SVISS - a novel transient gene silencing system for gene function discovery and validation in tobacco plants.

We developed a novel, two-component transient gene silencing system in which the satellite tobacco mosaic virus (STMV) is used as vector for the delivery of inhibitory RNA into tobacco plants and the tobacco mosaic virus strain U2 (TMV-U2) is used as helper virus for supplying replication and movement proteins in trans. The main advantage of the system is that by uncoupling virus replication components from silencing induction components, the intensity of silencing becomes more pronounced. We call this system satellite virus-induced silencing system (SVISS) and will demonstrate here its robustness, speed and effectiveness.

Efficient secretion of biologically active recombinant OB protein (leptin) in Escherichia coli, purification from the periplasm and characterization.

The genes encoding the mature forms of mouse (mOB) and human OB (hOB) protein (also called leptin) were fused to the secretion signal coding sequence of the Escherichia coli outer membrane protein A (sOMP A). The hybrid genes were preceded by a ribosome binding site (RBS) and were expressed under transcriptional control of both the lipoprotein promoter (Plpp) and the lac promoter-operator (POlac). The recombinant fusion proteins were efficiently expressed and exported into the periplasmic compartment of E.

Characterization of critical residues in the cytoplasmic domain of the human interleukin-5 receptor alpha chain required for growth signal transduction.

Interleukin (IL)-5 binds to a cell surface receptor composed of two polypeptide chains, alpha and beta, both belonging to the hemopoietic cytokine receptor family. Mouse cells expressing common mouse beta chain (AIC2B) that were transfected with human IL-5 receptor (R)alpha cDNA proliferated in response to picomolar concentrations of human IL-5, indicating that a functional receptor was reconstituted. We show that in these cells, human (h)IL-5 as well as mouse (m)IL-3 induce tyrosine phosphorylation of beta chain and JAK 2 kinase.

Characterization of the murine IL-5 receptor complex with the use of a panel of monoclonal antibodies. Relationship to the murine IL-3 receptor.

To obtain mAb against the murine IL-5R (mIL-5R), Wistar rats were immunized with B13 cells, a murine Ly-1+ (CD5+) pre-B cell line which is dependent on IL-3 or IL-5 for its growth. A first group of six mAb could immunoprecipitate, from detergent-lysed B13 cells, a 60-kDa polypeptide (p60) corresponding to the recently cloned mIL-5R alpha-chain. A second group of three mAb was able to immunoprecipitate a protein doublet of 130 to 140 kDa (p130 and p140) corresponding to the previously characterized mIL-3R and mIL-3R-like polypeptide, respectively.

Characterization of interleukin 5 receptors on eosinophilic sublines from human promyelocytic leukemia (HL-60) cells.

The T cell product interleukin 5 (IL-5) has been shown to be a key factor in the development and the maturation of the eosinophilic cell lineage. We report here on the detection of human IL-5 receptors on eosinophilic sublines of the promyelocytic leukemia HL-60. Sodium butyrate, which initiates differentiation to mature eosinophils, also induces the appearance of high affinity (Kd 1-5 X 10(-11) M) IL-5 binding sites on these cells.

Expression of human and murine interleukin-5 in eukaryotic systems.

A cDNA coding for murine interleukin-5 (IL-5) was isolated from the EL4.ExC5 cell line. With the exception of a single amino acid substitution at position 79 (Arg----His), it is identical to a published sequence.

Gradient separation of granulocytic progenitor cells (CFUc) from human blood mononuclear leukocytes.

Two different density-gradient techniques were compared for separation of human blood-derived granulocyte-macrophage progenitor cells (CFUc) from immunocompetent cells using albumin or Percoll as gradient media. Mean CFUc enrichment by means of discontinuous albumin gradient was twofold, whereas Percoll gradients yielded about 20- to 40-fold enrichment of CFUc in relation to lymphocytes. The pH of gradient media proved to have a major influence on separation quality.

Morphologic alterations in canine marrow of long-term survivors after 1200 R whole-body x-irradiation and autologous blood leukocyte engraftment.

The marrow matrix of total-body x-irradiated dogs (1200 R midline dose) was able to support effective hemopoiesis for several hundred days if the animals were transfused with their own mononuclear leukocytes collected from the blood prior to irradiation and preserved at ultralow temperatures. However, a lesion developed in the marrow, consisting of a fibrosis originating in conjunction with or from the endosteum. The fibrotic tissue substantially reduced the available marrow space in dogs with advanced lesions.

Regeneration of blood-forming organs after autologous leukocyte transfusion in lethally irradiated dogs. I. Distribution and cellularity of the bone marrow in normal dogs.

Marrow cellularity in adult beagles (1-2 yr old) is highest in centrally located bones, with values between 8000 and 12,000 nucleated cells per sq mm. It decreases gradually towards the peripheral parts of the body, reaching values below 1000 per sq mm in bones distal to the elbow and knee. The first tail segment always contains some active marrow.