Scientific Resources

PD 098059 has been shown previously to inhibit the dephosphorylated form of mitogen-activated protein kinase kinase-1 (MAPKK1) and a mutant MAPKK1(S217E,S221E),which has low levels of constitutive activity (Dudley,D. T.,Pang,L.,Decker,S. J.,Bridges,A. J.,and Saltiel,A. R. (1995) Proc. Natl. Acad. Sci. U.S.A. 92,7686-7689). Here we report that PD 098059 does not inhibit Raf-activated MAPKK1 but that it prevents the activation of MAPKK1 by Raf or MEK kinase in vitro at concentrations (IC50 = 2-7 microM) similar to those concentrations that inhibit dephosphorylated MAPKK1 or MAPKK1(S217E,S221E). PD 098059 inhibited the activation of MAPKK2 by Raf with a much higher IC50 value (50 microM) and did not inhibit the phosphorylation of other Raf or MEK kinase substrates,indicating that it exerts its effect by binding to the inactive form of MAPKK1. PD 098059 also acts as a specific inhibitor of the activation of MAPKK in Swiss 3T3 cells,suppressing by 80-90% its activation by a variety of agonists. The high degree of specificity of PD 098059 in vitro and in vivo is indicated by its failure to inhibit 18 protein Ser/Thr kinases (including two other MAPKK homologues) in vitro by its failure to inhibit the in vivo activation of MAPKK and MAP kinase homologues that participate in stress and interleukin-1-stimulated kinase cascades in KB and PC12 cells,and by lack of inhibition of the activation of p70 S6 kinase by insulin or epidermal growth factor in Swiss 3T3 cells. PD 098059 (50 microM) inhibited the activation of p42MAPK and isoforms of MAP kinase-activated protein kinase-1 in Swiss 3T3 cells,but the extent of inhibition depended on how potently c-Raf and MAPKK were activated by any particular agonist and demonstrated the enormous amplification potential of this kinase cascade. PD 098059 not only failed to inhibit the activation of Raf by platelet-derived growth factor,serum,insulin,and phorbol esters in Swiss 3T3 cells but actually enhanced Raf activity. The rate of activation of Raf by platelet-derived growth factor was increased 3-fold,and the subsequent inactivation that occurred after 10 min was prevented. These results indicate that the activation of Raf is suppressed and that its inactivation is accelerated by a downstream component(s) of the MAP kinase pathway. View Publication

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Autografts using untreated or in vitro manipulated bone marrow and peripheral blood stem cells represent promising approaches to the treatment of malignant diseases. In this work,the collagen gel culture technique was compared with agar and methylcellulose for its capacity to permit the growth of human granulomonocytic (day 14 CFU-GM; collagen vs agar or MTC) or erythroblastic (day 7 CFU-E and day 14 BFU-E; collagen versus methylcellulose) colonies in autologous transplantation products. Our results show that the collagen culture system always gave as many or more colonies than the other techniques. It also allowed harvesting of gels onto glass slides and subsequent May-Grünwald-Giemsa,cytochemical or immunocytochemical staining. We suggest that the collagen assay represents an interesting alternative to the widely used agar or methylcellulose systems for the culture of hematopoietic progenitors because of the equal or higher number of colonies detected,the easy phenotypical identification of colonies in stained gels,and the ability to store high-quality documentation. This technique is particularly attractive for use in the quality control of autologous bone marrow transplantation procedures. View Publication

Ionomycin,a new polyether antibiotic with a high affinity for calcium ions,is obtained in pure form from fermentation broths of Streptomyces conglobatus sp. nov. Trejo by solvent extraction. It is unique amongst known polyether antibiotics in that it has a UV absorption maximum at 300 nm. thereby distinguishing it from other antibiotics of its class. The Ca salt has the molecular formula C41H70O9Ca. Ionomycin is a narrow spectrum antibiotic being active against Gram-positive bacteria. View Publication

As described in the preceding paper,monoclonal antibodies have been raised by immunization of rats with mouse hematopoietic cells which detect a major cell-surface glycoprotein (Mr = 95 000) of mouse bone-marrow cells of the granulocytic series. While most of the monoclonal antibodies detect this molecule one bone-marrow and spleen cells of all mouse strains,two antibodies recognize alternative allelic forms of the molecule. One alloantigen is expressed in all the remaining inbred strains examined. The alloantigens are codominantly expressed on the cells of F1 mice. Backcrosses of DBA/2 and C57BL/6 with F1 mice (B6D2F1) confirmed that a single genetic locus is involved in the expression of the two antigenic forms and demonstrated linkage to Ly-m11 which has previously been mapped to mouse chromosome 2. These genetic mapping experiments and the biochemical properties of the glycoprotein suggested that it might be identical to a glycoprotein first identified on murine fibroblasts by Hughes and August and designated Pgp-1. This has been firmly established by exchange of monoclonal antibody reagents and sequential immunoprecipitations. View Publication

A murine leukocyte surface glycoprotein (Mr = 95 000) has been defined by means of xenogeneic monoclonal antibodies. In normal hematopoietic tissues,the glycoprotein is found in highest amounts in the bone marrow. Flow cytometric analysis shows that essentially all bone-marrow cells express the glycoprotein and that it is a major component of a subpopulation of cells containing predominantly granulocytic precursors. In contrast,only about 5 percent of thymocytes express sufficient glycoprotein to be detected by flow cytometric analysis,although under stringent conditions up to 20 percent of thymocytes are susceptible to complement-mediated cytotoxicity using a monoclonal antibody against the glycoprotein. Functional assays showed that both prothymocytes and colony forming unit-spleen express the glycoprotein which is broadly distributed on murine hematopoietic tumor cell lines. However,although some Thy-1+ (T) cell lymphomas express large amounts of the glycoprotein,others do not express detectable quantities of the molecule. The glycoprotein is not restricted to hematopoietic cells and can be detected on lung,kidney,brain,and liver as well as cultured fibroblasts. Monoclonal antibodies against the glycoprotein cross-react with an antigen present on human cells. As described in the accompanying paper,the glycoprotein exists in two antithetical allelic forms and we show that it is identical to a polymorphic surface molecule independently characterized by Colombatti and co-workers. View Publication

By using immunoelectron microscopy,we have localized the binding site on 50S Escherichia coli ribosomal subunits for puromycin,an antibiotic that interacts with the ribosomal peptidyltransferase center. This was achieved by affinity-labeling 50S subunits with N-bromoacetyl puromycin and treating the labeled subunits with an antibody specific for the N6,N6-dimethyladenosine moiety of puromycin. The position of the puromycin binding site was then revealed by localizing the attachment sites of the IgG molecules on the surfaces of the 50S subunits under the electron microscope: it was located at the interface between the subunits,on and around the wider lateral protuberance of the 50S subunit. This localizes directly the peptidyl transferase center on the surface of the large ribosomal subunit. View Publication

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Lymphoblasts from 93 children with acute lymphoblastic leukemia (ALL) were characterized by immunologic cell surface markers. These patients were treated on a single protocol,featuring adriamycin therapy during remission,and have been followed from 2 to 6.5 yr (median 4 yr). Three classes of patients were defined serologically: HTA+ Ia- CALLA-,Ia+ CALLA+ HTA-,and Ia+ CALLA- HTA-. Disease-free survival and sites of relapse were assessed within immunologic subsets. Similar to the findings of others,T-cell (HTA+ Ia-) patients fared poorly as compared to non-T-cell (Ia+ HTA-) patients (median disease-free survival was 12 and 47 mo. respectively; p = 0.0004). The majority of relapses in the HTA+ patients occurred at extramedullary sites. Late testicular relapse was rare among Ia+ patients. In addition,the common ALL antigen" (CALLA) may identify a relatively favorable subset within the Ia+ population. The prognostic value of the immunologic markers was compared with traditional clinical factors. There was much overlap between HTA+� View Publication

This report describes the establishment directly from normal preimplantation mouse embryos of a cell line that forms teratocarcinomas when injected into mice. The pluripotency of these embryonic stem cells was demonstrated conclusively by the observation that subclonal cultures,derived from isolated single cells,can differentiate into a wide variety of cell types. Such embryonic stem cells were isolated from inner cell masses of late blastocysts cultured in medium conditioned by an established teratocarcinoma stem cell line. This suggests that such conditioned medium might contain a growth factor that stimulates the proliferation or inhibits the differentiation of normal pluripotent embryonic cells,or both. This method of obtaining embryonic stem cells makes feasible the isolation of pluripotent cells lines from various types of noninbred embryo,including those carrying mutant genes. The availability of such cell lines should made possible new approaches to the study of early mammalian development. View Publication

A method has been described for the long-term culture of human bone marrow cells in liquid medium. Hematopoiesis,as measured by the production of granulocytic-macrophage progenitor cells (CFUc),continued for at least 20 weeks and was dependent upon the presence of a marrow-derived adherent layer of cells. As in the case of murine marrow liquid cultures,the adherent layer consisted of mononuclear phagocytic cells,endothelial cells,and lipid-laden adipocytes,the latter being essential for long-term hematopoiesis. Optimal growth conditions included McCoy's medium supplemented with fetal bovine serum,horse serum,and hydrocortisone and incubation at 33 degrees C. Horse serum in conjunction with hydrocortisone appeared essential for the growth of adipocytes. View Publication

Serotonin receptor affinity and photelectron spectral data were obtained on a number of substituted N,N-dimethyltryptamines. Evidence is presented that electron-donating substituents in the 5-position lead to enhanced behavioral disruption activity and serotonin receptor affinity as compared to unsubstituted N,N-dimethyltryptamine and analogues substituted in the 4- or 6-position. Some correlation was found between ionization potentials and behavioral activity,which may have implications concerning the mechanism of receptor binding. View Publication