Low-dose antigen promotes induction of FOXP3 in human CD4+ T cells

Low Ag dose promotes induction and persistence of regulatory T cells (Tregs) in mice, yet few studies have addressed the role of Ag dose in the induction of adaptive CD4(+)FOXP3(+) Tregs in humans. To this end, we examined the level of FOXP3 expression in human CD4(+)CD25(-) T cells upon activation with autologous APCs and varying doses of peptide. Ag-specific T cells expressing FOXP3 were identified by flow cytometry using MHC class II tetramer (Tmr). We found an inverse relationship between Ag dose and the frequency of FOXP3(+) cells for both foreign Ag-specific and self Ag-specific T cells. Through studies of FOXP3 locus demethylation and helios expression, we determined that variation in the frequency of Tmr(+)FOXP3(+) T cells was not due to expansion of natural Tregs, but instead, we found that induction, proliferation, and persistence of FOXP3(+) cells was similar in high- and low-dose cultures, whereas proliferation of FOXP3(-) T cells was favored in high Ag dose cultures. The frequency of FOXP3(+) cells positively correlated with suppressive function, indicative of adaptive Treg generation. The frequency of FOXP3(+) cells was maintained with IL-2, but not upon restimulation with Ag. Together, these data suggest that low Ag dose favors the transient generation of human Ag-specific adaptive Tregs over the proliferation of Ag-specific FOXP3(-) effector T cells. These adaptive Tregs could function to reduce ongoing inflammatory responses and promote low-dose tolerance in humans, especially when Ag exposure and tolerance is transient.     

Evidence that Cd101 is an autoimmune diabetes gene in nonobese diabetic mice

We have previously proposed that sequence variation of the CD101 gene between NOD and C57BL/6 mice accounts for the protection from type 1 diabetes (T1D) provided by the insulin-dependent diabetes susceptibility region 10 (Idd10), a <1 Mb region on mouse chromosome 3. In this study, we provide further support for the hypothesis that Cd101 is Idd10 using haplotype and expression analyses of novel Idd10 congenic strains coupled to the development of a CD101 knockout mouse. Susceptibility to T1D was correlated with genotype-dependent CD101 expression on multiple cell subsets, including Foxp3(+) regulatory CD4(+) T cells, CD11c(+) dendritic cells, and Gr1(+) myeloid cells. The correlation of CD101 expression on immune cells from four independent Idd10 haplotypes with the development of T1D supports the identity of Cd101 as Idd10. Because CD101 has been associated with regulatory T and Ag presentation cell functions, our results provide a further link between immune regulation and susceptibility to T1D.     

Infectivity period of mice inoculated with human adenoviruses

Due to non-productive infections, mice are not a good model to study some human adenoviruses. However, mice provide an excellent model to study the metabolic effects of human adenovirus, Ad36. Research interest in Ad36 is increasing rapidly, and consequently an increase in the use of mice as a model is anticipated. However, little is known about the transmission potential of Ad36 from infected mice to other laboratory animals or personnel. While underestimating the infectivity could promote inadvertent spread of Ad36, overstating it could drain valuable laboratory resources and animals. Therefore, we determined the duration of infectivity in female C57BL/6J mice that were experimentally infected with human adenoviruses Ad36 or Ad2. Other uninfected mice were co-housed for one week with the experimentally-infected animals, four or eight weeks postinfection. Additionally, uninfected mice were housed in the cages of mice that were infected with Ad36, 12 weeks earlier. The presence of viral DNA in tissues was used to indicate infection of mice. Although experimentally-infected mice harboured viral DNA at least up to 12 weeks, the horizontal transmission of infection was observed in co-housed mice only up to four weeks postinfection. Thus, Ad36-infected mice should be considered potentially infective for eight weeks and appropriate handling and barrier containment should be used. After eight week postinfection, horizontal transmission appears unlikely. This information may provide guidelines for animal handling, and experimental design using Ad36, which may increase safety for laboratory personnel and reduce the number of mice required for experiments.     

Nucleotides and Phosphorylation Bi-directionally Modulate Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) Binding to the N-Methyl-d-aspartate (NMDA) Receptor Subunit GluN2B

The Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII) and the NMDA-type glutamate receptor are key regulators of synaptic plasticity underlying learning and memory. Direct binding of CaMKII to the NMDA receptor subunit GluN2B (formerly known as NR2B) (i) is induced by Ca²⁺/CaM but outlasts this initial Ca²⁺-stimulus, (ii) mediates CaMKII translocation to synapses, and (iii) regulates synaptic strength. CaMKII binds to GluN2B around S1303, the major CaMKII phosphorylation site on GluN2B. We show here that a phospho-mimetic S1303D mutation inhibited CaM-induced CaMKII binding to GluN2B in vitro, presenting a conundrum how binding can occur within cells, where high ATP concentration should promote S1303 phosphorylation. Surprisingly, addition of ATP actually enhanced the binding. Mutational analysis revealed that this positive net effect was caused by four modulatory effects of ATP, two positive (direct nucleotide binding and CaMKII T286 autophosphorylation) and two negative (GluN2B S1303 phosphorylation and CaMKII T305/6 autophosphorylation). Imaging showed positive regulation by nucleotide binding also within transfected HEK cells and neurons. In fact, nucleotide binding was a requirement for efficient CaMKII interaction with GluN2B in cells, while T286 autophosphorylation was not. Kinetic considerations support a model in which positive regulation by nucleotide binding and T286 autophosphorylation occurs faster than negative modulation by GluN2B S1303 and CaMKII T305/6 phosphorylation, allowing efficient CaMKII binding to GluN2B despite the inhibitory effects of the two slower reactions.     

Cohesin mediates chromatin interactions that regulate mammalian β-globin expression

The β-globin locus undergoes dynamic chromatin interaction changes in differentiating erythroid cells that are thought to be important for proper globin gene expression. However, the underlying mechanisms are unclear. The CCCTC-binding factor, CTCF, binds to the insulator elements at the 5' and 3' boundaries of the locus, but these sites were shown to be dispensable for globin gene activation. We found that, upon induction of differentiation, cohesin and the cohesin loading factor Nipped-B-like (Nipbl) bind to the locus control region (LCR) at the CTCF insulator and distal enhancer regions as well as at the specific target globin gene that undergoes activation upon differentiation. Nipbl-dependent cohesin binding is critical for long-range chromatin interactions, both between the CTCF insulator elements and between the LCR distal enhancer and the target gene. We show that the latter interaction is important for globin gene expression in vivo and in vitro. Furthermore, the results indicate that such cohesin-mediated chromatin interactions associated with gene regulation are sensitive to the partial reduction of Nipbl caused by heterozygous mutation. This provides the first direct evidence that Nipbl haploinsufficiency affects cohesin-mediated chromatin interactions and gene expression. Our results reveal that dynamic Nipbl/cohesin binding is critical for developmental chromatin organization and the gene activation function of the LCR in mammalian cells.     

Using quantitative redox proteomics to dissect the yeast redoxome

To understand and eventually predict the effects of changing redox conditions and oxidant levels on the physiology of an organism, it is essential to gain knowledge about its redoxome: the proteins whose activities are controlled by the oxidation status of their cysteine thiols. Here, we applied the quantitative redox proteomic method OxICAT to Saccharomyces cerevisiae and determined the in vivo thiol oxidation status of almost 300 different yeast proteins distributed among various cellular compartments. We found that a substantial number of cytosolic and mitochondrial proteins are partially oxidized during exponential growth. Our results suggest that prevailing redox conditions constantly control central cellular pathways by fine-tuning oxidation status and hence activity of these proteins. Treatment with sublethal H(2)O(2) concentrations caused a subset of 41 proteins to undergo substantial thiol modifications, thereby affecting a variety of different cellular pathways, many of which are directly or indirectly involved in increasing oxidative stress resistance. Classification of the identified protein thiols according to their steady-state oxidation levels and sensitivity to peroxide treatment revealed that redox sensitivity of protein thiols does not predict peroxide sensitivity. Our studies provide experimental evidence that the ability of protein thiols to react to changing peroxide levels is likely governed by both thermodynamic and kinetic parameters, making predicting thiol modifications challenging and de novo identification of peroxide sensitive protein thiols indispensable.     

Multipotent progenitor cells derived from adult peripheral blood of swine have high neurogenic potential in vitro

Peripheral blood-derived multipotent adult progenitor cells (PBD-MAPCs) are a novel population of stem cells, isolated from venous blood of green fluorescent protein transgenic swine, which proliferate as multicellular non-adherent spheroids. Using a simple differentiation protocol, a large proportion of these cells developed one of five distinct neural cell phenotypes, indicating that these primordial cells have high neurogenic potential. Cells exhibiting neural morphologies developed within 48 h of exposure to differentiation conditions, increased in percentage over 2 weeks, and stably maintained the neural phenotype for three additional weeks in the absence of neurogenic signaling molecules. Cells exhibited dynamic neural-like behaviors including extension and retraction of processes with growth cone-like structures rich in filamentous actin, cell migration following a leading process, and various cell-cell interactions. Differentiated cells expressed neural markers, NeuN, β-tubulin III and synaptic proteins, and progenitor cells expressed the stem cell markers nestin and NANOG. Neurally differentiated PBD-MAPCs exhibited voltage-dependent inward and outward currents and expressed voltage-gated sodium and potassium channels, suggestive of neural-like membrane properties. PBD-MAPCs expressed early neural markers and developed neural phenotypes when provided with an extracellular matrix of laminin without the addition of cytokines or growth factors, suggesting that these multipotent cells may be primed for neural differentiation. PBD-MAPCs provide a model for understanding the mechanisms of neural differentiation from non-neural sources of adult stem cells. A similar population of cells, from humans or xenogeneic sources, may offer the potential of an accessible, renewable and non-tumorigenic source of stem cells for treating neural disorders.     

Early evolution within kinetoplastids (euglenozoa), and the late emergence of trypanosomatids

Many important relationships amongst kinetoplastids, including the position of trypanosomatids, remain uncertain, with limited taxon sampling of markers other than small subunit ribosomal RNA (SSUrRNA). We report gene sequences for cytosolic heat shock proteins 90 and/or 70 (HSP90, HSP70) from the potentially early-diverging kinetoplastids Ichthyobodo necator and Rhynchobodo sp., and from bodonid clades ‘2’ (Parabodonidae) and ‘3’ (Eubodonidae). Some of the new cytosolic HSP70 sequences represent a distinct paralog family (HSP70-B), which is related to yet another paralog known from trypanosomatids (HSP70-C). The (HSP70-B, HSP70-C) clade seemingly diverged before the separation between kinetoplastids and diplonemids. Protein phylogenies support the basal placement of Ichthyobodo within kinetoplastids. Unexpectedly, Rhynchobodo usually forms the next most basal group, separated from the clade ‘1’ bodonids with which it has been allied. Bootstrap support is often weak, but the possibility that Rhynchobodo represents a separate early-diverging lineage within core kinetoplastids deserves further testing. Trypanosomatids always fall remote from the root of kinetoplastids, forming a specific relationship with bodonid clades 2 (and 3), generally with strong bootstrap support. These protein trees with improved taxon sampling provide the best evidence to date for a ‘late’ emergence of trypanosomatids, contradicting recent SSUrRNA-based proposals for a relatively early divergence of this group.     

The subunit structure and dynamics of the 20S proteasome in chicken skeletal muscle

We have succeeded in purifying the 20S core proteasome particle from less than 1 g of skeletal muscle in a rapid process involving two chromatographic steps. The individual subunits were readily resolved by two-dimensional PAGE, and the identities of each of the 14 subunits were assigned by a combination of peptide mass fingerprinting and MS/MS/de novo sequencing. To assess the dynamics of proteasome biogenesis, chicks were fed a diet containing stable isotope-labeled valine, and the rate of incorporation of label into valine-containing peptides derived from each subunit was assessed by mass spectrometric analysis after two-dimensional separation. Peptides containing multiple valine residues from the 20S proteasome and other soluble muscle proteins were analyzed to yield the relative isotope abundance of the precursor pool, a piece of information that is essential for calculation of turnover parameters. The rates of synthesis of each subunit are rather similar, although there is evidence for high turnover subunits in both the alpha (nonproteolytic) and beta (proteolytic) rings. The variability in synthesis rate for the different subunits is consistent with a model in which some subunits are produced in excess, whereas others may be the rate-limiting factor in the concentration of 20S subunits in the cell. The ability to measure turnover rates of proteins on a proteome-wide scale in protein assemblies and in a complex organism provides a new dimension to the understanding of the dynamic proteome.