degradation or translational repression of mRNA. It has been reported that the downregulation of miR-199a plays an important role in cardiac ischemic tolerance. We examined the expression of miR-199a after 3-nitropropionic acid (3-NPA) preconditioning in rat brain. 3-NPA (20mg/kg), an irreversible inhibitor of succinate dehydrogenase, was injected intraperitoneally to induce ischemic tolerance in rats. For comparison, the control group received intraperitoneal injections of vehicle (0.9% sodium chloride). Quantitative RT-PCR assay was applied to detect the expression of miR-199a. Luciferase reporter assays and Western blotting were used to verify the target genes of miR-199a. In cortex and striatum, miR-199a was downregulated at two separate time intervals (the 2nd and 4th day), while in the hippocampus, it was downregulated on the 2nd day after 3-NPA preconditioning. The maximum reduction of miR-199a was 66.3% in striatum (4th day), 54.9% in hippocampus (2nd day), and 27.6% in cortex (2nd day). The level of sirt1 protein, a putative target of miR-199a and a known mediator of neuroprotective effect in brain ischemic tolerance, decreased significantly in hippocampal neurons by overexpression of miR-199a, while it increased with knockdown of miR-199a. Taking these results together, we hypothesize miR-199a may have a role in the formation of cerebral ischemic tolerance.