Purification and Characterization of an Inhibitor of Thymidine Uptake From Culture Supernatants of Human Tonsil Lymphocytes

Lymphocytes from human tonsils were cultured in the absence of serum for 3 days. In the presence of the concentrated culture supernatant the proliferative response of PBL, to con A, as measured by the uptake of (\u273)H-tdr, was significantly reduced. The suppressor substance was referred to as SMAL (suppressor of mitogen activated lymphocytes). The estimated molecular weight of SMAL under nondenaturing conditions was 100,000-300,000. SMAL also suppressed the incorporation of (\u273)H-tdr by a variety of mouse and human tumor cell lines. The activity of SMAL was sensitive to pronase and heating at 100(DEGREES)C for 30 minutes but insensitive to RNase. Treatment with DNase, however, enhanced the activity of SMAL. SMAL activity was also destroyed by treatment with 5% TCA, 0.4 M HCl or 60% acetonitrile, but resistant to 6 M urea or dialysis against pH 2 buffer for 24 hours. SMAL activity was precipitated in 40-80% ammonium sulfate saturation. When applied to a phenyl-sepharose column no activity was recovered. SMAL was not produced by heat-killed tonsil lymphocytes or lymphocytes-treated with cycloheximide. Maximal production occurred in the first 24 hours of culture, and progressively less was produced in subsequent 24-hour intervals. Both T- and B lymphocyte-enriched culture supernatants contained SMAL. SMAL adhered strongly to DEAE-cellulose, but less than two-fold purification was achieved. Using QMA-Accell anion exchange medium, a 5-fold purification of SMAL with higher specific activity was obtained with HPLC. Activity of SMAL was recovered after native polyacrylamide gel electrophoresis by electroblotting to DEAE-cellulose paper followed by eluting the bound materials with salt. Two active components, one corresponding to a large and/or less negatively charged molecule and another corresponding to a small and/or highly acidic molecule, were recovered. HPLC-purified SMAL at relatively low doses inhibited the uptake and phosphorylation of (\u273)H-tdr, without significant effect on cell proliferation. The inhibition of (\u273)H-tdr uptake was favored over that of (\u273)H-udr or (\u273)H-adr, and this effect was reversible. At relatively high doses of HPLC-purified SMAL, the growth of mouse thymoma EL-4 and human T cell leukemia CEM-CM(,3) cell lines was inhibited

Ikaros represses and activates PU.1 cell-type-specifically through the multifunctional Sfpi1 URE and a myeloid specific enhancer

Generation of myeloid and lymphoid cells from progenitors involves dynamic changes in transcription factor expression and use, and disruption of hematopoietic transcription factor function and expression can contribute to leukemic transformation. PU.1 and Ikaros are pivotal factors whose expression and utilization are dynamically altered during hematopoietic development. Here, we demonstrate that expression of PU.1, encoded by the Sfpi1 gene, is divergently regulated by Ikaros in distinct cell type-specific contexts. Chromatin immune precipitation analysis and functional perturbations revealed that Ikaros can directly repress or activate Sfpi1 transcription via different PU.1 cis-elements, with PU.1 and Ikaros collaborating at myeloid-specific elements but not at other elements. Our results thus shed light on how PU.1 and Ikaros can act as lineage competency factors to facilitate both myeloid and lymphoid developmental programs

Cell-Type-Specific Activation and Repression of PU.1 by a Complex of Discrete, Functionally Specialized cis-Regulatory Elements

The transcription factor PU.1 is critical for multiple hematopoietic lineages, but different leukocyte types require strictly distinct patterns of PU.1 regulation. PU.1 is required early for T-cell lineage development but then must be repressed by a stage-specific mechanism correlated with commitment. Other lineages require steady, low expression or upregulation. Until now, only the promoter plus a distal upstream regulatory element (URE) could be invoked to explain nearly all Sfpi1 (PU.1) activation and repression, including bifunctional effects of Runx1. However, the URE is dispensable for most Sfpi1 downregulation in early T cells, and we show that it retains enhancer activity in immature T-lineage cells even where endogenous Sfpi1 is repressed. We now present evidence for another complex of conserved noncoding elements that mediate discrete, cell-type-specific regulatory features of Sfpi1, including a myeloid cell-specific activating element and a separate, pro-T-cell-specific silencer element. These elements yield opposite, cell-type-specific responses to Runx1. T-cell-specific repression requires Runx1 acting through multiple nonconsensus sites in the silencer core. These newly characterized sites recruit Runx1 binding in early T cells in vivo and define a functionally specific scaffold for dose-dependent, Runx-mediated repression

A gene regulatory network armature for T lymphocyte specification

Choice of a T lymphoid fate by hematopoietic progenitor cells depends on sustained Notch–Delta signaling combined with tightly regulated activities of multiple transcription factors. To dissect the regulatory network connections that mediate this process, we have used high-resolution analysis of regulatory gene expression trajectories from the beginning to the end of specification, tests of the short-term Notch dependence of these gene expression changes, and analyses of the effects of overexpression of two essential transcription factors, namely PU.1 and GATA-3. Quantitative expression measurements of >50 transcription factor and marker genes have been used to derive the principal components of regulatory change through which T cell precursors progress from primitive multipotency to T lineage commitment. Our analyses reveal separate contributions of Notch signaling, GATA-3 activity, and down-regulation of PU.1. Using BioTapestry (www.BioTapestry.org), the results have been assembled into a draft gene regulatory network for the specification of T cell precursors and the choice of T as opposed to myeloid/dendritic or mast-cell fates. This network also accommodates effects of E proteins and mutual repression circuits of Gfi1 against Egr-2 and of TCF-1 against PU.1 as proposed elsewhere, but requires additional functions that remain unidentified. Distinctive features of this network structure include the intense dose dependence of GATA-3 effects, the gene-specific modulation of PU.1 activity based on Notch activity, the lack of direct opposition between PU.1 and GATA-3, and the need for a distinct, late-acting repressive function or functions to extinguish stem and progenitor-derived regulatory gene expression

Regulatory network discovery using heuristics

This thesis improves the GRN discovery process by integrating heuristic information via a co-regulation function, a post-processing procedure, and a Hub Network algorithm to build the backbone of the network.Doctor of Philosoph

Using partitioning and non-partitioning clustering algorithms for included proteins sequences in esophagus, stomach and colon cancer

A thorough recognition of the nature and duties of the genes is based upon having adequate information about the proteins. However, the proteomic projects follow a slow trend; therefore, solving the protein-related problems has become as one of the most important challenges in bio-informatics. Consequently, the presence of tools which can enhance the structural recognition, classification, and interpretation of proteins will be advantageous. Statistical methods are among the tools to help solve bio-informatics problems. These methods may be used to help predict the third structures of proteins, study proteins collectively, as well as extract new interactions among the protein collections. One of the very efficient and useful methods in the collective study of protein subsets is the cluster analysis. In the present study, the recognized protein sequences related to esophagus, stomach, and colon cancers are analyzed through partitioning, non-partitioning, and fuzzy clustering methods. Needleman-Wunsch global alignment algorithm was used to determine pair-wise similarities. The evaluations have shown that the clusters obtained through using the AGNES method have produced more powerful structures; yet, it can be said that the PAM clustering method, compared to other ones, has produced the best results in predicting ability of the 3D structure of the unknown protein sequences

The effects of booster sessions on self-management interventions for chronic musculoskeletal pain: a systematic review and meta-analysis of randomised controlled trials

Our objective was to investigate the effectiveness of booster sessions after self-management interventions as a means of maintaining self-management behaviours in the treatment of chronic musculoskeletal pain. We searched MEDLINE, EMBASE, Science Citation Index, Cochrane Central Register of Controlled Trials and PsycINFO. Two authors independently identified eligible trials and collected data. We calculated the odds ratio (OR) for the analyses of dichotomous data, and standardised mean differences (SMD) with 95% confidence interval (CI) for continuous variables. Our search identified 14 studies with a total of 1695 patients. All studies were at high risk of bias and provided very low quality evidence. For the primary outcomes, booster sessions had no evidence of an effect on improving patient-reported outcomes on physical function (SMD-0.13, 95%CI -0.32 to -0.06; P=0.18), pain-related disability (SMD-0.16, 95%CI -0.36 to 0.03; P=0.11) and pain self-efficacy (SMD 0.15, 95%CI -0.07 to 0.36; P=0.18). For the secondary outcomes, booster sessions caused a significant reduction in patient-reported pain catastrophising (SMD-0.42, 95%CI -0.64 to -0.19; P=0.0004), and no evidence of an effect on patient-reported pain intensity, depression, coping or treatment adherence. There is currently little evidence that booster sessions are an effective way to prolong positive treatment effects or improve symptoms of long-term musculoskeletal conditions following self-management interventions. However, the studies were few with high heterogeneity, high risk of bias and overall low quality of evidence. Our review argues against including booster sessions routinely to self-management interventions for the purpose of behaviour maintenance