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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

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

Neutral endopeptidase (NEP) may regulate peptide-induced inflammation in the respiratory tract. It is of interest to determine which respiratory resident cells express NEP. Trachea and bronchi from seven nonsmoking,nonasthmatic subjects were examined. NEP messenger ribonucleic acid (mRNA) was characterized by Northern blot hybridization of cultured human tracheobronchial epithelial and smooth muscle cells,and reverse transcriptase-polymerase chain reaction (RT-PCR) in trachea and bronchi. In situ hybridization with biotin- and 35S-labelled antisense complementary ribonucleic acid (cRNA) probes was used to determine the distribution of NEP mRNA in human bronchial mucosa. NEP-immunoreactive material was detected using MEK10 murine monoclonal antibodies and the immunogold method with silver enhancement. NEP mRNA was 4.5 kb in size in the cultured human smooth muscle and epithelial cells by Northern blot analysis. No evidence was found by RT-PCR for truncated,alternatively spliced NEP mRNAs,such as del exon 16 or del exons 5-18 in human bronchus. NEP mRNA was detected by in situ hybridization in epithelial cells,submucosal glands,bronchial smooth muscle and endothelium. NEP-immunoreactive material was identified in the epithelium,submucosal glands,bronchial smooth muscle,and endothelium,demonstrating an excellent correlation between the distribution of NEP mRNA and the cell surface protein. NEP mRNA and immunoreactive material were excluded from epithelial goblet cell and submucosal gland mucous cell vacuoles. We conclude that the various sites of NEP protein and mRNA expression correlate with the locations of peptide receptors and NEP enzyme function,and are consistent with the hypothesis that NEP may regulate peptide-induced inflammation in human bronchi. 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

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To study the role of the murine heat-stable Ag (HSA) in lymphocyte maturation,we generated transgenic mice in which the HSA cDNA was under the transcriptional control of the TCR V beta promoter and Ig mu enhancer. The HSA transgene was expressed during all stages of B lymphocyte maturation. Expression was first detected in the earliest lymphoid-committed progenitors,which normally do not express HSA,and subsequently reached the highest levels in pro- and pre-B cells. In bone marrow,the number of IL-7-responsive clonogenic progenitors was textless 4% of normal,whereas the frequency of earlier B lymphocyte-restricted precursors,detectable as Whitlock-Witte culture-initiating cells,was normal. Pro- and pre-B cells detected by flow cytometry were reduced by approximately 50% relative to controls. Mature splenic B cells were also reduced but to a lesser extent than in marrow,and their response to LPS stimulation was impaired. Reconstitution of SCID and BALB/c-nu/nu mice with HSA transgenic marrow indicated that the perturbations in B lymphopoiesis were not caused by a defective marrow microenvironment or by abnormal T cells. Our previous studies showed elevated HSA expression throughout thymocyte development,which resulted in a profound depletion of CD4+CD8+ double-positive and single-positive thymocytes. Together,these results indicate that HSA levels can determine the capacity of early T and B lymphoid progenitors to proliferate and survive. Therefore,HSA could serve as an important regulator during the early stages of B and T lymphopoiesis. 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

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Murine hematopoietic stem cells with varying proliferative capacity can be assayed by limiting dilution analysis of cobblestone area" (CA) formation on stromal layers in microlong-term bone marrow cultures. Cobblestone area forming cell (CAFC) frequency determined at early time points (day 7) correlates with mature stem cells measured as day 8 CFU-S� 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

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

Most primitive hematopoietic cells appear to be normally quiescent in vivo,whereas their leukemic counterparts in patients with chronic myeloid leukemia (CML) are maintained in a state of rapid turnover. This difference is also seen in the long-term culture system,where control of primitive hematopoietic progenitor proliferation is mediated by interactions of these cells with marrow-derived mesenchymal cells of the fibroblast lineage. We now show that exogenous addition of macrophage inflammatory protein 1 alpha (MIP-1 alpha) to normal long-term cultures can reversibly and specifically block the activation of primitive" (high proliferative potential)� View Publication