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We used reverse transcription-polymerase chain reaction (RT-PCR) to clone a rat complementary DNA that encoded the PVG rat granulocyte-macrophage colony-stimulating factor (GM-CSF). PCR products were cloned into a eukaryotic expression vector and transfected into the mouse myeloma cell line Sp2/0-Ag14. Cell culture supernatants of two of these transfectants supported proliferation of the growth factor-dependent cell line, DA-3, and promoted myeloid colony formation in rat and mouse bone marrow cell (BMC) cultures. The GM-CSF activity in these supernatants was neutralized by a polyclonal antibody to mouse GM-CSF. The cloning and expression of rat GM-CSF provides a valuable reagent for the study of the biology and clinical applications of the GM-CSFs.

An in vivo transplantation system has been used to evaluate the developmental capacities of specific mouse mammary epithelial cell populations. Specifically, mouse mammary epithelial cells with distinctly limited developmental potentials have been identified using this procedure. Two distinct epithelial cell progenitors have been identified by experiments designed to determine whether basal lobular and ductal phenotypes could develop independently under conditions imposed by a limiting dilution. The prediction that these separate epithelial progenitors must exist was based upon the results from transplantation experiments carried out in epithelium-divested mammary fat pads of syngeneic mice with mammary epithelium from two different transgenic mouse models. The results presented here demonstrate the following points: 1) lobular, i.e. secretory, progenitor cells are present as distinct entities among the mammary epithelial cells found in immature virgin female mice; 2) similarly, ductal epithelial progenitors are present within the same population; 3) lobular progenitors are present in greater numbers, although both cell populations are extremely small; 4) as expected, some inocula produce outgrowths with simultaneous development of both lobular and ductal phenotypes--it is not known whether this indicates cooperative interaction between the two epithelial progenitors or signals the presence of a third progenitor type capable of producing both ductular and lobular committed daughters; 5) these findings have important consequences in the design of experiments aimed at testing the effects of known and putative mammary oncogenes and tumor suppressor genes, using techniques which include cellular transformation in vitro followed by in vivo cultivation and evaluation.

The PDE2, cyclic GMP-stimulated, and the PDE4, cyclic AMP-specific enzymes provide the major, detectable cyclic AMP phosphodiesterase activities in murine thymocytes. In the absence of the cyclic GMP, PDE4 activity predominated (approximately 80% total) but in the presence of low (10 microM) cyclic GMP concentrations, PDE2 activity constituted the major PDE activity in thymocytes (approximately 80% total). The PDE4 selective inhibitor rolipram dose-dependently inhibited thymocyte PDE4 activity (IC50 approximately 65 nM). PDE2 was dose-dependently activated (EC50 approximately 1 microM) by cyclic GMP and inhibited by erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) (IC50 approximately 4 microM). EHNA was shown to serve as a selective inhibitor of PDE-2 activity as assessed from studies using separated PDE1, PDE2, PDE3 and PDE4 species from hepatocytes as well as human PDE2 and PDE4 enzymes. EHNA completely ablated the ability of cyclic GMP to activate PDE2 activity, whilst having a much smaller inhibitory effect on the unstimulated PDE2 activity. EHNA exhibited normal Michaelian kinetics of inhibition for the cyclic GMP-stimulated PDE2 activity with Hill plots near unity. Apparent negative co-operative effect were seen in the absence of cyclic GMP with Hill coefficients of approximately 0.3 for inhibition of PDE2 activity. Within 5 min of challenge of thymocytes with the lectin phytohaemagglutinin (PHA) there was a transient decrease (approximately 83%) in PDE-4 activity and in PDE2 activity (approximately 40%). Both anti-TCR antibodies also caused an initial reduction in the PDE4 activity which was followed by a sustained and profound increase in activity. In contrast to that observed with PHA, anti-TCR/CD3 antisera had little effect on PDE2 activity. It is suggested that, dependent upon the intracellular concentrations of cyclic GMP, thymocyte cyclic AMP metabolism can be expected to switch from being under the predominant control of PDE4 activity to that determined predominantly by PDE2 activity. These activities may be rapidly and differentially regulated following ligation of different cell surface receptors.

Three series of analogs were regioselectively prepared from a protected forskolin precursor to afford 7-carbamoyl-7-desacetylforskolins (series 1), 6-carbamoyl-7-desacetylforskolins (series 2), and 6-carbamoylforskolins (series 3). The analogs were pharmacologically evaluated for binding (IC50) to and activation (EC50) of type I adenylyl cyclase in membranes from stably transfected Sf9 cell lines expressing a single adenylate cyclase subtype. The following ranges were determined for the IC50's and EC50's of each individual series: series 1, IC50 = 43-1600 nM, EC50 = 0.5-9.6 microM; series 2, IC50 = 65-680 nM, EC50 = 0.63-6.5 microM; series 3, IC50 = 21-271 nM, EC50 = 0.5-8.1 microM (forskolin IC50 = 41 nM and EC50 = 0.5 microM). Activation paralleled binding; however, some analogs exhibited poor binding and good activation whereas others demonstrated good binding but poor activation. Steric bulk tended to diminish binding and activation when at the 6- or 7-position, although bulk was accommodated at the 6-position if the 7-site was reacetylated. Acylation of the 7-position by the carbamoyl linker or acetyl was important for obtaining good binding and activation; however, the effect was more pronounced with binding. For both binding and activation, small, linear, lipophilic substituents (propyl, allyl, isopropyl) are well tolerated at the 7-position but less so in the 6-position, even when the 7-site is reacetylated. Planar aromatic moieties (phenyl and 2-pyridinyl) demonstrated moderate to good potency for binding and activation when located at either the 6- or 7-positions. There is an overall trend toward increasing potency for both binding and activation with polar substituents.

Retinoids exert pleiotropic effects on the development of vertebrates through the action of retinoic acid receptors (RAR) and retinoid X receptors (RXR). We have investigated the effect of synthetic retinoids selective for RXR and RAR on the development of Xenopus and zebrafish embryos. In Xenopus, both ligands selective for RAR and RXR caused striking malformations along the anterior-posterior axis, whereas in zebrafish only ligands specific for RAR caused embryonic malformations. In Xenopus, RAR- and RXR-selective ligands regulated the expression of the Xlim-1, gsc, and HoxA1 genes similarly as all-trans-retinoic acid. Nevertheless, RXR-selective ligands activated only an RXR responsive reporter but not an RAR responsive reporter introduced by microinjection into the Xenopus embryo, consistent with our failure to detect conversion of an RXR-selective ligand to different derivatives in the embryo. These results suggest that Xenopus embryos possess a unique response pathway in which liganded RXR can control gene expression. Our observations further illustrate the divergence in retinoid responsiveness between different vertebrate species.

The classical definition of lymphohematopoietic stem cells (LHSC), the most primitive progenitors of all blood cells, requires that they have the capacity for self-renewal and for the long-term production of all blood cell lineages. However, other characteristics of LHSC have been debated. Our previous data suggested that mouse LHSC are very slowly proliferating cells that generate delayed multilineage engraftment, while radioprotection" (rapid engraftment that will prevent early death from radiation-induced marrow aplasia) results from more committed progenitors. Alternatively�

The mechanism of the anti-anginal effect of perhexiline is unclear but appears to involve a shift in cardiac metabolism from utilization of fatty acid to that of carbohydrate. We tested the hypothesis that perhexiline inhibits the enzyme carnitine palmitoyltransferase-1 (CPT-1), which controls access of long chain fatty acids to the mitochondrial site of beta-oxidation. Perhexiline produced a concentration-dependent inhibition of CPT-1 in rat cardiac and hepatic mitochondria in vitro, with half-maximal inhibition (IC50) at 77 and 148 mumol/L, respectively. Amiodarone, another drug with anti-anginal properties, also inhibited cardiac CPT-1 (IC50 = 228 mumol/L). The rank order of potency for inhibition was malonyl-CoA {\textgreater} 4-hydroxyphenylglyoxylate (HPG) = perhexiline {\textgreater} amiodarone = monohydroxy-perhexiline. Kinetic analysis revealed competitive inhibition of cardiac and hepatic CPT-1 by perhexiline with respect to palmitoyl-CoA but non-competitive inhibition with respect to carnitine. Curvilinear Dixon plots generated apparent inhibitory constant (Ki)" values for perhexiline which indicated a greater sensitivity of the cardiac than the hepatic enzyme to inhibition by perhexiline. Perhexiline inhibition of CPT-1 unlike that of malonyl-CoA and HPG was unaffected by pretreatment with the protease nagarse. These data establish for the first time that two agents with proven anti-anginal effects inhibit cardiac CPT-1. This action is likely to contribute to the anti-ischaemic effects of both perhexiline and amiodarone."

A high proportion of the CD34+CD38- cells in normal human marrow are defined as long-term culture-initiating cells (LTC-IC) because they can proliferate and differentiate when co-cultured with cytokine-producing stromal feeder layers. In contrast, very few CD34+CD38- cells will divide in cytokine-containing methylcellulose and thus are not classifiable as direct colony-forming cells (CFC), although most can proliferate in serum-free liquid cultures containing certain soluble cytokines. Analysis of the effects of 16 cytokines on CD34+CD38- cells in the latter type of culture showed that Flt3-ligand (FL), Steel factor (SF), and interleukin (IL)-3 were both necessary and sufficient to obtain an approximately 30-fold amplification of the input LTC-IC population within 10 d. As single factors, only FL and thrombopoietin (TPO) stimulated a net increase in LTC-IC within 10 d. Interestingly, a significantly increased proportion of the CFC produced from the TPO-amplified LTC-IC were erythroid. Increases in the number of directly detectable CFC of textgreater 500-fold were also obtainable within 10 d in serum-free cultures of CD34+CD38- cells. However, this required the presence of IL-6 and/or granulocyte/colony-stimulating factor and/or nerve growth factor beta in addition to FL, SF, and IL-3. Also, for this response, the most potent single-acting factor tested was IL-3, not FL. Identification of cytokine combinations that differentially stimulate primitive human hematopoietic cell self-renewal and lineage determination should facilitate analysis of the intracellular pathways that regulate these decisions as well as the development of improved ex vivo expansion and gene transfer protocols. View Publication

Mitogen-activated protein (MAP) kinase cascades represent one of the major signal systems used by eukaryotic cells to transduce extracellular signals into cellular responses. Four MAP kinase subgroups have been identified in humans: ERK, JNK (SAPK), ERK5 (BMK), and p38. Here we characterize a new MAP kinase, p38beta. p38beta is a 372-amino acid protein most closely related to p38. It contains a TGY dual phosphorylation site, which is required for its kinase activity. Like p38, p38beta is activated by proinflammatory cytokines and environmental stress. A comparison of events associated with the activation of p38beta and p38 revealed differences, most notably in the preferred activation of p38beta by MAP kinase kinase 6 (MKK6), whereas p38 was activated nearly equally by MKK3, MKK4, and MKK6. Moreover, in vitro and in vivo experiments showed a strong substrate preference by p38beta for activating transcription factor 2 (ATF2). Enhancement of ATF2-dependent gene expression by p38beta was approximately20-fold greater than that of p38 and other MAP kinases tested. The data reported here suggest that while closely related, p38beta and p38 may be regulated by differing mechanisms and may exert their actions on separate downstream targets.

Wortmannin at nanomolar concentrations is a potent and specific inhibitor of phosphoinositide (PI) 3-kinase and has been used extensively to demonstrate the role of this enzyme in diverse signal transduction processes. At higher concentrations, wortmannin inhibits the ataxia telangiectasia gene (ATM)-related DNA-dependent protein kinase (DNA-PKcs). We report here the identification of the site of interaction of wortmannin on the catalytic subunit of PI 3-kinase, p110alpha. At physiological pH (6.5 to 8) wortmannin reacted specifically with p110alpha. Phosphatidylinositol-4,5-diphosphate, ATP, and ATP analogs [adenine and 5'-(4-fluorosulfonylbenzoyl)adenine] competed effectively with wortmannin, while substances containing nucleophilic amino acid side chain functions had no effect at the same concentrations. This suggests that the wortmannin target site is localized in proximity to the substrate-binding site and that residues involved in wortmannin binding have an increased nucleophilicity because of their protein environment. Proteolytic fragments of wortmannin-treated, recombinant p110alpha were mapped with anti-wortmannin and anti-p110alpha peptide antibodies, thus limiting the target site within a 10-kDa fragment, colocalizing with the ATP-binding site. Site-directed mutagenesis of all candidate residues within this region showed that only the conservative Lys-802-to-Arg mutation abolished wortmannin binding. Inhibition of PI 3-kinase occurs, therefore, by the formation of an enamine following the attack of Lys-802 on the furan ring (at C-20) of wortmannin. The Lys-802-to-Arg mutant was also unable to bind FSBA and was catalytically inactive in lipid and protein kinase assays, indicating a crucial role for Lys-802 in the phosphotransfer reaction. In contrast, an Arg-916-to-Pro mutation abolished the catalytic activity whereas covalent wortmannin binding remained intact. Our results provide the basis for the design of novel and specific inhibitors of an enzyme family, including PI kinases and ATM-related genes, that play a central role in many physiological processes. View Publication

A 145-kDa tyrosine-phosphorylated protein that becomes associated with Shc in response to multiple cytokines has been purified from the murine hemopoietic cell line B6SUtA1. Amino acid sequence data were used to clone the cDNA encoding this protein from a B6SUtA1 library. The predicted amino acid sequence encodes a unique protein containing an N-terminal src homology 2 domain, two consensus sequences that are targets for phosphotyrosine binding domains, a proline-rich region, and two motifs highly conserved among inositol polyphosphate 5-phosphatases. Cell lysates immunoprecipitated with antiserum to this protein exhibited both phosphatidylinositol 3,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate polyphosphate 5-phosphatase activity. This novel signal transduction intermediate may serve to modulate both Ras and inositol signaling pathways. Based on its properties, we suggest the 145-kDa protein be called SHIP for SH2-containing inositol phosphatase.