Blunted expression of miR-199a-5p in regulatory T cells of patients with chronic obstructive pulmonary disease compared to unaffected smokers.

Chronic obstructive pulmonary disease (COPD) is characterized by an abnormal regulatory T cell (T(reg)) response and increases in T helper type 1 (Th1) and Th17 cell responses. It is unclear if dysregulation of microRNAs (miRNA) within T(reg) cells contributes to the abnormal inflammatory response in COPD. In this study, we aimed to compare the miRNA profile of COPD T(reg) cells with that of healthy controls and to explore the function of differentially expressed miRNAs. We first obtained T(reg) and T effector cells (Teff ) from peripheral blood of non-smokers, unaffected current smokers and COPD current smokers. Then, we assessed their miRNA expression by microarray analysis followed by real-time reverse transcription-polymerase chain reaction (RT-PCR) validation of particular miRNAs. Six and 96 miRNAs were expressed differentially in COPD T(reg) cells versus T(reg) cells of healthy non-smokers and healthy smokers, whereas no differences were found in miRNA expression in T(eff) cells. We found that miR-199a-5p was repressed by approximately fourfold in T(reg) cells of COPD patients compared to healthy smokers (P < 0·05). In addition, miR-199a-5p was over-expressed in T(reg) cells compared to Teff cells (P < 0·001) and had significant over-representation of its target genes in the T(reg) transcriptome, being associated with the transforming growth factor (TGF)-ß activation pathway (P < 0·01). We also confirmed the function of miR-199a5p in an in-vitro loss-of-function cell model running TaqMan® arrays of the human TGF-ß pathway. These findings suggest that the abnormal repression of miR-199a-5p in patients with COPD compared to unaffected smokers may be involved in modulating the adaptive immune balance in favour of a Th1 and Th17 response.

Virtual 2-D gel electrophoresis: visualization and analysis of the E. coli proteome by mass spectrometry.

Mass spectrometric surface analysis of isoelectric focusing gels provides an ultrasensitive approach to proteome analysis. This "virtual 2-D gel" approach, in which mass spectrometry is substituted for the size-based separation of SDS-PAGE, provides advantages in mass resolution and accuracy over classical 2-D gels and can be readily automated. Protein identities can be postulated from molecular mass (+/-0.1-0.2% for proteins of <50 kDa in size) and pI (+/-0.3 pH unit) and confirmed by MALDI in-source decay of the intact protein (providing sequence spanning up to 43 residues) or by peptide mass mapping following gel-wide chemical cleavage. Additionally, posttranslational modifications such as fatty acid acylation can be detected by the mass-resolved heterogeneity of component hydrocarbon chains. Sensitivity was evaluated by comparing the number of proteins detected by this method to equivalently loaded silver-stained 2-D gels. In the 5.7-6.0 pH range, E. coli is predicted to contain 435 proteins; virtual 2-D gels found 250 proteins ranging from >2 to <120 kDa in size present at levels to tens of femtomoles, as compared to the 100 proteins found by silver-staining 2-D gels. Extrapolating this result to the total theoretical proteome suggests that this technology is capable of detecting over 2500 E. coli proteins.

Predicting splice variant from DNA chip expression data.

Alternative splicing of premessenger RNA is an important layer of regulation in eukaryotic gene expression. Splice variation of a large number of genes has been implicated in various cell growth and differentiation processes. To measure tissue-specific splicing of genes on a large scale, we collected gene expression data from 11 rat tissues using a high-density oligonucleotide array representing 1600 rat genes. Expression of each gene on the chip is measured by 20 pairs of independent oligonucleotide probes. Two algorithms have been developed to normalize and compare the chip hybridization signals among different tissues at individual oligonucleotide probe level. Oligonucleotide probes (the perfect match [PM] probe of each probe pair), detecting potential tissue-specific splice variants, were identified by the algorithms. The identified candidate splice variants have been compared to the alternatively spliced transcripts predicted by an EST clustering program. In addition, 50% of the top candidates predicted by the algorithms were confirmed by RT-PCR experiment. The study indicates that oligonucleotide probe-based DNA chip assays provide a powerful approach to detect splice variants at genome scale.

Escherichia coli proteome analysis using the gene-protein database.

The gene-protein database of Escherichia coli is a collection of data, largely generated from the separation of complex mixtures of cellular proteins on two-dimensional (2-D) polyacrylamide gel electrophoresis. The database currently contains about 1600 protein spots. The data are comprised of both identification information for many of these proteins and data on how the level or synthesis rates of proteins vary under different growth conditions. Three projects are underway to further elucidate the E. coli proteome including a project to localize on 2-D gels all of the open reading framed encoded by the E. coli chromosome, a project to determine the condition(s) under which each open reading frame is expressed and a project to determine the abundance and location of each protein in the cell. Applications for proteome databases for cell modeling are discussed and examples of applications in therapeutic drug discovery are given.

Unique identification of proteins from small genome organisms: theoretical feasibility of high throughput proteome analysis.

We evaluate current levels of accuracy for estimation of molecular weight (Mr) and isoelectric point (pI) to proteins on two-dimensional (2-D) gels as well as the distribution and clustering of proteins in the predicted proteome of E. coli. We also examine the ability to find single candidates within the predicted proteome for matching to a protein seen on 2-D gels, based on the current level of accuracy. We discuss the levels of accuracy needed to match predicted proteins to observed proteins based solely on Mr and pI criteria obtained from genomic information, and propose methodology to achieve this level of accuracy. In addition, we will address the future goals of this work since the small genomes of bacteria provide a foundation and stepping stone to similar studies in higher organisms.

Identification of a control region for expression of the forespore-specific Bacillus subtilis locus spoVA.

The role of a 20 bp conserved region located 45-64 nucleotides 5' of the spoVA transcription start point in Bacillus subtilis and Bacillus licheniformis was investigated by deletion analysis and by mobility shift assay. Deletions 5' of this conserved sequence had little effect on expression of a spoVA-lacZ fusion, whereas deletions extending into the sequence reduced expression of the spoVA-lacZ fusion by 85%. The timing of expression of spoVA was not affected by deletion of the sequence. The region was shown by mobility shift assays to bind specifically to a protein. Binding activity was detected in protein extracts prepared from bacteria 1 h or more after they had started to sporulate, but not in extracts prepared from vegetative bacteria. Mutations in all known spoO loci were screened but none prevented appearance of the binding activity; nor did mutations in any of the stage II and III loci tested. It is concluded that the 20 bp conserved region is the binding site of an activator that is subject to temporal regulation independent of known spo loci.

Identification of the promoter and the transcriptional start site of the spoVA operon of Bacillus subtilis and Bacillus licheniformis.

The region upstream of the coding sequence of the spoVA operon was studied by several techniques to identify the promoter and to determine the start point for transcription of spoVA. The results of plasmid integration analysis in Bacillus subtilis showed that no more than 119 bp upstream of the coding sequence is needed for expression. A comparison of the sequence of this upstream region with the corresponding sequence from Bacillus licheniformis showed four stretches that were perfectly conserved, interspersed with poorly conserved stretches; the second and third of these conserved stretches appeared to represent the '-35' and '-10' regions of a promoter recognized by RNA polymerase containing sigma G. Primer extension analysis in B. subtilis revealed a spoVA transcript which had apparently initiated 6 bp downstream of the putative '-10' heptanucleotide CATACTA, that is, 27 bp upstream of the coding sequence. This transcript was observed 4 h and 5 h after the initiation of sporulation, but not at earlier times.