Natural genetic variation caused by transposable elements in humans

Transposons and transposon-like repetitive elements collectively occupy 44% of the human genome sequence. In an effort to measure the levels of genetic variation that are caused by human transposons, we have developed a new method to broadly detect transposon insertion polymorphisms of all kinds in humans. We began by identifying 606,093 insertion and deletion (indel) polymorphisms in the genomes of diverse humans. We then screened these polymorphisms to detect indels that were caused by de novo transposon insertions. Our method was highly efficient and led to the identification of 605 nonredundant transposon insertion polymorphisms in 36 diverse humans. We estimate that this represents 25-35% of approximately 2075 common transposon polymorphisms in human populations. Because we identified all transposon insertion polymorphisms with a single method, we could evaluate the relative levels of variation that were caused by each transposon class. The average human in our study was estimated to harbor 1283 Alu insertion polymorphisms, 180 L1 polymorphisms, 56 SVA polymorphisms, and 17 polymorphisms related to other forms of mobilized DNA. Overall, our study provides significant steps toward (i) measuring the genetic variation that is caused by transposon insertions in humans and (ii) identifying the transposon copies that produce this variation.     

Dual action of epidermal growth factor: extracellular signal-stimulated nuclear–cytoplasmic export and coordinated translation of selected messenger RNA

We report the first example of a coordinated dual action of epidermal growth factor (EGF) in stimulating the nuclear–cytoplasmic export and translation of a select messenger RNA (mRNA). The effect of EGF is mediated by the RNA-binding protein Grb7 (growth factor receptor–bound protein 7), which serves as an adaptor for a specific mRNA–protein export complex and a translational regulator. Using the κ–opioid receptor (OR [KOR]) as a model, we demonstrate that EGF activates nuclear SHP-2 (Src homology region 2–containing tyrosine phosphatase), which dephosphorylates Grb7 in the nucleus. Hypophosphorylated Grb7 binds to the KOR mRNA and recruits the Hu antigen R–exportin-1 (CRM1) complex to form a nuclear–cytoplasmic export complex that exports KOR mRNA. EGF also activates focal adhesion kinase in the cytoplasm to rephosphorylate Grb7, releasing KOR mRNA for active translation. In summary, this study uncovers a coordinated, dual activity of EGF in facilitating nuclear export of a specific mRNA–protein complex as well as translational activation of the exported mRNA.     

Molecular systematics of Helicoma, Helicomyces and Helicosporium and their teleomorphs inferred from rDNA sequences

Three genera of asexual, helical-spored fungi, Helicoma, Helicomyces and Helicosporium traditionally have been differentiated by the morphology of their conidia and conidiophores. In this paper we assessed their phylogenetic relationships from ribosomal sequences from ITS, 5.8S and partial LSU regions using maximum parsimony, maximum likelihood and Bayesian analysis. Forty-five isolates from the three genera were closely related and were within the teleomorphic genus Tubeufia sensu Barr (Tubeufiaceae, Ascomycota). Most of the species could be placed in one of the seven clades that each received 78% or greater bootstrap support. However none of the anamorphic genera were monophyletic and all but one of the clades contained species from more than one genus. The 15 isolates of Helicoma were scattered through the phylogeny and appeared in five of the clades. None of the four sections within the genus were monophyletic, although species from Helicoma sect. helicoma were concentrated in Clade A. The Helicosporium species also appeared in five clades. The four Helicomyces species were distributed among three clades. Most of the clades supported by sequence data lacked unifying morphological characters. Traditional characters such as the thickness of the conidial filament and whether conidiophores were conspicuous or reduced proved to be poor predictors of phylogenetic relationships. However some combinations of characters including conidium colour and the presence of lateral, tooth-like conidiogenous cells did appear to be predictive of genetic relationships.     

TRPV1+ sensory neurons control beta cell stress and islet inflammation in autoimmune diabetes

In type 1 diabetes, T cell-mediated death of pancreatic beta cells produces insulin deficiency. However, what attracts or restricts broadly autoreactive lymphocyte pools to the pancreas remains unclear. We report that TRPV1(+) pancreatic sensory neurons control islet inflammation and insulin resistance. Eliminating these neurons in diabetes-prone NOD mice prevents insulitis and diabetes, despite systemic persistence of pathogenic T cell pools. Insulin resistance and beta cell stress of prediabetic NOD mice are prevented when TRPV1(+) neurons are eliminated. TRPV1(NOD), localized to the Idd4.1 diabetes-risk locus, is a hypofunctional mutant, mediating depressed neurogenic inflammation. Delivering the neuropeptide substance P by intra-arterial injection into the NOD pancreas reverses abnormal insulin resistance, insulitis, and diabetes for weeks. Concordantly, insulin sensitivity is enhanced in trpv1(-/-) mice, whereas insulitis/diabetes-resistant NODxB6Idd4-congenic mice, carrying wild-type TRPV1, show restored TRPV1 function and insulin sensitivity. Our data uncover a fundamental role for insulin-responsive TRPV1(+) sensory neurons in beta cell function and diabetes pathoetiology.     

Phylogenetic relationships and convergence of helicosporous fungi inferred from ribosomal DNA sequences

Helicosporous fungi form elegant, coiled, and multicellular mitotic spores (conidia). In this paper, we investigate the phylogenetic relationships among helicosporous fungi in the asexual genera Helicoma, Helicomyces, Helicosporium, Helicodendron, Helicoon, and in the sexual genus Tubeufia (Tubeufiaceae, Dothideomycetes, and Ascomycota). We generated ribosomal small subunit and partial large subunit sequences from 39 fungal cultures. These and related sequences from GenBank were analyzed using parsimony, likelihood, and Bayesian analysis. Results showed that helicosporous species arose convergently from six lineages of fungi in the Ascomycota. The Tubeufiaceae s. str. formed a strongly supported monophyletic lineage comprising most species from Helicoma, Helicomyces, and Helicosporium. However, within the Tubeufiaceae, none of the asexual genera were monophyletic. Traditional generic characters, such as whether conidiophores were conspicuous or reduced, the thickness of the conidial filament, and whether or not conidia were hygroscopic, were more useful for species delimitation than for predicting higher level relationships. In spite of their distinctive, barrel-shaped spores, Helicoon species were polyphyletic and had evolved in different ascomycete orders. Helicodendron appeared to be polyphyletic although most representatives occurred within Leotiomycetes. We speculate that some of the convergent spore forms may represent adaptation to dispersal in aquatic environments.     

Small amounts of isotope-reinforced polyunsaturated fatty acids suppress lipid autoxidation

Polyunsaturated fatty acids (PUFAs) undergo autoxidation and generate reactive carbonyl compounds that are toxic to cells and associated with apoptotic cell death, age-related neurodegenerative diseases, and atherosclerosis. PUFA autoxidation is initiated by the abstraction of bis-allylic hydrogen atoms. Replacement of the bis-allylic hydrogen atoms with deuterium atoms (termed site-specific isotope-reinforcement) arrests PUFA autoxidation due to the isotope effect. Kinetic competition experiments show that the kinetic isotope effect for the propagation rate constant of Lin autoxidation compared to that of 11,11-D(2)-Lin is 12.8 ± 0.6. We investigate the effects of different isotope-reinforced PUFAs and natural PUFAs on the viability of coenzyme Q-deficient Saccharomyces cerevisiae coq mutants and wild-type yeast subjected to copper stress. Cells treated with a C11-BODIPY fluorescent probe to monitor lipid oxidation products show that lipid peroxidation precedes the loss of viability due to H-PUFA toxicity. We show that replacement of just one bis-allylic hydrogen atom with deuterium is sufficient to arrest lipid autoxidation. In contrast, PUFAs reinforced with two deuterium atoms at mono-allylic sites remain susceptible to autoxidation. Surprisingly, yeast treated with a mixture of approximately 20%:80% isotope-reinforced D-PUFA:natural H-PUFA are protected from lipid autoxidation-mediated cell killing. The findings reported here show that inclusion of only a small fraction of PUFAs deuterated at the bis-allylic sites is sufficient to profoundly inhibit the chain reaction of nondeuterated PUFAs in yeast.     

3,4-Diarylpiperidines as potent renin inhibitors

The discovery and SAR of a series of potent renin inhibitors possessing a novel 3,4-diarylpiperidine scaffold are described herein. The resulting compound 38 exhibit low nanomolar plasma renin IC(50), had a clean CYP 3A4 profile and displayed micromolar affinity for the hERG channel. Furthermore, it was found to be efficacious in the double transgenic rat hypertension model and show good to moderate oral bioavailability in two animal species.     

Nuclear targeting of IGF-1 receptor in orbital fibroblasts from Graves' disease: apparent role of ADAM17

Insulin-like growth factor-1 receptor (IGF-1R) comprises two subunits, including a ligand binding domain on extra- cellular IGF-1Rα and a tyrosine phosphorylation site located on IGF-1Rβ. IGF-1R is over-expressed by orbital fibroblasts in the autoimmune syndrome, Graves' disease (GD). When activated by IGF-1 or GD-derived IgG (GD-IgG), these fibroblasts produce RANTES and IL-16, while those from healthy donors do not. We now report that IGF-1 and GD-IgG provoke IGF-1R accumulation in the cell nucleus of GD fibroblasts where it co-localizes with chromatin. Nuclear IGF-1R is detected with anti-IGF-1Rα-specific mAb and migrates to approximately 110 kDa, consistent with its identity as an IGF-1R fragment. Nuclear IGF-1R migrating as a 200 kDa protein and consistent with an intact receptor was undetectable when probed with either anti-IGF-1Rα or anti-IGF-1Rβ mAbs. Nuclear redistribution of IGF-1R is absent in control orbital fibroblasts. In GD fibroblasts, it can be abolished by an IGF-1R-blocking mAb, 1H7 and by physiological concentrations of glucocorticoids. When cell-surface IGF-1R is cross-linked with (125)I IGF-1, (125)I-IGF-1/IGF-1R complexes accumulate in the nuclei of GD fibroblasts. This requires active ADAM17, a membrane associated metalloproteinase, and the phosphorylation of IGF-1R. In contrast, virally encoded IGF-1Rα/GFP fusion protein localizes equivalently in nuclei in both control and GD fibroblasts. This result suggests that generation of IGF-1R fragments may limit the accumulation of nuclear IGF-1R. We thus identify a heretofore-unrecognized behavior of IGF-1R that appears limited to GD-derived fibroblasts. Nuclear IGF-1R may play a role in disease pathogenesis.