技术资料

Monoclonal antibody HEA125 was used to study the tissue distribution of an epithelial cell surface glycoprotein of Mr 34,000 (Egp34). A large panel of normal and neoplastic tissues was examined for immunoreactivity with HEA125 by means of a sensitive immunoperoxidase technique. HEA125 labeled most epithelial cell types throughout the body but did not label any nonepithelial tissue. Major exceptions were epidermal keratinocytes,gastric parietal cells,hepatocytes,thymic cortical epithelial,and myoepithelial cells. Normal mesothelial cells were unreactive. In normal glandular epithelia and tubular adenocarcinomas exclusively the basolateral cell membranes were stained. HEA125 intensely reacted with all tested carcinoma specimens derived from colorectum,stomach,pancreas,liver,lung,mammary gland,ovary,thyroid,kidney,urinary bladder,and prostate including a number of anaplastic,diffusely infiltrating carcinomas. Metastatic lesions of these tumors were consistently positive. Generally,the staining of tumor cells was very homogeneous. The majority of squamous cell carcinomas were less strongly labeled than adenocarcinomas; keratinizing areas of the tumor masses were negative. Germ cell tumors and mesotheliomas of epithelioid type focally expressed the antigen. Egp34 was found to be absent from sarcomas,lymphomas,melanomas,and neurogenic tumors. Hence,HEA125 is a useful reagent for the distinction of carcinomas from nonepithelial neoplasms,even at very low degrees of histological differentiation. Furthermore,HEA125 allows the immunohistochemical detection of micrometastases originating from carcinomas. The antigen is detectable in formalin-fixed paraffin sections. View Publication

The amino acid sequence of a human placental bFGF was determined by a combination of protein and cDNA sequencing. The placental bFGF consists of 157 amino acid residues with a calculated molecular weight of 17,464 and is highly homologous to bovine pituitary bFGF. The human protein contains an amino terminal extension when compared to the sequence established for bovine bFGF (Esch et al.,1985) and to the sequence of the predicted translation product based on human bFGF cDNA clones (Abraham et al.,1986). View Publication

The glycoprotein hormone erythropoietin regulates the level of oxygen in the blood by modulating the number of circulating erythrocytes,and is produced in the kidney or liver of adult and the liver of fetal or neonatal mammals. Neither the precise cell types that produce erythropoietin nor the mechanisms by which the same or different cells measure the circulating oxygen concentration and consequently regulate erythropoietin production are known. Cells responsive to erythropoietin have been identified in the adult bone marrow,fetal liver or adult spleen. In cultures of erythropoietic progenitors,erythropoietin stimulates proliferation and differentiation to more mature red blood cells. Detailed molecular studies have been hampered,however,by the impurity and heterogeneity of target cell populations and the difficulty of obtaining significant quantities of the purified hormone. Highly purified erythropoietin may be useful in the treatment of various forms of anaemia,particularly in chronic renal failure. Here we describe the cloning of the human erythropoietin gene and the expression of an erythropoietin cDNA clone in a transient mammalian expression system to yield a secreted product with biological activity. View Publication

The acute lymphoblastic leukemia- (ALL) associated membrane antigen is a single glycosylated polypeptide of approximate m.w. of 100,000 (gp100),containing no intrachain disulfide linkages. Approximately 50% of gp100 will bind to lentil lectin,whereas 100% will bind to the lectin from Ricinus communis. Both lentil-binding and lentil nonbinding forms of the antigen appear to be identical by 2-dimensional isoelectric focusing/SDS polyacrylamide gel electrophoresis and peptide mapping. Carbohydrate,although contributing approximately 20 to 25% of the m.w.,appears not to be involved in the antigenic site of the ALL antigen as judged by precipitation of a molecule after tunicamycin treatment of cells or glycosidase digestion. Charge shift electrophoresis and labeling with the lipophilic nitrene reagent hexanoyl diiodo-N-(4-azido-2-nitrophenyl)-tyramine suggests that the cALL antigen is probably not an integral membrane protein; however,it remains tightly bound to the plasma membrane after subcellular fractionation. A glycoprotein of the same m.w. has been detected by immunoprecipitation on bone marrow cells of nonleukemic patients. serologic studies indicate that the cALL-associated antigen is found on the terminal transferase-positive lymphoid cells,and it therefore seems likely that the gp100 molecule is a normal gene products of lymphocyte precursors. View Publication

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The anti-My-10 mouse monoclonal antibody was raised against the immature human myeloid cell line KG-1a and was selected for nonreactivity with mature human granulocytes. Anti-My-10 immunoprecipitated a KG-1a cell surface protein with an apparent Mr of approximately 115 kD. We describe the binding of this antibody to human hematopoietic cell types and show that My-10 is expressed specifically on immature normal human marrow cells,including hematopoietic progenitor cells. My-10 is also expressed by leukemic marrow cells from a subpopulation of patients. Thus,this antibody allows the identification and purification of hematopoietic progenitor cells from normal human marrow and the subclassification of leukemia. 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

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

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Hypoxia-inducible factor 1 (HIF-1) is found in mammalian cells cultured under reduced O2 tension and is necessary for transcriptional activation mediated by the erythropoietin gene enhancer in hypoxic cells. We show that both HIF-1 subunits are basic-helix-loop-helix proteins containing a PAS domain,defined by its presence in the Drosophila Per and Sim proteins and in the mammalian ARNT and AHR proteins. HIF-1 alpha is most closely related to Sim. HIF-1 beta is a series of ARNT gene products,which can thus heterodimerize with either HIF-1 alpha or AHR. HIF-1 alpha and HIF-1 beta (ARNT) RNA and protein levels were induced in cells exposed to 1% O2 and decayed rapidly upon return of the cells to 20% O2,consistent with the role of HIF-1 as a mediator of transcriptional responses to hypoxia. View Publication

The requirement of complex sphingolipid biosynthesis for growth of neurons was examined in developing rat cerebellar Purkinje neurons using a dissociated culture system. Purkinje cells developed well-differentiated dendrites and axons after 2 weeks in a serum-free nutrient condition. Addition of 2 microM fumonisin B1,a fungal inhibitor of mammalian ceramide synthase,inhibited incorporation of [3H]galactose/glucosamine and [14C]-serine into complex sphingolipids of cultured cerebellar neurons. Under this condition,the expression of Purkinje cell-enriched sphingolipids,including GD1 alpha,9-O-acetylated LD1 and GD3,and sphingomyelin,was significantly decreased. After 2 weeks' exposure to fumonisin B1,dose-dependent measurable decreases in the survival and visually discernible differences in the morphology were seen in fumonisin-treated Purkinje cells. The Purkinje cell dendrites exhibited two types of anomalies; one population of cells developed elongated but less-branched dendrites after a slight time lag,but their branches began to degenerate. In some cells,formation of elongated dendrite trees was severely impaired. However,treatment with fumonisin B1 also led to the formation of spinelike protrusions on the dendrites of Purkinje cells as in control cultures. In contrast to the alterations observed in Purkinje cells,morphology of other cell types including granule neurons appeared to be almost normal after treatment with fumonisin B1. These observations indicated strongly that membrane sphingolipids participate in growth and maintenance of dendrites and in the survival of cerebellar Purkinje cells. Indeed,these effects of fumonisin B1 were reversed,but not completely,by the addition of 6-[[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino dcaproyl]sphingosine (C6-NBD-ceramide),a synthetic derivative of ceramide. Thus,we conclude that deprivation of membrane sphingolipids in a culture environment is responsible for aberrant growth of Purkinje cells. View Publication

Treatment of cells with a variety of growth factors triggers a phosphorylation cascade that leads to activation of mitogen-activated protein kinases (MAPKs,also called extracellular signal-regulated kinases,or ERKs). We have identified a synthetic inhibitor of the MAPK pathway. PD 098059 [2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one] selectively inhibited the MAPK-activating enzyme,MAPK/ERK kinase (MEK),without significant inhibitory activity of MAPK itself. Inhibition of MEK by PD 098059 prevented activation of MAPK and subsequent phosphorylation of MAPK substrates both in vitro and in intact cells. Moreover,PD 098059 inhibited stimulation of cell growth and reversed the phenotype of ras-transformed BALB 3T3 mouse fibroblasts and rat kidney cells. These results indicate that the MAPK pathway is essential for growth and maintenance of the ras-transformed phenotype. Further,PD 098059 is an invaluable tool that will help elucidate the role of the MAPK cascade in a variety of biological settings. View Publication