Posttranslational modification of gluten shapes TCR usage in celiac disease

Posttranslational modification of Ag is implicated in several autoimmune diseases. In celiac disease, a cereal gluten-induced enteropathy with several autoimmune features, T cell recognition of the gluten Ag is heavily dependent on the posttranslational conversion of Gln to Glu residues. Evidence suggests that the enhanced recognition of deamidated gluten peptides results from improved peptide binding to the MHC and TCR interaction with the peptide-MHC complex. In this study, we report that there is a biased usage of TCR Vβ6.7 chain among TCRs reactive to the immunodominant DQ2-α-II gliadin epitope. We isolated Vβ6.7 and DQ2-αII tetramer-positive CD4(+) T cells from peripheral blood of gluten-challenged celiac patients and sequenced the TCRs of a large number of single T cells. TCR sequence analysis revealed in vivo clonal expansion, convergent recombination, semipublic response, and the notable conservation of a non-germline-encoded Arg residue in the CDR3β loop. Functional testing of a prototype DQ2-α-II-reactive TCR by analysis of TCR transfectants and soluble single-chain TCRs indicate that the deamidated residue in the DQ2-α-II peptide poses constraints on the TCR structure in which the conserved Arg residue is a critical element. The findings have implications for understanding T cell responses to posttranslationally modified Ags.     

Use of lactose to induce expression of soluble NifA protein domains of Herbaspirillum seropedicae in Escherichia coli

Overexpression and purification are procedures used to allow functional and structural characterization of proteins. Many overexpressed proteins are partially or completely insoluble, and can not be easily purified. The NifA protein is an enhancer-binding protein involved in activating the expression of nif and some fix genes. The NifA protein from many organisms is usually insoluble when over-expressed, and therefore difficult to work with in vitro. In this work we have overexpressed the central + C-terminal and the central domains of the Herbaspirrilum seropedicae NifA protein in an Escherichia coli background. Expression was induced with either IPTG or lactose. The data showed that induction with lactose promoted a significantly higher percentage of these proteins in the soluble fraction than with IPTG. This probably reflects a slower kinetics of induction by lactose.     

Exercise Improves Body Fat,Lean Mass,and Bone Mass in Breast Cancer Survivors

Given the negative effects of a breast cancer diagnosis and its treatments on body weight and bone mass, we investigated the effects of a 6‐month randomized controlled aerobic exercise intervention vs. usual care on body composition in breast cancer survivors. Secondary aims were to examine the effects stratified by important prognostic and physiologic variables. Seventy‐five physically inactive postmenopausal breast cancer survivors were recruited through the Yale–New Haven Hospital Tumor Registry and randomly assigned to an exercise (n = 37) or usual care (n = 38) group. The exercise group participated in 150 min/week of supervised gym‐ and home‐based moderate‐intensity aerobic exercise. The usual care group was instructed to maintain their current physical activity level. Body composition was assessed at baseline and 6‐months through dual‐energy X‐ray absorptiometry (DXA) by one radiologist blinded to the intervention group of the participants. On an average, exercisers increased moderate‐intensity aerobic exercise by 129 min/week over and above baseline levels compared with 45 min/week among usual care participants (P < 0.001). Exercisers experienced decreases in percent body fat (P = 0.0022) and increases in lean mass (P = 0.047) compared with increases in body fat and decreases in lean mass in usual care participants. Bone mineral density (BMD) was also maintained among exercisers compared with a loss among usual care participants (P = 0.043). In summary, moderate‐intensity aerobic exercise, such as brisk walking, produces favorable changes in body composition that may improve breast cancer prognosis.     

Cell-type-specific repression of internal ribosome entry site activity by double-stranded RNA-binding protein 76

Translation of picornavirus plus-strand RNA genomes occurs via internal ribosomal entry at highly structured 5' untranslated regions. In addition to canonical translation factors, translation rate is likely influenced by supplementary host and viral trans-acting factors. We previously reported that insertion of a heterologous human rhinovirus type 2 internal ribosomal entry site (IRES) into the poliovirus (PV) genome, generating the chimeric virus PV-RIPO, selectively abrogates viral translation and propagation in neurons, which eliminate poliovirus's signature neuropathogenicity. While severely deficient in cells of neuronal lineage, the rhinovirus IRES promotes efficient propagation of PV-RIPO in cancer cells. Tumor-specific IRES function can be therapeutically exploited to direct viral cytotoxicity to cancer cells. Neuron-glioma heterokaryon analysis implicates neuronal trans-dominant inhibition in this effect, suggesting that host trans-acting factors repress IRES function in a cell-type-specific manner. We identified a set of proteins from neuronal cells with affinity for the rhinovirus IRES, including double-stranded RNA-binding protein 76 (DRBP76). DRBP76 associates with the IRES in neuronal but not in malignant glioma cells. Moreover, DRBP76 depletion in neuronal cells enhances rhinovirus IRES-driven translation and virus propagation. Our observations suggest that cell-type-specific association of DRBP76 with the rhinovirus IRES represses PV-RIPO translation and propagation in neuronal cells.     

The expression of nifB gene from Herbaspirillum seropedicae is dependent upon the NifA and RpoN proteins

The putative nifB promoter region of Herbaspirillum seropedicae contained two sequences homologous to NifA-binding site and a -24/-12 type promoter. A nifB::lacZ fusion was assayed in the backgrounds of both Escherichia coli and H. seropedicae. In E. coli, the expression of nifB::lacZ occurred only in the presence of functional rpoN and Klebsiella pneumoniae nifA genes. In addition, the integration host factor (IHF) stimulated the expression of the nifB::lacZ fusion in this background. In H. seropedicae, nifB expression occurred only in the absence of ammonium and under low levels of oxygen, and it was shown to be strictly dependent on NifA. DNA band shift experiments showed that purified K. pneumoniae RpoN and E. coli IHF proteins were capable of binding to the nifB promoter region, and in vivo dimethylsulfate footprinting showed that NifA binds to both NifA-binding sites. These results strongly suggest that the expression of the nifB promoter of H. seropedicae is dependent on the NifA and RpoN proteins and that the IHF protein stimulates NifA activation of nifB promoter.     

Cytological Detection of the c25H Deletion Involving the Albino (c) Locus on Chromosome 7 in the Mouse

A deletion of the albino (c) locus on mouse chromosome 7 has been demonstrated using Q- and G-banding methods in a mouse heterozygous for the radiation-induced lethal albino allele, c(25H). The deletion, which is thought to be 1-6 cM long, represents about 7.6% of the length of the metaphase chromosome.     

Topographic mapping from the retina to the midbrain is controlled by relative but not absolute levels of EphA receptor signaling

Topographic maps are a fundamental feature of sensory representations in nervous systems. The formation of one such map, defined by the connection of ganglion cells in the retina to their targets in the superior colliculus of the midbrain, is thought to depend upon an interaction between complementary gradients of retinal EphA receptors and collicular ephrin-A ligands. We have tested this hypothesis by using gene targeting to elevate EphA receptor expression in a subset of mouse ganglion cells, thereby producing two intermingled ganglion cell populations that express distinct EphA receptor gradients. We find that these two populations form separate maps in the colliculus, which can be predicted as a function of the net EphA receptor level that a given ganglion cell expresses relative to its neighbors.     

Diversification of the Structural Determinants of Fibroblast Growth Factor-Heparin Interactions: IMPLICATIONS FOR BINDING SPECIFICITY*

The functions of a large number (>435) of extracellular regulatory proteins are controlled by their interactions with heparan sulfate (HS). In the case of fibroblast growth factors (FGFs), HS binding determines their transport between cells and is required for the assembly of high affinity signaling complexes with their cognate FGF receptor. However, the specificity of the interaction of FGFs with HS is still debated. Here, we use a panel of FGFs (FGF-1, FGF-2, FGF-7, FGF-9, FGF-18, and FGF-21) spanning five FGF subfamilies to probe their specificities for HS at different levels as follows: binding parameters, identification of heparin-binding sites (HBSs) in the FGFs, changes in their secondary structure caused by heparin binding and structures in the sugar required for binding. For interaction with heparin, the FGFs exhibit K_D values varying between 38 nm (FGF-18) and 620 nm (FGF-9) and association rate constants spanning over 20-fold (FGF-1, 2,900,000 m⁻¹ s⁻¹ and FGF-9, 130,000 m⁻¹ s⁻¹). The canonical HBS in FGF-1, FGF-2, FGF-7, FGF-9, and FGF-18 differs in its size, and these FGFs have a different complement of secondary HBS, ranging from none (FGF-9) to two (FGF-1). Differential scanning fluorimetry identified clear preferences in these FGFs for distinct structural features in the polysaccharide. These data suggest that the differences in heparin-binding sites in both the protein and the sugar are greatest between subfamilies and may be more restricted within a FGF subfamily in accord with the known conservation of function within FGF subfamilies.     

The double-stranded RNA binding protein 76:NF45 heterodimer inhibits translation initiation at the rhinovirus type 2 internal ribosome entry site

Poliovirus (PV) plus-strand RNA genomes initiate translation in a cap-independent manner via an internal ribosome entry site (IRES) in their 5' untranslated region. Viral translation is codetermined by cellular IRES trans-acting factors, which can influence viral propagation in a cell-type-specific manner. Engineering of a poliovirus recombinant devoid of neuropathogenic properties but highly lytic in malignant glioma cells was accomplished by exchange of the cognate poliovirus IRES with its counterpart from human rhinovirus type 2 (HRV2), generating PV-RIPO. Neuroblast:glioma heterokaryon analyses revealed that loss of neurovirulence is due to trans-dominant repression of PV-RIPO propagation in neuronal cells. The double-stranded RNA binding protein 76 (DRBP76) was previously identified to bind to the HRV2 IRES in neuronal cells and to inhibit PV-RIPO translation and propagation (M. Merrill, E. Dobrikova, and M. Gromeier, J. Virol. 80:3347-3356, 2006). The results of size exclusion chromatography indicate that DRBP76 heterodimerizes with nuclear factor of activated T cells, 45 kDa (NF45), in neuronal but not in glioma cells. The DRBP76:NF45 heterodimer binds to the HRV2 IRES in neuronal but not in glioma cells. Ribosomal profile analyses show that the heterodimer preferentially associates with the translation apparatus in neuronal cells and arrests translation at the HRV2 IRES, preventing PV-RIPO RNA assembly into polysomes. Results of this study suggest that the DRBP76:NF45 heterodimer selectively blocks HRV2 IRES-driven translation initiation in neuron-derived cells.