Inhibition of murine monocyte proliferation by a colony-stimulating factor-1 antisense oligodeoxynucleotide. Evidence for autocrine regulation.

Using a combination of v-myc and v-ras oncogenes, we have established a growth factor-independent monocyte cell line from murine fetal liver (FL-ras/myc). Biologic and molecular characterization demonstrated that the gene for the macrophage growth factor CSF-1 and the c-fms proto-oncogene (CSF-1 receptor) are expressed in this cell line, thus suggesting autocrine regulation as a possible mechanism for the unregulated growth of these cells. To study this possibility, we used 1) mAb, to neutralize the CSF-1 protein produced by the cell line, and 2) antisense oligomers, to inhibit CSF-1 gene products by specific base-pairing of complementary nucleic acids.

Enhanced therapeutic efficacy against an ovarian tumor xenograft of immunotoxins used in conjunction with recombinant alpha-interferon.

The antitumor effects of two immunotoxins were evaluated in vitro and in vivo against the human ovarian carcinoma cell line, OVCAR-3. The immunotoxins used were composed of recombinant ricin A chain (rRTA) covalently attached to a monoclonal antibody directed toward the human transferrin receptor (45412/rRTA, also called 454A12 MAB-rRTA by Cetus Corporation) or Pseudomonas exotoxin coupled to an anticarcinoma monoclonal antibody (NR-LU-10/PE). Preliminary characterization of the NR-LU-10 antigen by immunoprecipitation and cellular fluorescence demonstrated two dominant cell surface polypeptide moieties with molecular weights of 40,000 and 45,000 and a minor component with a molecular weight of 33,000.

Test of cure for genital Chlamydia trachomatis infection in women.

Convenient, reliable tests of cure for genital chlamydial infections have not been evaluated. Cervical appearance, endocervical Gram stain, enzyme immunoassay, and culture for Chlamydia trachomatis were evaluated during a pretreatment visit and at two subsequent randomized test-of-cure visits for 64 nongravid women with endocervical C trachomatis of 3544 patients screened. There were no useful correlations between C trachomatis resolution and cervical appearance.

Cell shape and increased free cytosolic calcium [Ca2+]i induced by growth factors.

Growth factors stimulate DNA synthesis of neoplastic cells but not of non-neoplastic cells in suspension cultures. Similarly, growth ceases in dense monolayers of non-neoplastic cells, while crowded neoplastic cells continue to grow. The mechanism of these important phenotypic changes is unknown; the block in growth stimulation could occur in early events of signal transduction at the plasma membrane or in a late step in the final steps of gene activation and induction of DNA synthesis.

Haemopoietic growth factor regulation of protein kinases and genes associated with cell proliferation.

Haemopoietic growth factors stimulate a number of common biochemical and molecular events despite the high specificity of individual ligand-receptor interactions. Analysis of three distinct colony-stimulating factors (CSFs), interleukin 3 (IL-3), granulocyte-CSF and granulocyte macrophage-CSF, and the lymphocytotropic growth factor IL-2 revealed remarkably similar distal subcellular biochemical signals, although the mode of initial membrane signal transduction may differ significantly. Both early progenitor cell growth factors, such as IL-3, and late-acting factors, such as CSF-1, stimulate tyrosine and serine/threonine substrate phosphorylations.

Hematopoietic growth-factor signal transduction and regulation of gene expression.

Hemopoietic cells have an absolute requirement for survival and proliferation for specific growth factors. The growth factors maintain the critical vitality of the cells by stimulating adenosine triphosphate (ATP) synthesis and hexose transport. Intracellular alkalinization also occurs rapidly through the stimulation of the Na+/H+ antiporter.

Novel characteristics of a 33KDa protein (pp33) rapidly phosphorylated in IL3 dependent cells by stimulation with IL3.

We report the novel properties of a 33 KDa cellular protein rapidly phosphorylated by stimulation of growth by IL3 in IL3 dependent lines. Although pp33 is readily soluble in SDS, SDS-solubilised pp33 is insoluble in non-ionic detergents and is excluded from electrophoretic analysis (IEF, NEPHGE) employing such detergents. Native pp33 is not extracted by non-ionic detergents with or without cation chelation.

IL-2 regulation of tyrosine kinase activity is mediated through the p70-75 beta-subunit of the IL-2 receptor.

The stimulation of activated human T lymphocytes with IL-2 results in increased tyrosine kinase activity. IL-2 treatment of Tac+ T cells stimulates the rapid phosphorylation of multiple protein substrates at M of 116, 100, 92, 70 to 75, 60, 56, 55, 33, and 32 kDa. Phosphorylation on tyrosine residues was detected by immunoaffinity purification of protein substrates with Sepharose linked antiphosphotyrosine mAb, 1G2.

Two-dimensional analysis of interleukin 2-regulated tyrosine kinase activation mediated by the p70-75 beta subunit of the interleukin 2 receptor.

The proliferation of activated T lymphocytes is dependent on the interaction of the polypeptide growth factor interleukin 2 (IL 2) with its heterodimeric receptor, which consists of a p55 alpha subunit and a p70-75 beta subunit. Previously, it was shown that IL 2 stimulates rapid serine phosphorylation of several membrane and cytysolic proteins. Here, using anti-phosphotyrosine antibodies to purify phosphotyrosyl proteins and two-dimensional gel analysis, we show that IL 2 stimulates rapid tyrosine phosphorylation of a variety of cellular proteins, including pp180, pp92, and pp42 in activated human T lymphocytes.

Activation of HIV-enhancer binding activity by mild detergents in human T cells.

Binding activity to the HIV enhancer, a sequence highly homologous to the NF-KB enhancer, is inducible by PMA in human Jurkat T cells. We here show that, similar to what is observed for NF-KB in B cells, mild detergents, such as Deoxycholate, reveal a cryptic binding activity in protein extracts from non activated cells. The complex revealed by detergent treatment correspond to the PMA inducible, highly sequence specific species.

In situ detection of a heat-shock regulatory element binding protein using a soluble synthetic enhancer sequence.

In various studies, enhancer binding proteins have been successfully absorbed out by competing sequences inserted into plasmids, resulting in the inhibition of the plasmid expression. Theoretically, such a result could be achieved using synthetic enhancer sequences not inserted into plasmids. In this study, a double stranded DNA sequence corresponding to the human heat shock regulatory element was chemically synthesized.

Interleukin 3 and phorbol ester stimulate tyrosine phosphorylation of overlapping substrate proteins.

FDC-P1 is a murine myeloid cell line that requires interleukin 3 (IL3) for survival and proliferation. While the biological effects of IL3 have been well described, the biochemical mechanisms of IL3 actions have only recently been examined. We have investigated whether IL3 or PMA stimulates phosphorylation of proteins on tyrosine as well as on serine/threonine residues as previously described [(1986) Blood 68, 906-913; (1987) Biochem.

Comparative analysis of IL-2 and IL-3 induced tyrosine phosphorylation.

IL2 and IL3 are polypeptide growth factors that support the survival and proliferation of, respectively, activated T lymphocytes and a range of myeloid cell types. We have examined the involvement of tyrosine phosphorylation in IL2 and IL3 mediated signal transduction. Phosphotyrosyl proteins were immunoaffinity purified and analyzed by single and two dimensional gel electrophoresis.

The molecular basis of immune cytokine action.

The polypeptide hormones governing the proliferation and differentiation of the mature immune system and hematopoiesis are collectively referred to as lymphokines. We have examined a number of biochemical and molecular events stimulated by several unique lymphokines which exhibit proliferative activity on lymphoid and myeloid cell lines. Interleukin-2 (IL-2) and several members of the colony-stimulating factors (IL-3, G-CSF, and GM-CSF) stimulate similar patterns of cellular phosphorylation including the prominent phosphorylation of a 68-kDa substrate present in numerous distinct lineage cell lines.

Interleukin 3 stimulation of tyrosine kinase activity in FDC-P1 cells.

Interleukin 3 stimulates the proliferation of FDC-P1, a murine myeloid cell line, however the biochemical events subsequent to binding of IL3 have only recently begun to be investigated. We have previously described the activation of protein kinase C (PK-C) and serine/threonine phosphorylation of a 68 kd protein following IL3 treatment of FDC-P1 cells. Here we have used an anti-phosphotyrosine antibody to purify proteins containing phosphotyrosine following IL3 administration to FDC-P1 cells.

Mitogen and lymphokine stimulation of heat shock proteins in T lymphocytes.

We have examined the effects of phytohemagglutinin (PHA) and the polypeptide growth factor interleukin 2 (IL-2) on the synthesis of the 70- and 90-kDa heat shock proteins (HSP70 and HSP90, respectively) in human T lymphocytes. Resting T cells (G0) stimulated with PHA responded with a generalized increase in protein synthesis that included HSP70. Gel blot analysis indicated that steady-state levels of HSP70 mRNA were not specifically modulated by PHA.

Specific inhibition of c-myc protein biosynthesis using an antisense synthetic deoxy-oligonucleotide in human T lymphocytes.

C-myc protein expression in human T cells was specifically inhibited by a 15-mer deoxy-oligonucleotide complementary to the 5' end of the human c-myc gene second exon. The oligonucleotide penetrates the cells without any treatment, with a plateau of cell association reached in 2 h. The oligonucleotide specifically blocked the de novo synthesis of c-myc protein, induced by PHA in human resting peripheral T cells, without impairing the overall synthesis of other proteins, as shown by two-dimensional analysis of [35S]methionine pulse-labeled proteins.

Molecular events associated with the action of haemopoietic growth factors.

Haemopoietic growth factors stimulate a number of consensus biochemical and molecular events regardless of the specificity detailed by unique ligand and receptor structures. Analysis of three distinct colony stimulating factors, CSFs (IL-3, G-CSF, GM-CSF) and the lymphocytotropic growth factor IL-2 reveal remarkable similar distal subcellular biochemical signals although initial membrane 'signal transduction' may differ significantly. Both early progenitor cell growth factors, such as IL-3, and late acting factors such as CSF-1, stimulate tyrosine and serine-threonine substrate phosphorylations.

Biochemical and molecular events controlled by lymphokine growth factors.

The polypeptide hormones that govern the proliferation and differentiation of the mature immune system and hematopoiesis are collectively referred to as lymphokines. We have examined a number of biochemical and molecular events stimulated by several unique lymphokines that exhibit proliferative activity on lymphoid and myeloid cell lines. IL-2 and several members of the colony-stimulating factors (multi-CSF, G-CSF, and GM-CSF) stimulate a similar pattern of cellular phosphorylation, including the prominent phosphorylation of a 68-kD substrate present in numerous distinct lineage cell lines.

Characterization of CD4 glycoprotein determinant-HIV envelope protein interactions: perspectives for analog and vaccine development.

The CD4 surface determinant, previously associated as a phenotypic marker for helper/inducer subsets of T lymphocytes, has now been critically identified as the binding/entry protein for human immunodeficiency viruses (HIV). The human CD4 molecule is readily detectable on monocytes, T lymphocytes, and brain tissues. Soluble HIV (HTLV IIIB) envelope protein (gp120) binds native or recombinant CD4 with equal affinity estimated to be 4 to 8 nM kDa.

Leaf photosynthesis and conductance of selected triticum species at different water potentials.

Leaf gas exchange characteristics of a desert annual (Triticum kotschyi [Boiss.] Bowden) and the wheat cultivar TAM W-101 (Triticum aestivum L. em Thell) were compared over a range of leaf water potentials from -0.

Chromatographic resolution of soluble and particulate protein phosphatases from Dictyostelium discoideum.

We have examined protein phosphatase activities that are present during the cellular differentiation of Dictyostelium. Utilizing differential centrifugation, ion exchange, gel filtration, and concanavalin A affinity chromatography we found a number of distinct protein phosphatase activities. Three peaks of soluble Kemptide phosphatase activity and a very broad and heterogeneous soluble histone phosphatase activity were resolved by anion exchange chromatography.

Extirpation of the fibular sesamoid simplified.

This technique allows the surgeon to maintain a firm grasp on the fibular sesamoid during its dissection, thus minimizing surgical trauma to the surrounding tissue and decreasing the chance of an inadvertent tenotomy of the flexor hallucis longus tendon. The authors have used this technique in 30 sesamoidectomies and found it to lower the frustration level significantly. The only instances in which it does not work as well as expected is with severely osteoporotic sesamoids, and partite sesamoids with fragments too small to pierce with the Kirschner wire.