Scientific Resources

The My-10 glycoprotein is an hematopoietic cell surface antigen expressed specifically by undifferentiated (blast) cells,constituting 1%-4% of normal adult bone marrow leukocytes. We used several immunological and in vitro culture methods to analyze the expression of this unique antigen on a variety of lymphohematopoietic progenitor cells. Colony-forming cells (CFC) for granulocyte-monocyte colonies (CFC-GM) and erythroid colonies (BFU-E) were predominantly My-10 positive. CFC with higher proliferative potential were more strongly My-10 positive than CFC with lower proliferative potential,and those for mixed-lineage and blast cell colonies were even more uniformly My-10 positive. Cells maintaining CFC-GM number in short-term marrow culture (pre-CFC) were found to be My-10 positive,as were lymphoid precursors defined by their content of intranuclear terminal deoxynucleotidyl transferase. More mature erythroid precursors (CFU-E) were heterogeneous for antigen expression and lost My-10 antigen progressively,in parallel with advancing maturational stage. The My-10 antigen permits rapid identification and purification of hematopoietic progenitor cells for further study or potential clinical application. The disappearance of the My-10 antigen,moreover,may be a probe for differentiation-linked cellular events. View Publication

The proliferation and differentiation effects of the synthetic retinoid TTNPB and of 13-cis retinoic acid (RA) on hemopoietic progenitors from bone marrow of myelodysplastic syndrome (MDS) patients were compared. The addition of TTNPB or RA to culture plates containing MDS patient's marrow cells stimulated myeloid colony (CFU-C) growth and caused a significant increase in granulocytic colonies (CFU-G). In the presence of RA the increase in CFU-G was statistically insignificant. Cellular differentiation studies in liquid suspension culture revealed that the two retinoic acid analogues cause a marked decrease in immature granulocytes and an increase in mature granulocytes. There was further an increase in the number of cells that reacted positively with monoclonal antibodies (McAb) binding specifically to granulocytes (B4,3,B13,9 and Leu M4) and a decrease in the percentage of cells reacting with the McAb against Ia-like determinants. These findings indicate that TTNPB is as active as RA in stimulating the growth of hemopoietic progenitors from MDS patients and in enhancing granulocytic differentiation in liquid culture. 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

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

In dissociated-cell cultures prepared from the embryonic rat hippocampus,neurons establish both axons and dendrites,which differ in geometry,in ultrastructure,and in synaptic polarity. We have used immunocytochemistry with monoclonal antibodies to study the regional distribution of beta-tubulin and micro-tubule-associated protein 2 (MAP2) in hippocampal cultures and their localization during early stages of axonal and dendritic development. After development for a week or more in culture,when axons and dendrites were well-differentiated,the distribution of these two proteins was quite different. Beta-tubulin was present throughout the nerve cell,in soma,dendrites,and axon. It was also present in all classes of non-neuronal cells,astrocytes,fibroblasts,and a presumptive glial progenitor cell. In contrast,MAP2 was preferentially localized to nerve cells; within neurons,MAP2 was present in soma and dendrites,but little or no immunostaining was detectable in axons. Both beta-tubulin and MAP2 were present in nerve cells at the time of plating. From the earliest stages of process extension,beta-tubulin was present in all neuronal processes,both axons and dendrites. Surprisingly,MAP2 was also initially present in both axons and dendrites,extending as far as the axonal growth cone. With subsequent development,MAP2 staining was selectively lost from the axon so that after 1 week in vitro little or no axonal staining remained. Taken together with earlier results (Cáceres et al.,1984a),these data indicate that the establishment of neuronal polarity,as manifested by the molecular differentiation of the axonal and dendritic cytoskeleton,occurs largely under endogenous control,even under culture conditions in which cell interactions are greatly restricted.(ABSTRACT TRUNCATED AT 250 WORDS) View Publication

Plasmacytoma growth factor (PCT-GF),a putative macrophage-derived lymphokine essential for the in vitro viability and proliferation of early generation plasmacytomas,was purified from conditioned medium of the murine macrophage cell line P388D1. The purification of PCT-GF was accomplished by a batch concentration on trimethylsilyl-controlled pore glass beads,followed by: gel filtration chromatography; hydrophobic interaction HPLC; and reverse-phase HPLC. SDS-PAGE analysis of the purified PCT-GF revealed a single band of Mr 23,000. The amino terminal sequence of PCT-GF was established as NH2-Pro-Thr-Ser-Gln-Val-Arg-Arg-Gly-Asp-Phe-Thr-Glu-Asp-Thr-Thr-Pro-Asn- Arg-Pro-Val-Tyr-Thr. No significant homology was found between this sequence and proteins in the National Biomedical Research Foundation database,suggesting that PCT-GF is a new lymphokine unrelated to previously described growth and differentiation factors. View Publication

Complementary DNA clones encoding the human kidney epidermal growth factor (EGF) precursor have been isolated and sequenced. They predict the sequence of a 1,207 amino acid protein which contains EGF flanked by polypeptide segments of 970 and 184 residues at its NH2- and COOH-termini,respectively. The structural organization of the human EGF precursor is similar to that previously described for the mouse protein and there is 66% identity between the two sequences. Transfection of COS-7 cells with the human EGF precursor cDNA linked to the SV40 early promoter indicate that it can be synthesized as a membrane protein with its NH2-terminus external to the cell surface. The human EGF precursor gene is approximately 110 kilobase pairs and has 24 exons. Its exon-intron organization revealed that various domains of the EGF precursor are encoded by individual exons. Moreover,15 of the 24 exons encode protein segments that are homologous to sequences in other proteins. Exon duplication and shuffling appear to have played an important role in determining the present structure of this protein. View Publication

A cDNA clone encoding a novel hematopoietic growth factor activity produced by a gibbon T cell line has been identified using a mammalian cell expression cloning system. The sequence of this cDNA proved to have significant homology to the sequence encoding murine interleukin 3 (IL-3). The human gene,which was readily identified because of its high degree of homology to the gibbon sequence,also displayed significant homology with the murine IL-3 sequence. The recombinant gibbon IL-3 protein proved to have multipotent colony stimulating activity when tested with normal human bone marrow cells,proving that this primate hematopoietin is not only structurally but also functionally related to murine IL-3. View Publication

Granulocyte colony-stimulating factor (G-CSF) is a member of the CSF family of hormone-like glycoproteins that regulate haematopoietic cell proliferation and differentiation,and G-CSF almost exclusively stimulates the colony formation of granulocytes from committed precursor cells in semi-solid agar culture. Recently,Nomura et al. have established a human squamous carcinoma cell line (designated CHU-2) from a human oral cavity tumour which produces large quantities of CSF constitutively,and the CSF produced by CHU-2 cells has been purified to homogeneity from the conditioned medium. We have now determined the partial amino-acid sequence of the purified G-CSF protein,and by using oligonucleotides as probes,have isolated several clones containing G-CSF complementary DNA from the cDNA library prepared with messenger RNA from CHU-2 cells. The complete nucleotide sequences of two of these cDNAs were determined and the expression of the cDNA in monkey COS cells gave rise to a protein showing authentic G-CSF activity. Furthermore,Southern hybridization analysis of DNA from normal leukocytes and CHU-2 cells suggests that the human genome contains only one gene for G-CSF and that some rearrangement has occurred within one of the alleles of the G-CSF gene in CHU-2 cells. View Publication

A simple and efficient procedure for the construction of bifunctional molecules is described and their use in a variety of applications documented. This procedure is based on our observation that mouse IgG1 monoclonal antibodies,when mixed with equimolar amounts of a high-affinity rat monoclonal antibody specific for mouse IgG1,yield uniform cyclic tetramolecular complexes each consisting of two mouse and two rat antibodies as shown by gel electrophoresis and electron microscopy. When solutions of two mouse antibodies (e.g. a and b) are mixed prior to the formation of complexes with the rat antibody,stable bispecific (a X b) complexes together with monospecific (a X a and b X b) complexes are obtained. Bispecific complexes prepared in this way were able to efficiently bind peroxidase to cell surface antigens,and to bind red blood cells to selected nucleated cell types present in heterogeneous populations. Tetrameric antibody complexes are more easily prepared than bispecific antibodies or bifunctional antibodies produced by transfection of myelomas with recombinant genes. They also have the advantage that the antigen-binding properties of the bivalent monoclonal antibodies are not compromised. Tetrameric antibody complexes thus represent a powerful new type of cross-linking reagent that may have a wide spectrum of applications in biology and medicine. View Publication

The work described in this paper demonstrates that the cellular binding of transforming growth factor beta,epidermal growth factor,platelet-derived growth factor,and fibroblast growth factor is reduced as cell density is increased. The reduction in transforming growth factor beta binding was observed in five different cell lines. Examination of several of the cell lines,under conditions where transforming growth factor beta binding is reduced,revealed that epidermal growth factor binding,platelet-derived growth factor binding,and fibroblast growth factor binding are also reduced. In the case of NRK-49F cells,the reduction in transforming growth factor beta binding results from a decrease in the number of high-affinity receptors and not from a change in receptor affinity. Similarly,it was determined that the reduction in epidermal growth factor binding is due to a selective reduction in the high-affinity receptors for epidermal growth factor. Overall,the data suggest that the effect of cell density on growth factor binding,which we refer to as density-induced down regulation of growth factor receptors,differs both from down regulation induced by a specific growth factor and from receptor transmodulation. View Publication

Twenty-four patients with acute promyelocytic leukemia (APL) were treated with all-trans retinoic acid (45 to 100 mg/m2/day). Of these,eight cases had been either nonresponsive or resistant to previous chemotherapy; the other 16 cases were previously untreated. All patients attained complete remission without developing bone marrow hypoplasia. Bone marrow suspension cultures were studied in 15 of the 24 patients. Fourteen of these patients had morphological maturation in response to the retinoic acid (1 mumol/L). Chloroacetate esterase and alpha-naphthyl acetate esterase staining as well as electronmicroscopic examination confirmed that retinoic acid-induced cells differentiated to granulocytes with increased functional maturation (as measured by nitroblue tetrazolium reduction,NBT). The single nonresponder to retinoic acid in vitro was resistant to treatment with retinoic acid but attained complete remission after addition of low-dose cytosine arabinoside (ara-C). During the course of therapy,none of the patients showed any abnormalities in the coagulation parameters we measured,suggesting an absence of any subclinical disseminated intravascular coagulation. The only side effects consisted of mild dryness of the lips and skin,with occasional headaches and digestive symptoms. Eight patients have relapsed after 2 to 5 months of complete remission. The others remain in complete remission at 1+ to 11+ months and are still being followed up. We conclude that all-trans retinoic acid is an effective inducer for attaining complete remission in APL. View Publication