CCR4 participation in Th type 1 (mycobacterial) and Th type 2 (schistosomal) anamnestic pulmonary granulomatous responses

CCR4 is purported to be a Th type 2 (Th2) cell-biased receptor but its functional role is unclear. Recent studies suggest that chemokine receptor expression and function are more complex in vivo and raise doubts regarding restricted CCR4 expression by Th2 cells. To address these issues, we analyzed the role of CCR4 in highly polarized models of Th type 1 (Th1) and Th2 cell-mediated pulmonary granulomas, respectively, elicited by i.v. challenge of primed mice with either mycobacterial purified protein derivative or schistosomal egg Ag-coated beads. CCR4 agonists were expressed during both responses, correlating with a shift of CCR4+ CD4+ T cells from blood to lungs. CCL22 dominated in draining nodes during the Th1 response. Analysis of CD4+ effector T cells revealed CCR4 expression and CCR4-mediated chemotaxis by both IFN-gamma and IL-4 producers. Studies of CCR4 knockout (CCR4(-/-)) mice showed partial impairment of the local type-2 cytokine response and surprisingly strong impairment of the Th1 response with abrogated IFN-gamma production during secondary but not primary challenge. Adoptive transfer indicated CCR4(-/-)CD4+ Th1 cell function was defective but this could not be reconstituted with wild-type (CCR4(+/+)) CD4+ T cells indicating involvement of another CCR4+ population. Coculture of CCR4(+/+)CD4+ T cells and CCR4(-/-) dendritic cells revealed intact IL-2 but impaired IFN-gamma production, pointing to a role for CCR4+ dendritic cells in effector cell expression. Therefore, CCR4 is not Th2-restricted and was required for sustenance and expression of the Th1 effector/memory response to mycobacterial Ags.     

Control of memory CD4 T cell recall by the CD28/B7 costimulatory pathway

The CD28/B7 costimulatory pathway is generally considered dispensable for memory T cell responses, largely based on in vitro studies demonstrating memory T cell activation in the absence of CD28 engagement by B7 ligands. However, the susceptibility of memory CD4 T cells, including central (CD62L(high)) and effector memory (T(EM); CD62L(low)) subsets, to inhibition of CD28-derived costimulation has not been closely examined. In this study, we demonstrate that inhibition of CD28/B7 costimulation with the B7-binding fusion molecule CTLA4Ig has profound and specific effects on secondary responses mediated by memory CD4 T cells generated by priming with Ag or infection with influenza virus. In vitro, CTLA4Ig substantially inhibits IL-2, but not IFN-gamma production from heterogeneous memory CD4 T cells specific for influenza hemagglutinin or OVA in response to peptide challenge. Moreover, IL-2 production from polyclonal influenza-specific memory CD4 T cells in response to virus challenge was completely abrogated by CTLA4Ig with IFN-gamma production partially inhibited. When administered in vivo, CTLA4Ig significantly blocks Ag-driven memory CD4 T cell proliferation and expansion, without affecting early recall and activation. Importantly, CTLA4Ig treatment in vivo induced a striking shift in the phenotype of the responding population from predominantly T(EM) in control-treated mice to predominantly central memory T cells in CTLA4Ig-treated mice, suggesting biased effects of CTLA4Ig on T(EM) responses. Our results identify a novel role for CD28/B7 as a regulator of memory T cell responses, and have important clinical implications for using CTLA4Ig to abrogate the pathologic consequences of T(EM) cells in autoimmunity and chronic disease.     

Growth of Escherichia coli MG1655 on LB medium: monitoring utilization of amino acids, peptides, and nucleotides with transcriptional microarrays

Analysis of gene expression data related to assimilation and biosynthesis of nitrogen-containing compounds amino acids, peptides, and nucleotides was used to monitor availability of these nutrients to Escherichia coli MG1655 growing on Luria-Bertani medium. The data indicate that free amino acids and nucleotides only transiently support the nitrogen requirement for growth and are no longer available by 3.5 h of fermentation. The resulting shortage of available nitrogen sources induces the Ntr response, which involves induction of the glnALG, glnK-amtB, dppABCDF, and oppABCDF operons as well as the genes coding for outer membrane proteins, porins OmpA and OmpC, and proteases OmpP and OmpT. The increased uptake of peptides facilitated by the products of dppABCDF, oppABCDF, ompA, ompC, ompP, and ompT alleviates nitrogen limitation of the growth.     

Human apoB overexpression and a high-cholesterol diet differently modify the brain APP metabolism in the transgenic mouse model of atherosclerosis

Epidemiological and biochemical data suggest a link between the cholesterol metabolism, the amyloid precursor protein (APP) processing and the increased cerebral beta-amyloid (Abeta) deposition in Alzheimer's disease (AD). The individual and combined effects of a high-cholesterol (HC) diet and the overexpression of the human apoB-100 gene were therefore examined on the cerebral expression and processing of APP in homozygous apoB-100 transgenic mice [Tg (apoB(+/+))], a validated model of atherosclerosis. When fed with 2% cholesterol for 17 weeks, only the wild-type mice exhibited significantly increased APP695 (123%) and APP770 (138%) mRNA levels in the cortex. The HC diet-induced hypercholesterolemia significantly increased the APP isoform levels in the membrane-bound fraction, not only in the wild-type animals (114%), but also in the Tg apoB(+/+) group (171%). The overexpression of human apoB-100 gene by the liver alone reduced the brain APP isoform levels in the membrane-bound fraction (78%), whereas the levels were increased by the combined effect of HC and the overexpression of the human apoB-100 gene (134%). The protein kinase C and beta-secretase protein levels were not altered by the individual or combined effects of these two factors. Our data indicate that the two atherogenic factors, the HC diet and the overexpression of the human apoB-100 gene by the liver, could exert different effects on the processing and expression of APP in the mice brain.     

Combining a polar resin and a pseudo-proline to optimize the solid-phase synthesis of a 'difficult sequence'.

This paper describes the optimization of a synthesis of a difficult sequence related to a 12-mer sequence of a Pan DR epitope (PADRE). Elongation was followed by on-line monitoring of the N(alpha)-Fmoc removal adapted for the batch methodology. Studying the intrinsic factors related to the peptide-resin, such as substitution level, resin nature and backbone protecting group, has led to an increase in the elongation yield and purity of the crude peptide. Optimal elongation was obtained by combining a polar resin such as PEGA and a pseudo-proline as the backbone protecting group.     

Effect of drought stress at supraoptimal temperature on polyamine concentrations in transgenic soybean with increased proline levels

The effect of drought stress at supraoptimal temperature on free proline and polyamine levels was compared in wild type and transgenic soybean (Glycine max cv. Ibis) plants having increased proline levels. Since glutamate and arginine are precursors of both proline and polyamines, it was assumed that the genetic manipulation of proline levels would also affect the polyamine levels. The proline and spermine concentrations increased, while the putrescine concentration generally decreased or did not change after the treatments in both genotypes. Following drought higher proline and lower spermine levels were detected in the transgenic plants compared to the wild type ones, which could be explained by the increased use of their common precursors for proline biosynthesis in the transgenic plants.     

Polo-like kinase and survivin are esophageal tumor-specific promoters

For developing successful cancer gene therapy strategies, tumor-specific gene delivery is essential. In this study, we used esophageal cancer (EC) cells to identify and evaluate esophageal tumor-specific gene promoters. Four genes (polo-like kinase-1/PLK, survivin/BIRC5, karyopherin alpha 2/KPNA2, and pituitary tumor transforming gene protein 1/PTTG1) were identified by a microarray analysis as highly expressed in EC cell lines vs. five normal organ tissues (liver, lung, kidney, brain, and heart). By quantitative RT-PCR, the average mRNA expression levels of these four genes in 20 primary ECs were 2.7-fold (PLK), 6.1-fold (survivin), 2.6-fold (KPNA2), and 2.4-fold (PTTG1) higher than that of each gene in 24 different normal organs. By dual luciferase assay, the promoter activity of PLK and survivin in EC cell lines was 18.9-fold and 28.5-fold higher, respectively, than in normal lung and renal cells. The promoters of PLK and survivin could be useful tools for developing EC-specific gene therapy vectors.     

Cortactin affects cell migration by regulating intercellular adhesion and cell spreading

Cortactin is an F-actin binding protein that stabilizes F-actin networks and promotes actin polymerization by activating the Arp2/3 complex. Overexpression of cortactin, as observed in several human cancers, stimulates cell migration, invasion, and experimental metastasis; however, the underlying mechanism is not understood. To investigate the importance of cortactin in cell migration, we downregulated its expression using RNA interference (RNAi). Stable downregulation of cortactin in HBL100 breast epithelial cells resulted in (i) decreased cell migration and invasion, (ii) enhanced cell-cell adhesion, and (iii) accelerated cell spreading. These phenotypic changes were reversed by expression of RNAi-resistant mouse cortactin. Cortactin colocalized with cadherin and beta-catenin in adherens junctions, consistent with its role in intercellular adhesion. Remarkably, cortactin deficiency did not affect lamellipodia formation. Instead, downregulation of cortactin in human squamous carcinoma cells that overexpress cortactin changed the cytoskeletal organization. We conclude that increased levels of cortactin, as found in human carcinomas, promote cell migration and invasion by reducing cell spreading and intercellular adhesive strength.     

Comprehensive identification of phosphorylation sites in postsynaptic density preparations

In the mammalian central nervous system, the structure known as the postsynaptic density (PSD) is a dense complex of proteins whose function is to detect and respond to neurotransmitter released from presynaptic axon terminals. Regulation of protein phosphorylation in this molecular machinery is critical to the activity of its components, which include neurotransmitter receptors, kinases/phosphatases, scaffolding molecules, and proteins regulating cytoskeletal structure. To characterize the phosphorylation state of proteins in PSD samples, we combined strong cation exchange (SCX) chromatography with IMAC. Initially, tryptic peptides were separated by cation exchange and analyzed by reverse phase chromatography coupled to tandem mass spectrometry, which led to the identification of phosphopeptides in most SCX fractions. Because each of these individual fractions was too complex to characterize completely in single LC-MS/MS runs, we enriched for phosphopeptides by performing IMAC on each SCX fraction, yielding at least a 3-fold increase in identified phosphopeptides relative to either approach alone (SCX or IMAC). This enabled us to identify at least one site of phosphorylation on 23% (287 of 1,264) of all proteins found to be present in the postsynaptic density preparation. In total, we identified 998 unique phosphorylated peptides, mapping to 723 unique sites of phosphorylation. At least one exact site of phosphorylation was determined on 62% (621 of 998) of all phosphopeptides, and approximately 80% of identified phosphorylation sites are novel.     

O-linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry

O-GlcNAc is a widespread dynamic carbohydrate modification of cytosolic and nuclear proteins with features analogous to phosphorylation. O-GlcNAc acts critically in many cellular processes, including signal transduction, protein degradation, and regulation of gene expression. However, the study of its specific regulatory functions has been limited by difficulties in mapping sites of O-GlcNAc modification. We report methods for direct enrichment and identification of in vivo O-GlcNAc-modified peptides through lectin weak affinity chromatography (LWAC) and mass spectrometry. The effectiveness of this strategy on complex peptide mixtures was demonstrated through enrichment of 145 unique O-GlcNAc-modified peptides from a postsynaptic density preparation. 65 of these O-GlcNAc-modified peptides were sequenced and belonged to proteins with diverse functions in synaptic transmission. Beta-elimination/Michael addition, MS(3) on O-GlcNAc neutral loss ions, and electron capture dissociation were shown to facilitate analysis of O-GlcNAc-modified peptides/sites from lectin weak affinity chromatography enriched postsynaptic density samples. Bassoon and Piccolo, proteins critical to synapse assembly and vesicle docking, were extensively modified by O-GlcNAc. In some cases, O-GlcNAc was mapped to peptides previously identified as phosphorylated, indicating potential interplay between these modifications. Shared substrate amino acid context was apparent in subsets of O-GlcNAc-modified peptides, including "PVST" and a novel "TTA" motif (two hydroxyl-containing amino acids adjacent to an alanine). The results suggest specific roles for O-GlcNAc modification in synaptic transmission, establish a basis for site-specific regulatory studies, and provide methods that will facilitate O-GlcNAc proteome analysis across a wide variety of cells and tissues.