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Significant activity of phosphatidylinositol 3-kinase (PI 3-kinase) was detected in the membrane fractions,or in the immunoprecipitates prepared with anti-phosphotyrosine antibodies,from rat adipocytes that had been incubated with insulin for 20 min. The PI 3-kinase activity in these preparations as well as in the whole cell lysates of adipocytes not treated with insulin was inhibited by the addition of wortmannin,a fungal metabolite,to the enzyme assay mixture. The inhibition was dependent on the inhibitor concentration with IC50 being less than 10 nM and perfect inhibition at 100 nM. The effect of insulin to induce membrane PI 3-kinase activity was mostly abolished,but its effects to tyrosine-phosphorylate the beta-subunit of the insulin receptor or other cellular substrate proteins including insulin-receptor-substrate-1 were not at all antagonized,by wortmannin added to the cell incubation medium. Insulin stimulation of cellular 2-deoxyglucose uptake and inhibition of isoproterenol-induced lipolysis observable in adipocytes under the same conditions were also antagonized by wortmannin added in the same concentration range as used for the inhibition of insulin-susceptible PI 3-kinase. It is concluded,therefore,that activation of wortmannin-sensitive PI 3-kinase plays a pivotal role in the intracellular signaling pathways arising from the insulin receptor autophosphorylation and leading to certain metabolic responses. View Publication

Phosphatidylinositol (PtdIns) 3-kinase is an enzyme implicated in growth factor signal transduction by associating with receptor and nonreceptor tyrosine kinases,including the platelet-derived growth factor receptor. Inhibitors of PtdIns 3-kinase could potentially give a better understanding of the function and regulatory mechanisms of the enzyme. Quercetin,a naturally occurring bioflavinoid,was previously shown to inhibit PtdIns 3-kinase with an IC50 of 1.3 microgram/ml (3.8 microM); inhibition appeared to be directed at the ATP-binding site of the kinase. Analogs of quercetin were investigated as PtdIns 3-kinase inhibitors,with the most potent ones exhibiting IC50 values in the range of 1.7-8.4 micrograms/ml. In contrast,genistein,a potent tyrosine kinase inhibitor of the isoflavone class,did not inhibit PtdIns 3-kinase significantly (IC50 textgreater 30 micrograms/ml). Since quercetin has also been shown to inhibit other PtdIns and protein kinases,other chromones were evaluated as inhibitors of PtdIns 3-kinase without affecting PtdIns 4-kinase or selected protein kinases. One such compound,2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (also known as 2-(4-morpholinyl)-8-phenylchromone,LY294002),completely and specifically abolished PtdIns 3-kinase activity (IC50 = 0.43 microgram/ml; 1.40 microM) but did not inhibit PtdIns 4-kinase or tested protein and lipid kinases. Analogs of LY294002 demonstrated a very selective structure-activity relationship,with slight changes in structure causing marked decreases in inhibition. LY294002 was shown to completely abolish PtdIns 3-kinase activity in fMet-Leu-Phe-stimulated human neutrophils,as well as inhibit proliferation of smooth muscle cells in cultured rabbit aortic segments. Since PtdIns 3-kinase appears to be centrally involved with growth factor signal transduction,the development of specific inhibitors against the kinase may be beneficial in the treatment of proliferative diseases as well as in elucidating the biological role of the kinase in cellular proliferation and growth factor response. View Publication

Analysis of [3H]phorbol-12,13-dibutyrate (PDBu) binding was performed with protein kinase C (PKC)-alpha,-beta 1,-gamma,-delta,-epsilon,-eta,and -zeta produced in Sf9 insect cells using the baculovirus expression system. With the exception of PKC-zeta,all of the PKC isozymes bound [3H]PDBu with high affinity (Kd textless 1 nM),either in the presence or in the absence of calcium. Scatchard analysis using 100% phosphatidylserine vesicles revealed slightly lower affinity for the calcium-independent isozymes (PKC-delta,-epsilon,and -eta) than for the calcium-dependent isozymes (PKC-alpha,-beta,and -gamma). Competition for [3H]PDBu binding by different classes of PKC activators showed that 12-deoxyphorbol esters,mezerein,and octahydromezerein likewise possessed lower affinity for the calcium-independent isozymes. The mezerein analog thymeleatoxin was the most marked example,being almost 20-fold less potent for binding to PKC-epsilon and -eta than to PKC-beta 1. In contrast,the indole alkaloids (-)-indolactam V and (-)-octylindolactam V and the postulated endogenous activator 1,2-diacylglycerol bound with similar affinities to all of the PKC isoforms,suggesting that different residues/configurations in the binding sites of the different PKC isozymes might be involved in interaction with the pharmacophore of the activators. The seven PKC isozymes also showed clearly different substrate specificities with exogenous peptide and protein substrates. The heterogeneous behavior of the different members of the PKC family with ligands and substrates may contribute to the heterogeneity of PKC-mediated pathways at the cellular level. View Publication

We have systematically optimized the concentrations of 20 components of a previously published serum-free medium (Brewer and Cotman,Brain Res 494: 65-74,1989) for survival of rat embryonic hippocampal neurons after 4 days in culture. This serum-free medium supplement,B27,produced neuron survival above 60%,independent of plating density above 160 plated cells/mm2. For isolated cells (textless 100 cells/mm2),survival at 4 days was still above 45%,but could be rescued to the 60% level at 40 cells/mm2 by simply applying a coverslip on top of the cells. This suggests a need for additional trophic factors. High survival was achieved with osmolarity lower than found in Dulbecco's Modified Eagle's Medium (DMEM),and by reducing cysteine and glutamine concentrations and by the elimination of toxic ferrous sulphate found in DME/F12. Neurobasal is a new medium that incorporates these modifications to DMEM. In B27/Neurobasal,glial growth is reduced to less than 0.5% of the nearly pure neuronal population,as judged by immunocytochemistry for glial fibrillary acidic protein and neuron-specific enolase. Excellent long-term viability is achieved after 4 weeks in culture with greater than 90% viability for cells plated at 640/mm2 and greater than 50% viability for cells plated at 160/mm2. Since the medium also supports the growth of neurons from embryonic rat striatum,substantia nigra,septum,and cortex,and neonatal dentate gyrus and cerebellum (Brewer,in preparation),support for other neuron types is likely. B27/Neurobasal should be useful for in vitro studies of neuronal toxicology,pharmacology,electrophysiology,gene expression,development,and effects of growth factors and hormones. View Publication

Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA),a potential inhibitor of adenosine deaminase (ADA),was tested as an inhibitor of the soluble cyclic nucleotide phosphodiesterase (PDE) isoenzymes from pig and human myocardium. Four soluble PDE activities were resolved from human papillary muscle extracts using anion exchange chromatography (DEAE Sepharose CL-6B). These activities were designated PDE I-IV according to the nomenclature of Beavo. PDE I was stimulated by Ca(2+)-calmodulin and PDE II by cGMP (1 microM). PDE III was inhibited by cGMP (1 microM) as well as SK&F 94120,and PDE IV by both rolipram and Ro 20-1724. Enzyme kinetics and inhibition constants were similar with the PDE isoenzymes from pig heart. However,porcine myocardium lacked Ca(2+)-calmodulin-stimulated soluble PDE I activity. The present data reveal that EHNA exerted a concentration-dependent inhibition of the cGMP-stimulated PDE II (cGs-PDE) (IC50: 0.8 microM (human),2 microM (pig)) but did not inhibit the other PDE isoenzymes (IC50 textgreater 100 microM). These findings indicate that EHNA is a potent and,as far as cytosolic PDEs are concerned,selective inhibitor of cGMP-stimulated PDEs. The compound may lend itself for the rational design of other isozyme selective PDE II inhibitors and for examining the specific biological functions of cGs-PDEs. EHNA may be used in systems in which inhibition of ADA is of no concern. Conversely,dual inhibition of both ADA and cGs-PDE by EHNA may cause accumulation of two inhibitory metabolites,adenosine and cGMP,which may act in synergy to mediate diverse pharmacological responses,including antiviral,antitumour and antiarrhythmic effects. View Publication

The synthetic cytokine (Synthokine) SC-55494 is a high-affinity interleukin-3 (IL-3) receptor ligand that stimulates greater in vitro multilineage hematopoietic activity than native IL-3,while inducing no significant increase in inflammatory activity relative to native IL-3. The aim of this study was to investigate the in vivo hematopoietic response of rhesus monkeys receiving Synthokine after radiation-induced marrow aplasia. Administration schedule and dose of Synthokine were evaluated. All animals were total-body irradiated (TBI) with 700 cGy 60Co gamma radiation on day 0. Beginning on day 1,cohorts of animals (n = 5) received Synthokine subcutaneously (SC) twice daily with 25 micrograms/kg/d or 100 micrograms/kg/d for 23 days or 100 micrograms/kg/d for 14 days. Control animals (n = 9) received human serum albumin SC once daily at 15 micrograms/kg/d for 23 days. Complete blood counts were monitored for 60 days postirradiation and the durations of neutropenia (NEUT; absolute neutrophil count [ANC] textless 500/microL) and thrombocytopenia (THROM; platelet count textless 20,000/microL) were assessed. Synthokine significantly (P textless .05) reduced the duration of THROM versus the HSA-treated animals regardless of dose or protocol length. The most striking reduction was obtained in the animals receiving 100 micrograms/kg/d for 23 days (THROM = 3.5 v 12.5 days in HSA control animals). Although the duration of NEUT was not significantly altered,the depth of the nadir was significantly lessened in all animal cohorts treated with Synthokine regardless of dose versus schedule length. Bone marrow progenitor cell cultures indicated a beneficial effect of Synthokine on the recovery of granulocyte-macrophage colony-forming units that was significantly higher at day 24 post-TBI in both cohorts treated at 25 and 100 micrograms/kg/d for 23 days relative to the control animals. Plasma pharmacokinetic parameters were evaluated in both normal and irradiated animals. Pharmacokinetic analysis performed in irradiated animals after 1 week of treatment suggests an effect of repetitive Synthokine schedule and/or TBI on distribution and/or elimination of Synthokine. These data show that the Synthokine,SC55 94,administered therapeutically post-TBI,significantly enhanced platelet recovery and modulated neutrophil nadir and may be clinically useful in the treatment of the myeloablated host. View Publication

Fumonisin B1 (FB1),a mycotoxin produced by the fungus Fusarium moniliforme,which is a common contaminant of corn,is suspected to be a cause of human esophageal cancer. FB1 is hepatotoxic and hepatocarcinogenic in rats,and although the mechanisms involved have not been clarified,the latter is associated with a weak initiating activity. The effects of FB1 on the activity of protein serine/threonine phosphatases (PPs) (PP1,PP2A,PP2B,PP2C and PP5/T/K/H) were investigated in the present study. Inhibition of dephosphorylation was noted for all five PPs with IC50 values of 80 microM-3000 microM. Among the five PPs examined,PP5 was most sensitive with an IC50 of 80 microM. This concentration is comparable to that estimated to be reached in the rat body by feeding FB1 to obtain hepatic tumors. Inhibition of PP5 could thus play important roles in the toxicity and carcinogenic action of FB1. View Publication

The site-directed recombinase Cre can be employed to delete or express genes in cell lines or animals. Clearly,the ability to control remotely the activity of this enzyme would be highly desirable. To this end we have constructed expression vectors for fusion proteins consisting of the Cre recombinase and a mutated hormone-binding domain of the murine oestrogen receptor. The latter still binds the anti-oestrogen drug tamoxifen but no longer 17 beta-oestradiol. We show here that in embryonic stem cells expressing such fusion proteins,tamoxifen can efficiently induce Cre-mediated recombination,thereby activating a stably integrated LacZ reporter gene. In the presence of either 10 microM tamoxifen or 800 nM 4-hydroxy-tamoxifen,recombination of the LacZ gene is complete within 3-4 days. By placing a tamoxifen-binding domain on both ends of the Cre protein,the enzymatic activity of Cre can be even more tightly controlled. Transgenic mice expressing such an tamoxifen-inducible Cre enzyme may thus provide a new and useful genetic tool to mutate or delete genes at specific times during development or in adult animals. View Publication

All-trans retinoic acid (ATRA) is successfully used in the cyto-differentiating treatment of acute promyelocytic leukemia (APL). Paradoxically,APL cells express PML-RAR,an aberrant form of the retinoic acid receptor type alpha (RAR alpha) derived from the leukemia-specific t(15;17) chromosomal translocation. We show here that AM580,a stable retinobenzoic derivative originally synthesized as a RAR alpha agonist,is a powerful inducer of granulocytic maturation in NB4,an APL-derived cell line,and in freshly isolated APL blasts. After treatment of APL cells with AM580 either alone or in combination with granulocyte colony-stimulating factor (G-CSF),the compound induces granulocytic maturation,as assessed by determination of the levels of leukocyte alkaline phosphatase,CD11b,CD33,and G-CSF receptor mRNA,at concentrations that are 10- to 100-fold lower than those of ATRA necessary to produce similar effects. By contrast,AM580 is not effective as ATRA in modulating the expression of these differentiation markers in the HL-60 cell line and in freshly isolated granulocytes obtained from the peripheral blood of chronic myelogenous leukemia patients during the stable phase of the disease. In NB4 cells,two other synthetic nonselective RAR ligands are capable of inducing LAP as much as AM580,whereas RAR beta- or RAR gamma-specific ligands are totally ineffective. These results show that AM580 is more powerful than ATRA in modulating the expression of differentiation antigens only in cells in which PML-RAR is present. Binding experiments,using COS-7 cells transiently transfected with PML-RAR and the normal RAR alpha,show that AM580 has a lower affinity than ATRA for both receptors. However,in the presence of PML-RAR,the synthetic retinoid is a much better transactivator of retinoic acid-responsive element-containing promoters than the natural retinoid,whereas,in the presence of RAR alpha,AM580 and ATRA have similar activity. This may explain the strong cyto-differentiating potential of AM580 in PML-RAR-containing leukemic cells. View Publication

In cultures of embryonic striatum,we previously reported that EGF induces the proliferation of single precursor cells,which give rise to spheres of undifferentiated cells that can generate neurons and glia. We report here that,in vitro,these embryonic precursor cells exhibit properties and satisfy criteria representative of stem cells. The EGF-responsive cell was able to generate the three major phenotypes of the mammalian CNS--neurons,astrocytes,and oligodendrocytes. Approximately 90% of both primary spheres and secondary expanded clones,derived from the primary spheres,contained all three cell types. The increase in frequency of EGF-generated spheres,from 1% in primary culture to close to 20% in secondary culture,and the large number of clonally derived secondary spheres that could be generated from a single primary sphere indicate that EGF induces both renewal and expansion of the precursor cell itself. In population studies,the EGF-responsive cells were carried through 10 passages,resulting in a 10(7)-fold increase in cell number,without losing their proliferative and multilineage potential. Thus,this study describes the first demonstration,through clonal and population analyses in vitro,of a mammalian CNS stem cell that proliferates in response to an identified growth factor (EGF) and produces the three principal cell types of the CNS. 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. View Publication

1. The effect of suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) on the stimulation of phospholipase C in 1321N1 cells transfected with the human P2U-purinoceptor (h-P2U-1321N1 cells) or with the turkey P2Y-purinoceptor (t-P2Y-1321N1 cells) was investigated. 2-Methylthioadenosine triphosphate (2MeSATP) was used as the agonist at t-P2Y-1321N1 cells and uridine triphosphate (UTP) at h-P2U-1321N1 cells. 2. Suramin caused a parallel shift to the right of the concentration-response curves for 2MeSATP in the t-P2Y-1321N1 cells,yielding a Schild plot with a slope of 1.16 +/- 0.08 and a pA2 value of 5.77 +/- 0.11. 3. Suramin also caused a shift to the right of concentration-response curves for UTP in the h-P2U-1321N1 cells,and on Schild plots gave a slope different from unity (1.57 +/- 0.19) and an apparent pA2 value of 4.32 +/- 0.13. Suramin was therefore a less potent antagonist at the P2U-purinoceptor than the P2Y-purinoceptor. 4. In the presence of the ectonucleotidase inhibitor,ARL 67156 (6-N,N-diethyl-beta,gamma-dibromomethylene-D-ATP) there was no significant difference in the EC50 or shapes of curves with either cell type,and no difference in pA2 values for suramin. 5. PPADS caused an increase in the EC50 for 2MeSATP in the t-P2Y-1321N1 cells. The Schild plot had a slope different from unity (0.55 +/- 0.15) and an X-intercept corresponding to an apparent pA2 of 5.98 +/- 0.65. 6. PPADS up to 30 microM had no effect on the concentration-response curve for UTP with the h-P2U-1321N1 cells. 7. In conclusion,suramin and PPADS show clear differences in their action at the 2 receptor types,in each case being substantially more effective as an antagonist at the P2Y-purinoceptor than at the P2U-purinoceptor. Ectonucleotidase breakdown had little influence on the nature of the responses at the two receptor types,or in their differential sensitivity to suramin. View Publication