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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.

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.

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.

Here, we have studied the activity of a novel protein-tyrosine kinase inhibitor that is selective for the Src family of tyrosine kinases. We have focused our study on the effects of this compound on T cell receptor-induced T cell activation, a process dependent on the activity of the Src kinases Lck and FynT. This compound is a nanomolar inhibitor of Lck and FynT, inhibits anti-CD3-induced protein-tyrosine kinase activity in T cells, demonstrates selectivity for Lck and FynT over ZAP-70, and preferentially inhibits T cell receptor-dependent anti-CD3-induced T cell proliferation over non-T cell receptor-dependent phorbol 12-myristate 13-acetate/interleukin-2 (IL-2)-induced T cell proliferation. Interestingly, this compound selectively inhibits the induction of the IL-2 gene, but not the granulocyte-macrophage colony-stimulating factor or IL-2 receptor genes. This compound offers a useful new tool for examining the role of the Lck and FynT tyrosine kinases versus ZAP-70 in T cell activation as well as the role of other Src family kinases in receptor function.

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.

Recombinant retroviruses offer many advantages for the genetic modification of human hematopoietic cells, although their use in clinical protocols has thus far given disappointing results. There is therefore an important need to develop new strategies that will allow effectively transduced primitive hematopoietic target populations to be both rapidly characterized and isolated free of residual nontransduced but biologically equivalent cells. To address this need, we constructed a murine stem cell virus (MSCV)-based retroviral vector containing the 228-bp coding sequence of the murine heat-stable antigen (HSA) and generated helper virus-free amphotropic MSCV-HSA producer cells by transfection of GP-env AM12 packaging cells. Light density and, in some cases, lineage marker-negative (lin-) normal human marrow or mobilized peripheral blood cells preactivated by exposure to interleukin-3 (IL-3), IL-6, and Steel factor in vitro for 48 hours were then infected by cocultivation with these MSCV-HSA producer cells for a further 48 hours in the presence of the same cytokines. Fluorescence-activated cell sorting (FACS) analysis of the cells 24 hours later showed 21% to 41% (mean, 27%) of those that were still CD34+ to have acquired the ability to express HSA. The extent of gene transfer to erythroid and granulopoietic progenitors (burst-forming unit-erythroid and colony-forming unit-granulocyte-macrophage), as assessed by the ability of these cells to form colonies of mature progeny in the presence of normally toxic concentrations of G418, averaged 11% and 12%, respectively, in 6 experiments. These values could be increased to 100% and 77%, respectively, by prior isolation of the CD34+HSA+ cell fraction and were correspondingly decreased to an average of 2% and 5%, respectively, in the CD34+HSA- cells. In addition, the extent of gene transfer to long-term culture-initiating cells (LTC-IC) was assessed by G418 resistance. The average gene transfer to LTC-IC-derived colony-forming cells in the unsorted population was textless or = 7% in 4 experiments. FACS selection of the initially CD34+HSA+ cells increased this value to 86% and decreased it to 3% for the LTC-IC plated from the CD34+HSA- cells. Transfer of HSA gene expression to a phenotypically defined more primitive subpopulation of CD34+ cells, ie, those expressing little or no CD38, could also be shown by FACS analysis of infected populations 24 hours after infection. These findings underscore the potential use of retroviral vectors encoding HSA for the specific identification and non-toxic selection immediately after infection of retrovirally transduced populations of primitive human hematopoietic cells. In addition, such vectors should facilitate the subsequent tracking of their marked progeny using multiparameter flow cytometry.

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.

BACKGROUND: At therapeutic concentrations, the antineoplastic agent taxol selectively perturbs mitotic spindle microtubules. Taxol has recently been shown to induce apoptosis, similar to the mechanism of cell death induced by other antineoplastic agents. However, taxol has shown efficacy against drug-refractory cancers, raising the possibility that this pharmacological agent may trigger an alternative apoptotic pathway. MATERIALS AND METHODS: The kinetics and IC50 of mitotic (M) block, aberrant mitosis, and cytotoxicity following taxol treatment were analyzed in human cell lines as well as normal mouse embryo fibroblasts (MEFs) and MEFs derived from p53-null mice. Apoptosis was followed by DNA gel electrophoresis and by in situ DNA end-labeling (TUNEL). RESULTS: Taxol induced two forms of cell cycle arrest: either directly in early M at prophase or, for those cells progressing through aberrant mitosis, arrest in G1 as multimininucleated cells. TUNEL labeling revealed that DNA nicking occurred within 30 min of the arrest in prophase. In contrast, G1-arrested, multimininucleated cells became TUNEL positive only after several days. In the subset of cells that became blocked directly in prophase, both wt p53-expressing and p53-null MEFs responded similarly to taxol, showing rapid onset of DNA nicking and apoptosis. However, p53-null MEFs progressing through aberrant mitosis failed to arrest in the subsequent G1 phase or to become TUNEL positive, and remained viable. CONCLUSIONS: Taxol induces two forms of cell cycle arrest, which in turn induce two independent apoptotic pathways. Arrest in prophase induces rapid onset of a p53-independent pathway, whereas G1-block and the resulting slow (3-5 days) apoptotic pathway are p53 dependent. 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.

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�