Genetic mapping and functional studies of a natural inhibitor of the insulin receptor tyrosine kinase: the mouse ortholog of human [alpha subscript 2]-HS glycoprotein

Fetuin/α2-HS glycoprotein (α2-HSG) homologs have been identified in several species including rat, sheep, pig, rabbit, guinea pig, cattle, mouse and human. Multiple physiological roles for these homologs have been suggested, including ability to bind to hydroxyapatite crystals and to specifically inhibit the tyrosine kinase (TK) activity of the insulin receptor (IR). In this study we report the identification, cloning, and characterization of the mouse Ahsg gene and its function as an IR-TK inhibitor. Genomic clones derived from a mouse Svj 129 genomic library were sequenced in order to characterize the intron-exon organization of the mouse Ahsg gene, including an 875 bp subclone containing 154 bp upstream from the transcription start site, the first exon, and part of the first intron. A second genomic subclone harboring a 3.45 kb BgI II fragment contained exons 2, 3, and 4 in addition to two adjacent elements within the first intron-a repetitive element of the B1 family (92 bp) and a 271 bp tract of (T,C)n *(A,G)n. We have mapped mouse Ahsg at 16 cM adjacent to the Dagk3 gene on chromosome 16 by genotyping interspecific backcross panels between C57BL/6J and Mus spretus. The position is syntenic with human chromosome 3q27, where the human AHSG gene resides. Using recombinant mouse α2-HSG expressed from a recombinant baculovirus, we demonstrate that mouse α2-HSG inhibits insulin-stimulated IR autophosphorylation and IR-TKA in vitro. In addition, mouse α2-HSG (25 μg/ml) completely abolishes insulin-induced DNA synthesis in H-35 rat hepatoma cells. Based on the sequence data and functional analysis, we conclude that the mouse Ahsg gene is the true ortholog of the human AHSG gene

Effect of antiorthostatic BedRest (BR) on GastroIntestinal Motility (GIM) of normal subjects

The combined effects of postural changes, fluid shifts and diuresis associated with the absence of the gravity vector may decrease gastrointestinal motility (GIM) during space flight. GIM can be estimated from the mouth to cecum transit time (MCTT) of orally administered lactulose (LAC); this test is used to assess changes in GIM in normal subjects and in patients with GI pathology and related disease conditions. Since bedrest (BR) mimics some of the physiological changes that occur during space flight, the effect of ten days of BR on GIM was evaluated from the MCTT of LAC. Methods: Subjects were 12 nonsmoking males between the ages of 35 and 50. After an 8-10 hour fast, subjects ingested Cephulac (registered) (20 g solution) with a low-fiber breakfast on four different days (45, 30, 25, and 20) before BR and on three separate days (4, 7, and 10) during BR. Breath-H2 concentrations were measured before and at 10 minute intervals for 4 hours after breakfast using a Quintron breathalyzer and MCTT was determined from these data. Results: MCTT ranged between 10 and 122 minutes during ambulation and 80 to 120 minutes during BR with means of 79 minutes and 122 minutes respectively. Conclusion: Mean MCTT during BR was 54 percent longer than during ambulation, suggesting that absorption and availability of orally administered medications and nutrients may be delayed or impaired as a result of decreased GIM during bedrest

cGMP-independent nitric oxide signaling and regulation of the cell cycle

BACKGROUND: Regulatory functions of nitric oxide (NO(•)) that bypass the second messenger cGMP are incompletely understood. Here, cGMP-independent effects of NO(• )on gene expression were globally examined in U937 cells, a human monoblastoid line that constitutively lacks soluble guanylate cyclase. Differentiated U937 cells (>80% in G0/G1) were exposed to S-nitrosoglutathione, a NO(• )donor, or glutathione alone (control) for 6 h without or with dibutyryl-cAMP (Bt(2)cAMP), and then harvested to extract total RNA for microarray analysis. Bt(2)cAMP was used to block signaling attributable to NO(•)-induced decreases in cAMP. RESULTS: NO(• )regulated 110 transcripts that annotated disproportionately to the cell cycle and cell proliferation (47/110, 43%) and more frequently than expected contained AU-rich, post-transcriptional regulatory elements (ARE). Bt(2)cAMP regulated 106 genes; cell cycle gene enrichment did not reach significance. Like NO(•), Bt(2)cAMP was associated with ARE-containing transcripts. A comparison of NO(• )and Bt(2)cAMP effects showed that NO(• )regulation of cell cycle genes was independent of its ability to interfere with cAMP signaling. Cell cycle genes induced by NO(• )annotated to G1/S (7/8) and included E2F1 and p21/Waf1/Cip1; 6 of these 7 were E2F target genes involved in G1/S transition. Repressed genes were G2/M associated (24/27); 8 of 27 were known targets of p21. E2F1 mRNA and protein were increased by NO(•), as was E2F1 binding to E2F promoter elements. NO(• )activated p38 MAPK, stabilizing p21 mRNA (an ARE-containing transcript) and increasing p21 protein; this increased protein binding to CDE/CHR promoter sites of p21 target genes, repressing key G2/M phase genes, and increasing the proportion of cells in G2/M. CONCLUSION: NO(• )coordinates a highly integrated program of cell cycle arrest that regulates a large number of genes, but does not require signaling through cGMP. In humans, antiproliferative effects of NO(• )may rely substantially on cGMP-independent mechanisms. Stress kinase signaling and alterations in mRNA stability appear to be major pathways by which NO(• )regulates the transcriptome

Yellow sea mine hunting using the Navy's CASS/GRAB model

The purpose of this work is to determine the necessity of a near real time ocean modeling capability such as the Naval Oceanographic Office's (NAVOCENANO) Modular Ocean Data Assimilation System (MODAS) model in shallow water (such as the Yellow Sea) mine hunting applications using the Navy's Comprehensive Acoustic Simulation System/Gaussian Ray Bundle (CASS/GRAB) Mode. Sound speed profiles inputted into CASS/GRAB were calculated from observational (MOODS) and climatological (GDEM) data sets for different seasons and regions of four different bottom types (sand, gravel, mud and rock). The CASS/GRAB model outputs were compared to the outputs from corresponding MODAS data sets. The results of the comparisons domonstrated in many cases a significant acoustic difference between the alternate profiles. These results demonstrated that there is a need for a predictive modeling capability such as MODAS to address the mine warfare (MIW) needs in the Yellow Sea region. There were some weaknesses detected in the profiles the MODAS model produces in the Yellow Sea, which must be resolved before it can reliably address the MIW needs in the regionNAVOCEANO, Stennis Space Center, MS 39522-5001Approved for public release; distribution is unlimited

Protocadherin-18 Is a Novel Differentiation Marker and an Inhibitory Signaling Receptor for CD8+ Effector Memory T Cells

CD8+ tumor infiltrating T cells (TIL) lack effector-phase functions due to defective proximal TCR-mediated signaling previously shown to result from inactivation of p56lck kinase. We identify a novel interacting partner for p56lck in nonlytic TIL, Protocadherin-18 (‘pcdh18’), and show that pcdh18 is transcribed upon in vitro or in vivo activation of all CD8+ central memory T cells (CD44+CD62LhiCD127+) coincident with conversion into effector memory cells (CD44+CD62LloCD127+). Expression of pcdh18 in primary CD8+ effector cells induces the phenotype of nonlytic TIL: defective proximal TCR signaling, cytokine secretion, and cytolysis, and enhanced AICD. pcdh18 contains a motif (centered at Y842) shared with src kinases (QGQYQP) that is required for the inhibitory phenotype. Thus, pcdh18 is a novel activation marker of CD8+ memory T cells that can function as an inhibitory signaling receptor and restrict the effector phase

Meta-analysis of genome-wide association studies of asthma in ethnically diverse North American populations.

Asthma is a common disease with a complex risk architecture including both genetic and environmental factors. We performed a meta-analysis of North American genome-wide association studies of asthma in 5,416 individuals with asthma (cases) including individuals of European American, African American or African Caribbean, and Latino ancestry, with replication in an additional 12,649 individuals from the same ethnic groups. We identified five susceptibility loci. Four were at previously reported loci on 17q21, near IL1RL1, TSLP and IL33, but we report for the first time, to our knowledge, that these loci are associated with asthma risk in three ethnic groups. In addition, we identified a new asthma susceptibility locus at PYHIN1, with the association being specific to individuals of African descent (P = 3.9 × 10(-9)). These results suggest that some asthma susceptibility loci are robust to differences in ancestry when sufficiently large samples sizes are investigated, and that ancestry-specific associations also contribute to the complex genetic architecture of asthma

Whole-Genome Sequencing of Individuals from a Founder Population Identifies Candidate Genes for Asthma

Asthma is a complex genetic disease caused by a combination of genetic and environmental risk factors. We sought to test classes of genetic variants largely missed by genome-wide association studies (GWAS), including copy number variants (CNVs) and low-frequency variants, by performing whole-genome sequencing (WGS) on 16 individuals from asthma-enriched and asthma-depleted families. The samples were obtained from an extended 13-generation Hutterite pedigree with reduced genetic heterogeneity due to a small founding gene pool and reduced environmental heterogeneity as a result of a communal lifestyle. We sequenced each individual to an average depth of 13-fold, generated a comprehensive catalog of genetic variants, and tested the most severe mutations for association with asthma. We identified and validated 1960 CNVs, 19 nonsense or splice-site single nucleotide variants (SNVs), and 18 insertions or deletions that were out of frame. As follow-up, we performed targeted sequencing of 16 genes in 837 cases and 540 controls of Puerto Rican ancestry and found that controls carry a significantly higher burden of mutations in IL27RA (2.0% of controls; 0.23% of cases; nominal p = 0.004; Bonferroni p = 0.21). We also genotyped 593 CNVs in 1199 Hutterite individuals. We identified a nominally significant association (p = 0.03; Odds ratio (OR) = 3.13) between a 6 kbp deletion in an intron of NEDD4L and increased risk of asthma. We genotyped this deletion in an additional 4787 non-Hutterite individuals (nominal p = 0.056; OR = 1.69). NEDD4L is expressed in bronchial epithelial cells, and conditional knockout of this gene in the lung in mice leads to severe inflammation and mucus accumulation. Our study represents one of the early instances of applying WGS to complex disease with a large environmental component and demonstrates how WGS can identify risk variants, including CNVs and low-frequency variants, largely untested in GWAS

Neuromuscular recovery from botulism involves multiple forms of compensatory plasticity

IntroductionBotulinum neurotoxin (BoNT) causes neuroparalytic disease and death by blocking neuromuscular transmission. There are no specific therapies for clinical botulism and the only treatment option is supportive care until neuromuscular function spontaneously recovers, which can take weeks or months after exposure. The highly specialized neuromuscular junction (NMJ) between phrenic motor neurons and diaphragm muscle fibers is the main clinical target of BoNT. Due to the difficulty in eliciting respiratory paralysis without a high mortality rate, few studies have characterized the neurophysiological mechanisms involved in diaphragm recovery from intoxication. Here, we develop a mouse model of botulism that involves partial paralysis of respiratory muscles with low mortality rates, allowing for longitudinal analysis of recovery.Methods and resultsMice challenged by systemic administration of 0.7 LD50 BoNT/A developed physiological signs of botulism, such as respiratory depression and reduced voluntary running activity, that persisted for an average of 8–12 d. Studies in isolated hemidiaphragm preparations from intoxicated mice revealed profound reductions in nerve-elicited, tetanic and twitch muscle contraction strengths that recovered to baseline 21 d after intoxication. Despite apparent functional recovery, neurophysiological parameters remained depressed for 28 d, including end plate potential (EPP) amplitude, EPP success rate, quantal content (QC), and miniature EPP (mEPP) frequency. However, QC recovered more quickly than mEPP frequency, which could explain the discrepancy between muscle function studies and neurophysiological recordings. Hypothesizing that differential modulation of voltage-gated calcium channels (VGCC) contributed to the uncoupling of QC from mEPP frequency, pharmacological inhibition studies were used to study the contributions of different VGCCs to neurophysiological function. We found that N-type VGCC and P/Q-type VGCC partially restored QC but not mEPP frequency during recovery from paralysis, potentially explaining the accelerated recovery of evoked release versus spontaneous release. We identified additional changes that presumably compensate for reduced acetylcholine release during recovery, including increased depolarization of muscle fiber resting membrane potential and increased quantal size.DiscussionIn addition to identifying multiple forms of compensatory plasticity that occur in response to reduced NMJ function, it is expected that insights into the molecular mechanisms involved in recovery from neuromuscular paralysis will support new host-targeted treatments for multiple neuromuscular diseases