Interleukin-3 (IL-3) is a cytokine produced by activated T-cells and mast cells that is active on a broadrange of hematopoietic cells and in the nervous system and appears to be important in several chronic inflammatory diseases. In this study, alanine substitutions were used to investigate the role of residues of the human ␤-common (h␤c) receptor and the murine IL-3-specific (␤ IL-3 ) receptor in IL-3 binding. We show that the domain 1 residues, Tyr 15 and Phe 79 , of the h␤c receptor are important for high affinity IL-3 binding and receptor activation as shown previously for the related cytokines, interleukin-5 and granulocyte-macrophage colony-stimulating factor, which also signal through this receptor subunit. From the x-ray structure of h␤c, it is clear that the domain 1 residues cooperate with domain 4 residues to form a novel ligand-binding interface involving the two protein chains of the intertwined homodimer receptor. We demonstrate by ultracentrifugation that the ␤ IL-3 receptor is also a homodimer. Its high sequence homology with h␤c suggests that their structures are homologous, and we identified an analogous binding interface in ␤ Interleukin-3 (IL-3) 1 is a cytokine produced by activated T-cells and mast cells that has been shown to stimulate renewal of pluripotent hematopoietic stem cells and to be a potent regulator of many hematopoietic cell lineages (1-3). Its role appears to be in stimulating inducible hematopoiesis in response to parasite infections (4), and it has also been implicated in the pathogenesis of several chronic inflammatory diseases, including asthma (1), and neurodegenerative disorders, such as multiple sclerosis (5). The effect of IL-3 on human cells is mediated by a receptor system composed of a ligand-specific ␣ subunit and a ␤ subunit (denoted ␤c) that is also part of the receptor systems for the related cytokines, interleukin-5 (IL-5) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (6 -9). Signaling through the ␤c receptor requires the formation of a high affinity complex involving each cytokine and its respective ␣ subunit (7-10). Whereas the ␣ subunits bind their ligands with low affinity, ␤c does not measurably bind any of the ligands alone. Upon receptor activation, the cytoplasmic portion of the ␤c subunit, which lacks any intrinsic kinase activity (11), initiates a number of signaling pathways including the Janus kinase 2/signal transducers and activators of transcription, phosphatidylinositol 3-kinase, and Ras/mitogen-activated protein kinase pathways (reviewed in Ref. 12).Mice also possess a ␤c subunit (m␤c) but have an additional IL-3-specific ␤ receptor (␤ IL-3 ). ␤ IL-3 differs from the m␤c subunit in its ability to bind murine IL-3 (mIL-3) directly (13), although the presence of the mIL-3 ␣ subunit is absolutely required for signaling (14). The properties of mIL-3-responsive precursor cells from gene knock-out mice lacking expression of the ␤ IL-3 subunit indicate that this subunit plays an important role in the response to mIL-3 stimulation (15)....

Interleukin-3 (IL-3) promotes both self-renewal and differentiation of early multipotential progenitors and is involved in inducible hematopoiesis in response to infections. Here we report new insights into these processes with the identification of a new isoform (SP2) of IL-3 receptor ␣ (IL-3R␣), present in mouse and human hematopoietic cells, which lacks domain 1 of the full-length receptor (SP1). Binding assays with ␤ IL-3 mutants showed that mouse SP2 uses a different high affinity binding mode to SP1, although both mouse and human SP2 and SP1 can stimulate IL-3-dependent growth. In IL-3-dependent differentiation models, human SP2 and SP1 gave differential effects on lineage commitment or self-renewal dependent on the cellular context, suggesting that different modes of ectodomain binding may modulate intracellular signaling. In a multipotential factor dependent cell-Paterson mix, the transcription factors C/EBP␣ and PU.1 and microRNAs miRNA-15a, -223, and -181a were up-regulated in cells undergoing SP2-supported differentiation compared with SP1-supported self-renewal. Similarly in M1 cells, SP2 promoted differentiation compared with SP1 and gave up-regulation of PU.1 and miRNA-155 and -223. These findings suggest that IL-3-promoted lineage commitment uses similar mechanisms to those of steady-state hematopoiesis. Both the SP1 and SP2 isoforms activated the Jak2/STAT5, Akt, and Erk1/2 signaling pathways in M1 cells, although the activation was more prolonged for the SP2 isoform.

There is a single major alcohol dehydrogenase (ADH) and a single major aldehyde dehydrogenase (AldDH) in Aspergillus nidulans . Both ADH and AldDH are induced by ethanol and by acetaldehyde and both are subject to carbon catabolite repression. ADH and AldDH are necessary for the utilization of ethanol and of threonine, indicating that both compounds are utilized via acetaldehyde. ADH and AldDH each give a single major activity band on gel electrophoresis. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of cell extracts shows at least two similar ADH polypeptides of approximate relative molecular mass (r. m. m.) 41000 and two similar AldDH polypeptides of approximate r. m. m. 57000. The in vitro translation of mRNA from induced, carbon derepressed wild-type cells gives up to three ADH polypeptides in the r. m. m. range 39000-43000 and an AldDH polypeptide of approximate r. m. m. 57000. The mRNA from uninduced, carbon repressed wild-type cells does not direct the synthesis of the ADH and AldDH polypeptides. This indicates that the regulation of ADH and AldDH is at the level of transcription and/or post-transcriptional modification. The probable explanation of the multiple ADH polypeptides is post-transcriptional modification of the mRNA. Allyl alcohol mutants were made by using diepoxyoctane and γ-rays as mutagens. There are two classes, alcA and alcR . Neither class can utilize ethanol or threonine as a carbon source. The alcA mutants lack normal ADH and are recessive. Of the 47 alcA mutants examined 39 do not make the ADH polypeptides while eight do so. Therefore alcA is the structural gene for ADH. The two alcA mutants tested do not make functional mRNA for ADH. The alcR mutants lack both ADH and AldDH and are recessive. No alcR mutants make the ADH or the AldDH polypeptides. The three alcR mutants tested do not make functional ADH or AldDH mRNA. The mutant alcR 125 is a nonsense mutant, which establishes that alcR codes for a protein. The alcA and alcR genes are adjacent on chromosome VII and a preliminary fine-structure map of the alcA gene has been made. Three mutants that cannot utilize ethanol or threonine and have ADH, but lack AldDH, define a gene AldA on chromosome VIII. The aldA 23 mutant makes the AldDH polypeptides, the other two aldA mutants do not. Therefore aldA is probably the structural gene for AldDH. Our current hypothesis is that alcA and aldA are the structural genes for ADH and AldDH respectively and alcR is a transacting regulatory gene coding for a protein whose function is necessary for the expression of the alcA and aldA genes.

The modulation of urokinase plasminogen activator receptor (uPAR) gene expression by tumor necrosis factor alpha (TNF~), phorbol ester (PMA) and amiloride was studied in three colon cancer cell lines, uPAR mRNA and protein were induced by TNF~ and by PMA but were inhibited by amiloride at concentrations of 0.1 to 1 mM in the presence or absence of TNFc~ and PMA. Nuclear run-on transcription assay indicated that the effects of amiloride and TNF~ were mediated at least in part at the transcriptional level, whereas PMA may act in part via a posttranscriptional mechanism. These results suggested that uPAR gene expression is modulated by multiple signal transduction pathways.

A collect~on of 124 isolates of Aeromonas salmonicida ssp. salmonicida from Denmark, Norway, Scotland, and North America was plasmid profiled. All stralns contained at least 1 large plasmid in the range of 60 to 150 kb, and all stralns possessed 2 low-molecular-weight plasmids of 5.2 a n d 5.4 k b Two a d d~t~o n a l low-molecular-weight plasmlds of 5.6 and 6.4 kb were frequently encountered. A total of 23 different plasrnids were demonstrated, 12 of them being found In more than one country. Forty d~fferent plasmld profiles were detected. Seven profiles, representing 75 strains (60 %), were demonstrated in lsolates from more than one country. One profile, w~t h 5 plasmlds of 150, 6.4, 5.6, 5.4, and 5 2 kb, proved to be the most common one among strains from North Amerlca (25 'X), Denmark (33 "%J, and Norway (50 ':h), but ~t proved to be only the third most common one among Scott~sh strains (10 'Z,). Plasmid profiling as an epidemiological t y p~n g method for Aeromonds salmonlclda ssp. salmonlcida was evaluated. The numerical index of discriminatory power (D) was calculated, resulting In a relat~vely high D value of 0.88. However, the results of the present study suggest that plasmid profiling may be of llmited value as an epidemiological marker within Aeromonas salrnonicida ssp. salrnonicida.