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A study was conducted on the proximity relationship between the rifampicin binding domain and the phosphodiester bond. A model was developed which fixed the distances of the Escherichia coli RNA polymerase subsites from rifampicin, assuming that they are properly oriented for phosphodiester synthesis. The model clearly shows that rifampicin can inhibit the translocation event without affecting the first phosphodiester bond.

Background: The R620W variant in protein tyrosine phosphatase non-receptor 22 (PTPN22) is associated with rheumatoid arthritis (RA). The PTPN22 gene has alternatively spliced transcripts and at least two of the splice forms have been confirmed to encode different PTPN22 (LYP) proteins, but detailed information regarding expression of these is lacking, especially with regard to autoimmune diseases. Methods: We have investigated the mRNA expression of known PTPN22 splice forms with TaqMan real-time PCR in relation to ZNF592 as an endogenous reference in peripheral blood cells from three independent cohorts with RA patients (n = 139) and controls (n = 111) of Caucasian origin. Polymorphisms in the PTPN22 locus (25 SNPs) and phenotypic data (gender, disease activity, ACPA and RF status) were used for analysis. Additionally, we addressed possible effects of methotrexate treatment on PTPN22 expression. Results: We found consistent differences in the expression of the PTPN22 splice forms in unstimulated peripheral blood mononuclear cells between RA patients and normal controls. This difference was more pronounced when comparing the ratio of splice forms and was not affected by methotrexate treatment. Conclusions: Our data show that RA patients and healthy controls have a shift in balance of expression of splice forms derived from the PTPN22 gene. This balance seems not to be caused by treatment and may be of importance during immune response due to great structural differences in the encoded PTPN22 proteins. [ABSTRACT FROM AUTHOR]

Background: Asthma exacerbations remain a major unmet clinical need. The difficulty in obtaining airway tissue and bronchoalveolar lavage samples during exacerbations has greatly hampered study of naturally occurring exacerbations. This study was conducted to determine if mRNA profiling of peripheral blood mononuclear cells (PBMCs) could provide information on the systemic molecular pathways involved during asthma exacerbations. Methodology/Principal Findings: Over the course of one year, gene expression levels during stable asthma, exacerbation, and two weeks after an exacerbation were compared using oligonucleotide arrays. For each of 118 subjects who experienced at least one asthma exacerbation, the gene expression patterns in a sample of peripheral blood mononuclear cells collected during an exacerbation episode were compared to patterns observed in multiple samples from the same subject collected during quiescent asthma. Analysis of covariance identified genes whose levels of expression changed during exacerbations and returned to quiescent levels by two weeks. Heterogeneity among visits in expression profiles was examined using K-means clustering. Three distinct exacerbation-associated gene expression signatures were identified. One signature indicated that, even among patients without symptoms of respiratory infection, genes of innate immunity were activated. Antigen-independent T cell activation mediated by IL15 was also indicated by this signature. A second signature revealed strong evidence of lymphocyte activation through antigen receptors and subsequent downstream events of adaptive immunity. The number of genes identified in the third signature was too few to draw conclusions on the mechanisms driving those exacerbations. Conclusions/Significance: This study has shown that analysis of PBMCs reveals systemic changes accompanying asthma exacerbation and has laid the foundation for future comparative studies using PBMCs. [ABSTRACT FROM AUTHOR]

Neuropathic pain resulting from nerve lesions or dysfunction represents one of the most challenging neurological diseases to treat. A better understanding of the molecular mechanisms responsible for causing these maladaptive responses can help develop novel therapeutic strategies and biomarkers for neuropathic pain. We performed a miRNA expression profiling study of dorsal root ganglion (DRG) tissue from rats four weeks post spinal nerve ligation (SNL), a model of neuropathic pain. TaqMan low density arrays identified 63 miRNAs whose level of expression was significantly altered following SNL surgery. Of these, 59 were downregulated and the ipsilateral L4 DRG, not the injured L5 DRG, showed the most significant downregulation suggesting that miRNA changes in the uninjured afferents may underlie the development and maintenance of neuropathic pain. TargetScan was used to predict mRNA targets for these miRNAs and it was found that the transcripts with multiple predicted target sites belong to neurologically important pathways. By employing different bioinformatic approaches we identified neurite remodeling as a significantly regulated biological pathway, and some of these predictions were confirmed by siRNA knockdown for genes that regulate neurite growth in differentiated Neuro2A cells. In vitro validation for predicted target sites in the 39-UTR of voltage-gated sodium channel Scn11a, alpha 2/ delta1 subunit of voltage-dependent Ca-channel, and purinergic receptor P2rx ligand-gated ion channel 4 using luciferase reporter assays showed that identified miRNAs modulated gene expression significantly. Our results suggest the potential for miRNAs to play a direct role in neuropathic pain. [ABSTRACT FROM AUTHOR]

Background A phase 2a, double-blind, placebo-controlled, multicenter study was conducted to evaluate safety, tolerability, and pilot efficacy of immunization with β-amyloid(1-42) in patients with Alzheimer disease. Six immunizations were planned but were halted when meningoencephalitis was recognized as an adverse event in 6% of immunized patients. Objective To identify biomarkers associated with both the risk of meningoencephalitis and antibody responsiveness. Participants One hundred fifty-three patients with mild to moderate Alzheimer disease. Main Outcome Measure Association between response to immunization and preimmunization expression levels of 8239 messenger RNA transcripts expressed in peripheral blood mononuclear cells that had been collected at the screening visit. Results Expression patterns of genes related to apoptosis and proinflammatory pathways (tumor necrosis factor pathway in particular) were identified as biomarkers of risk for the development of meningoencephalitis. Expression patterns of genes related to protein synthesis, protein trafficking, DNA recombination, DNA repair, and cell cycle were strongly associated with IgG response to immunization. Conclusions Candidate biomarkers associated with risk of immunotherapy-related meningoencephalitis were detected in blood collected prior to treatment. In addition, a different set of biomarkers were identified that were associated with the desired outcome of IgG response. [ABSTRACT FROM AUTHOR]

Escherichia coli RNA polymerase has two sites, the i and i + 1, for the binding of the first two substrates. The i site is template- and Mg2+-independent and purine-nucleotide-specific, whereas the i + 1 site is template- and Mg2+-dependent and shows no nucleotide preference. The specificity of the i site for purine nucleotides is well in accord with the fact that most promoters initiate with a purine nucleotide. But there are a few promoters that initiate with a pyrimidine nucleotide. Dinucleotide synthesis at these promoters is completely inhibited by rifampicin. Earlier studies have failed to identify an i site for pyrimidine nucleotides. In this paper, using a fluorescent analog of UTP, namely uridine 5'-[γ-(5-sulfonic acid)naphthylamidate]-triphosphate, abbreviated as UTP[AmNS], we are able to show its binding to RNA polymerase, with a Kd of 0.8 μM, in the absence of Mg2+ and template. This suggests the presence of an i pyrimidine nucleotide site. The fact that UTP-[AmNS] is capable of initiating RNA synthesis from the i site is further evidenced by the abortive transcription analyses at the lac promoter. Fluorescence titration studies performed in the presence and absence of purine initiator molecules indicate that this site is different from the i purine site. Scatchard analysis of the above data indicates the presence of a single binding site for UTP[AmNS] in the absence of Mg2+. Moreover UTP[AmNS] binds to the core enzyme with a Kd of 3.0 μM implying that, unlike the i purine nucleotide site, the sigma protein confers a tighter binding of UTP-[AmNS] to the low-Kd site. Forster's energy transfer measurements using UTP[AmNS] as the donor and rifampicin as the acceptor have been used for estimation of the distance of the i pyrimidine nucleotide site from the rifampicin site. From these measurements, we infer that there is no direct interference of rifampicin with the first [ABSTRACT FROM AUTHOR]

On amino acid starvation, Escherichia coli cells exhibit an adaptive facility termed the stringent response. This is characterized by the production of high levels of a regulatory nucleotide, ppGpp, and concomitant curtailment in rRNA synthesis. Various studies reported earlier indicated that RNA polymerase is the site of action of ppGpp although a direct demonstration of the interaction of ppGpp with E. coli RNA polymerase is still lacking. Here we report the labelling of PpGpp with a fluorescent probe, 1-aminonapthalene-5- sulphonate (AmMS), at the terminal phosphates. AmNS-ppGpp responded much like a ppGpp molecule in an in vitro total transcription assay at selective promoters. Fluorescence titralion of the tryptophan emission of RNA polymerase by AmNS-ppGpp indicated a unique binding site in the absence of template DNA. Competition experiments showed that unlabelled ppGpp binds to the enzyme at the same site. Sigma factor seems to have no effect on this binding. The titration profile is also characterized by a single slope in the Scatchard analysis. The presence of GTP or GDP does not influence the binding of AmNS-ppGpp with RNA polymerase. Forster's distance measurement was carried out which placed AmNS-ppGpp 27Å away from the rifampicin-binding domain of RNA polymerase. [ABSTRACT FROM AUTHOR]

Abstract: Acidic mammalian chitinase (AMCase), an enzyme implicated in the pathology of asthma, is capable of chitin cleavage at a low pH optimum. The corresponding gene (CHIA) can be found in genome databases of a variety of mammals, but the enzyme properties of only the human and mouse proteins were extensively studied. We wanted to compare enzymes of closely related species, such as humans and macaques. In our attempt to study macaque AMCase, we searched for CHIA-like genes in human and macaque genomes. We found that both genomes contain several additional CHIA-like sequences. In humans, CHIA-L1 (hCHIA-L1) is an apparent pseudogene and has the highest homology to CHIA. To determine which of the two genes is functional in monkeys, we assessed their tissue expression levels. In our experiments, CHIA-L1 expression was not detected in human stomach tissue, while CHIA was expressed at high levels. However, in the cynomolgus macaque stomach tissue, the expression pattern of these two genes was reversed: CHIA-L1 was expressed at high levels and CHIA was undetectable. We hypothesized that in macaques CHIA-L1 (mCHIA-L1), and not CHIA, is a gene encoding an acidic chitinase, and cloned it, using the sequence of human CHIA-L1 as a guide for the primer design. We named the new enzyme MACase (Macaca Acidic Chitinase) to emphasize its differences from AMCase. MACase shares a similar tissue expression pattern and pH optimum with human AMCase, but is 50 times more active in our enzymatic activity assay. DNA sequence of the mCHIA-L1 has higher percentage identity to the human pseudogene hCHIA-L1 (91.7%) than to hCHIA (84%). Our results suggest alternate evolutionary paths for human and monkey acidic chitinases. [Copyright &y& Elsevier]