We have developed a simple and rapid method for preparing DNA-binding protein extracts from mammalian cells. The protocol is derived from the large scale procedure of Dignam et al. (1) and utilizes hypotonic lysis followed by high salt extraction of nuclei. The technique described by Dignam has several drawbacks, including the need for large numbers of cells and lengthy incubation and dialysis steps. It is labor-intensive and precludes preparation of multiple samples simultaneously. Our aim in developing this micropreparation procedure was to easily and rapidly extract DNA-binding proteins from small numbers of cells. Frequently, the quantity of cells available for extraction of DNA-binding proteins is limiting, as in analysis of clinical samples, of multiple clones of transfected cells, or of COS cell pools transiently transfected with a cDNA expression library. Ideally, such a technique would allow processing of many samples simultaneously and quickly on the benchtop. The method described in this report accomplishes these goals. In addition, it gives an excellent yield of DNA-binding proteins, comparable to that of the large scale Dignam protocol with minimal proteolysis. We typically start with between 5X10 and 10 cells. All centrifugations of less than 30 seconds are carried out in a room temperature microfuge; between steps, the samples are placed on ice. Adherent cells are scraped into 1.5 ml of cold phosphatebuffered saline (PBS); non-adherent cells are pelleted and resuspended in 1.5 ml cold PBS. The cell suspension is then transferred to a microfuge tube. Cells are pelleted for 10 seconds and resuspended in 400 yX cold Buffer A (10 mM HEPES-KOH pH 7.9 at 4°C, 1.5 mm MgCl2, 10 mM KC1, 0.5 mM dithiothreitol, 0.2 mM PMSF) by flicking the tube. The cells are allowed to swell on ice for 10 minutes, and then vortexed for 10 seconds. Samples are centrifuged for 10 seconds, and the supernatant fraction is discarded. The pellet is resuspended in 20—100 y\ (according to starting number of cells) of cold Buffer C (20 mM HEPES-KOH pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM PMSF) and incubated on ice for 20 min for high-salt extraction. Cellular debris is removed by centrifugation for 2 minutes at 4C and the supernatant fraction (containing DNA binding proteins) is stored at -70°C. The yield is 50 to 75 /tg protein per 10 cells.

/pdf/a-rapid-micropreparation-technique-for-extraction-of-dna-3oj1p90299.pdf

A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells.

The colony-stimulating factors (CSFs) are a group of cytokines central to the hematopoiesis of blood cells, the modulation of their functional responses, as well as the maintenance of homeostasis and overall immune competence. This group consists of the macrophage-CSF (M-CSF), granulocyte-CSF (G-CSF), granulocyte/macrophage-CSF (GM-CSF), and multi-CSF (IL-3). M-CSF and G-CSF are relatively lineage-specific, having a role in the proliferation, differentiation, and survival of macrophages, neutrophils, and their precursors. In contrast, GM-CSF and multi-CSF function at earlier stages of lineage commitment regulating the expansion and maturation of primitive hematopoietic progenitors. Colony stimulating factor production and degradation are strictly controlled, thus allowing for effective modulation of their biological functions in steady-state conditions as well as under periods of stress. Moreover, the mechanisms behind their expression and that of their cognate receptors ensures that their actions are tightly coordinated, within the context of a network of complex but finely tuned regulatory pathways derived from a variety of local and endocrine hematopoietic regulators. In this review we present some of the most salient information on CSF biology collected over the last three decades. We examine the gene and protein structure of each of the four CSFs and their corresponding receptors, and consider the main determinants behind their biological activities. The components responsible for their functional redundancy as well as the mechanisms that mediate their specificity are also discussed. Although most of available knowledge about CSFs is on human and mouse CSFs, an attempt was made to integrate recent findings in other systems in order to highlight a more widespread role for CSFs throughout evolution.

Regulation of myeloid development and function by colony stimulating factors.

The mechanisms regulating the development and function of the immune system are diverse and complicated. The signaling pathways and target genes that become activated upon cell-surface stimulation are currently being defined, and transcription factor activator protein 1 (AP-1) is proving to be an important regulator of nuclear gene expression in leukocytes. In vitro and in vivo studies have demonstrated that AP-1 expression is induced after a diverse range of stimuli and that AP-1 contributes to the regulation of a large number of genes. In this review we will examine the role of AP-1 during leukocyte activation and differentiation in the immune system.

https://jlb.onlinelibrary.wiley.com/doi/pdf/10.1002/jlb.63.2.139

Transcriptional regulation in the immune system: all roads lead to AP-1.

Background:Volatile anesthetics modulate lymphocyte function during surgery, and this compromises postoperative immune competence. The current work was undertaken to examine whether volatile anesthetics induce apoptosis in human T lymphocytes and what apoptotic signaling pathway might be used.Method

Volatile anesthetics induce caspase-dependent, mitochondria-mediated apoptosis in human T lymphocytes in vitro

Over the past decade, there has been an exponential increase in our knowledge of how cytokines regulate signal transduction, cell cycle progression, differentiation and apoptosis. Research has focused on different biochemical and genetic aspects of these processes. Initially, cytokines were identified by clonogenic assays and purified by biochemical techniques. This soon led to the molecular cloning of the genes encoding the cytokines and their cognate receptors. Determining the structure and regulation of these genes in normal and malignant hematopoietic cells has furthered our understanding of neoplastic transformation. Furthermore, this has allowed the design of modified cytokines which are able to stimulate multiple receptors and be more effective in stimulating the repopulation of hematopoietic cells after myelosuppressive chemotherapy. The mechanisms by which cytokines transduce their regulatory signals have been evaluated by identifying the involvement of specific protein kinase cascades and their downstream transcription factor targets. The effects of cytokines on cell cycle regulatory molecules, which either promote or arrest cell cycle progression, have been more recently examined. In addition, the mechanisms by which cytokines regulate apoptotic proteins, which mediate survival vs death, are being elucidated. Identification and characterization of these complex, interconnected pathways has expanded our knowledge of leukemogenesis substantially. This information has the potential to guide the development of therapeutic drugs designed to target key intermediates in these pathways and effectively treat patients with leukemias and lymphomas. This review focuses on the current understanding of how hematopoietic cytokines such as IL-3, as well as its cognate receptor, are expressed and the mechanisms by which they transmit their growth regulatory signals. The effects of aberrant regulation of these molecules on signal transduction, cell cycle regulatory and apoptotic pathways in transformed hematopoietic cells are discussed. Finally, anti-neoplastic drugs that target crucial constituents in these pathways are evaluated.

Signal transduction, cell cycle regulatory, and anti-apoptotic pathways regulated by IL-3 in hematopoietic cells : possible sites for intervention with anti-neoplastic drugs

The human interleukin 3 (IL-3) promoter is comprised of several cis-acting DNA sequences that modulate T-cell expression of IL-3. These are located within 315 nucleotides upstream of the mRNA start site. Transient expression of reporter genes linked to serially deleted sequences of the IL-3 promoter has allowed mapping of two activator sequences and an interposed repressor sequence. The proximal regulatory region is specific to IL-3 and prerequisite for efficient transcription. Its effect is enhanced by a second, more distal activating sequence consisting of an AP-1 binding site. Between the two activators lies a transcriptional silencer, which is a potent repressor in the absence of the AP-1 site. DNA-nuclear protein binding experiments demonstrate specific complex formation within each of these functional regions. Thus, both positive and negative regulatory elements appear to control expression of the human IL-3 gene in activated T cells.

/pdf/positive-and-negative-elements-regulate-human-interleukin-3-j22soqx7wn.pdf

Heather S. Murphy

Papers

Positive and negative elements regulate human interleukin 3 expression.