Showing papers by "Kettering University published in 1989"
TL;DR: Results indicate that cyclosporin A either directly inhibits the function of nuclear proteins critical to T lymphocyte activation or inhibits the action of a more proximal member of the signal transmission cascade leading from the antigen receptor to the nucleus.
Abstract: One action of cyclosporin A thought to be central to many of its immunosuppressive effects is its ability to inhibit the early events of T lymphocyte activation such as lymphokine gene transcription in response to signals initiated at the antigen receptor. Cyclosporin A was found to specifically inhibit the appearance of DNA binding activity of NF-AT, AP-3, and to a lesser extent NF-kappa B, nuclear proteins that appear to be important in the transcriptional activation of the genes for interleukin-2 and its receptor, as well as several other lymphokines. In addition, cyclosporin A abolished the ability of the NF-AT binding site to activate a linked promoter in transfected mitogen-stimulated T lymphocytes and in lymphocytes from transgenic mice. These results indicate that cyclosporin A either directly inhibits the function of nuclear proteins critical to T lymphocyte activation or inhibits the action of a more proximal member of the signal transmission cascade leading from the antigen receptor to the nucleus.
661 citations
TL;DR: Two nearly identical, linked genes that encode these transcripts have been cloned for Fc gamma RIII(CD16), one of which (III-1) is allelic for NA- 1 and NA-2, and the allelic sites have been mapped to two single nucleotides in the extracellular domain.
Abstract: A low affinity receptor for IgG immune complexes, Fc gamma RIII(CD16), is expressed on human NK cells as an integral membrane glycoprotein anchored through a transmembrane peptide; on polymorphonuclear neutrophils (PMN) the receptor is anchored through a phosphatidylinositol (PI) linkage. The protein on NK cells has a molecular mass 6-10 kD larger than that on PMN, and, unlike the latter, is resistant to PI-specific phospholipase C (PI-PLC). Fc gamma RIII(CD16) transcripts isolated from PMN and NK cells of single donors revealed multiple single nucleotide differences, one of which converts an in frame UGA termination codon to a CGA codon. The resulting open reading frame encodes a longer cytoplasmic domain for Fc gamma RIII(CD16) in NK cells, contributing to its transmembrane anchor. Two nearly identical, linked genes that encode these transcripts have been cloned for Fc gamma RIII(CD16), one of which (III-1) is allelic for NA-1 and NA-2. The allelic sites have been mapped to two single nucleotides in the extracellular domain. These genes are transcribed in a cell type-specific fashion to generate the alternatively anchored forms of this receptor.
580 citations
TL;DR: It is reported here that in vitro and in the presence of ATP, T antigen assembles a double hexamer, centred on the core origin and extending beyond it by 12 bp in each direction, which initiates replication at the coreorigin.
Abstract: Simian virus 40 (SV40) replicates in nuclei of human and monkey cells. One viral protein, large tumour (T) antigen, is required for the initiation of DNA replication. The development of in vitro replication systems which retain this property has facilitated the identification of the cellular components required for replication. T antigen recognizes the pentanucleotide 5'-GAGGC-3' which is present in four copies within the 64 base-pairs (bp) of the core origin. In the presence of ATP it binds with increased affinity forming a distinctive, bilobed structure visible in electron micrographs. As a helicase, it unwinds SV40 DNA bidirectionally from the origin. We report here that in vitro and in the presence of ATP, T antigen assembles a double hexamer, centred on the core origin and extending beyond it by 12 bp in each direction. The assembly of this dodecamer initiates an untwisting of the duplex by 2-3 turns. In the absence of ATP, a tetrameric structure is the largest found at the core origin. In the absence of DNA, but in the presence of ATP or its non-hydrolysable analogues, T antigen assembles into hexamers. This suggests that ATP effects an allosteric change in the monomer. The change alters protein-protein interactions and allows the assembly of a double hexamer, which initiates replication at the core origin.
333 citations
TL;DR: The isolation of a cDNA encoding a novel serine protease that is present in HL-60 cells and is down-regulated during induced differentiation of these cells is described and this protease myeloblastin is named.
300 citations
TL;DR: The structural heterogeneity of the human low affinity receptor for IgG, FcRII(CD32), has been elucidated through the isolation, characterization, and expression of cDNA clones derived from myeloid and lymphoid RNA that predict amino acid sequences consistent with integral membrane glycoproteins with single membrane spanning domains.
Abstract: The structural heterogeneity of the human low affinity receptor for IgG, FcRII(CD32), has been elucidated through the isolation, characterization, and expression of cDNA clones derived from myeloid and lymphoid RNA. These clones predict amino acid sequences consistent with integral membrane glycoproteins with single membrane spanning domains. The extracellular domains display sequence homology to other Fc gamma Rs and members of the Ig supergene family. A minimum of three genes (Fc gamma RIIa, IIa', and Fc gamma RIIb) encode these transcripts, which demonstrate highly related extracellular and membrane spanning domains. IIa/IIa' differ substantially in the intracytoplasmic domain from IIb. Alternative splicing of the IIb gene generates further heterogeneity in both NH2- and COOH-terminal domains of the predicted proteins. Comparison to the murine homologues of these molecules reveals a high degree of conservation between the products of one of these genes, Fc gamma RIIb, and the murine beta gene in primary sequence, splicing pattern, and tissue distribution. In contrast, the sequence of IIa' indicates its relationship to the beta-like genes, with mutation giving rise to a novel cytoplasmic domain, while IIa is a chimera of both alpha- and beta-like genes. Expression of these cDNA molecules by transfection results in the appearance of IgG binding molecules that bear the epitopes defined by the FcRII(CD32) mAbs previously described.
299 citations
TL;DR: The structural requirements for GPtdIns anchoring are investigated through the study of two closely related pro-teins which exhibit alternative membrane attachment, and analysed a series of III-1 and III-2 mutants in transient expression assays, showing that Ser 203 in the GPTDIns attachment domain is the dominant residue in deter-mining whether the molecule can be GPtdins-anchored.
Abstract: Cell-surface proteins are associated with the lipid bilayer either as membrane-spanning molecules or as glycosyl phosphatidylinositol (GPtdIns)-linked proteins. Proteins destined for GPtdIns anchoring are synthesized as precursors with a hydrophobic C-terminal transmembrane domain, which is removed during the processing of these proteins in the endoplasmic reticulum (ref. 1). We have investigated the structural requirements for GPtdIns anchoring through the study of two closely related proteins which exhibit alternative membrane attachment. The IgG Fc receptor, Fc gamma RIII, is GPtdIns-linked on neurophils (III-1) whereas on natural killer (NK) cells and macrophages it is found as a transmembrane-anchored molecule (III-2), able to mediate antibody-dependent cellular cytotoxicity and phagocytosis. At the primary structural level, the III-1 gene differs from that encoding III-2 by only nine nucleotide substitutions, which result in six amino-acid differences, and the absence of 21 amino acids at the C terminus. We have analysed a series of III-1 and III-2 mutants in transient expression assays, and show that Ser 203 in the GPtdIns attachment domain is the dominant residue in determining whether the molecule can be GPtdIns-anchored. As in the case of its murine homologue, Fc gamma RII alpha, surface expression of the III-2 molecule is dependent on co-expression of a second subunit, the gamma chain of F epsilon RI. Our data also suggest that gamma chain can associate with the III-1 precursor, preventing GPtdIns attachment, favouring instead a transmembrane form.
180 citations
TL;DR: Analysis of avian leukosis virus-induced B-cell lymphomas for multiple, stage-specific oncogene activations indicates that c-myc and c-bic may act synergistically during lymphomagenesis and that c -bic is involved in late stages of tumor progression.
Abstract: We have examined avian leukosis virus-induced B-cell lymphomas for multiple, stage-specific oncogene activations. Three targets for viral integration were identified: c-myb, c-myc, and a newly identified locus termed c-bic. The c-myb and c-myc genes were associated with different lymphoma phenotypes. The c-bic locus was a target for integration in one class of lymphomas, usually in conjunction with c-myc activation. The data indicate that c-myc and c-bic may act synergistically during lymphomagenesis and that c-bic is involved in late stages of tumor progression.
178 citations
TL;DR: Results of serial cytogenetic investigations on the pretreatment and posttreatment mediastinal yolk sac tumor and immature teratoma biopsies and two separate leukemic bone marrow aspirates from a patient who developed acute nonlymphocytic leukemia show that in this case the leukemia was derived from the malignant germ cell clone.
Abstract: The biological basis for acute leukemia associated with mediastinal germ cell tumors has remained unexplained due to lack of critical data that would illuminate the genetic relationship between the two tumors in a given patient. Here we present results of serial cytogenetic investigations on the pretreatment and posttreatment mediastinal yolk sac tumor and immature teratoma biopsies and two separate leukemic bone marrow aspirates from a patient who developed acute nonlymphocytic leukemia 11 months after the initial diagnosis of the germ cell tumor. Presence of an i(12p) in all tumor clones and trisomy 21 in one clone in the posttreatment mediastinal tumor and all leukemic clones establishes the common origin of all tumor clones and shows that in this case the leukemia was derived from the malignant germ cell clone.
112 citations
TL;DR: The results indicate that the t(3;22)(q27;q11) is the third most common recurring translocation in diffuse non-Hodgkin's lymphoma.
102 citations
TL;DR: Site-directed mutagenesis of the vaccinia virus gene encoding a type I DNA topoisomerase implicates Tyr-274 as the active-site residue that forms a covalent adduct with DNA during cycles of DNA-strand breakage and reunion.
Abstract: Site-directed mutagenesis of the vaccinia virus gene encoding a type I DNA topoisomerase implicates Tyr-274 as the active-site residue that forms a covalent adduct with DNA during cycles of DNA-strand breakage and reunion. Replacement of Tyr-274 by phenylalanine results in loss of the ability of the enzyme to relax negatively supercoiled DNA as well as to form the covalent DNA-protein intermediate. Substitution of phenylalanine for tyrosine at nine other sites in the protein has no apparent effect on enzyme activity. Amino acid sequence alignment reveals Tyr-274 to be homologous to Tyr-727 and Tyr-771, respectively, of the type I topoisomerases from Saccharomyces cerevisiae and Saccharomyces pombe; Tyr-727 and Tyr-771 have been shown to represent the active-site tyrosines of those enzymes. Sequence comparison of the active-site regions defines a motif Ser-Lys-Xaa-Xaa-Tyr common to the viral and cellular type I topoisomerases, including the human enzyme.
77 citations
TL;DR: Coordinate regulation of HLA class II gene expression during development and coinduction of class II genes by soluble factors suggests that common trans-acting factor(s) control expression of these genes.
TL;DR: It is construed that vaccinia DNA topoisomerase I acts to promote illegitimate recombination in E. coli by promoting int-independent prophage excision.
Abstract: Vaccinia virus encapsidates a Mr 32,000 type IDNA topoisomerase. Although the vaccinia gene encoding the topoisomerase is essential for virus growth, the role of the enzyme in vivo remains unclear. In the present study, the physiologic consequences of vaccinia topoisomerase action have been examined in a heterologous system, Escherichia coli. The vaccinia topoisomerase gene was inducibly expressed in an int-lambda lysogen BL21(DE3) using a T7 RNA polymerase-based transcription system. Expression of active topoisomerase in this context resulted in recA-dependent lysogenic induction as well as cell lysis. Surprisingly, topoisomerase expression also effected a 200-fold increase in the titer of infectious lambda phage, apparently by promoting int-independent prophage excision. This effect was not observed during lysogenic induction with nalidixic acid. Restriction analysis of genomic DNA from plaque-purified excisants revealed (in 10 of 10 cases) gross alterations of the DNA structure around the att site relative to the structure of the parental phage DE3. It is construed therefore that vaccinia DNA topoisomerase I acts to promote illegitimate recombination in E. coli.
TL;DR: In this article, a single base substitution in the acceptor splice site at the 3′ end of the first intron, changing the normal AG dinucleotide to AA, was found to result in a larger than normal DRB4 gene transcript.
Abstract: The HLA-DRβ protein, DRβIV, encoded by the DRB4 gene, is found on class II+ cells of all DR4, DR9, and most DR7 individuals. However, in some DR7 individuals (DR7, Dw11), the DRβIV protein cannot be detected. To investigate the molecular mechanism responsible for this defect in expression, two overlapping genomic clones encoding the defective DRB4 allele (DRB4-null) were isolated. Although restriction fragment length analysis demonstrated no obvious alterations in the DRB4-null gene, nucleotide sequence analysis revealed a single base substitution in the acceptor splice site at the 3′ end of the first intron, changing the normal AG dinucleotide to AA. The nucleotide sequences of all the exons and remaining splice junctions were identical to those of the normal DRB4 gene. The effect of the altered splice junction was evident from RNA blot analysis where inactivation of the normal splice site was found to result in a larger than normal DRB4 gene transcript. Thus, defective expression of the DRβIV protein results from incorrect processing of the mRNA from the DRB4-null allele.
TL;DR: Evidence is presented that the multiprotein primosome per se can act as a DNA helicase in the absence of the DNA polymerase III holoenzyme.
Journal Article•
TL;DR: Human IL3 is a multilineage hemopoietic growth factor whose activities appear to encompass and extend beyond those of GM-CSF.
TL;DR: Two patients with metastatic gastrointestinal adenocarcinoma in the breast are reported, each with a histologic pattern similar to the primary neoplasm and an absence of in situ carcinoma which characterizes primary breast cancer.
Abstract: Two patients with metastatic gastrointestinal adenocarcinoma in the breast are reported. Metastases in the breast are usually painless upper outer quadrant masses. On mammography they are typically well-circumscribed lesions without microcalcifications. A breast mass in a patient with a history of cancer, even if clinically or mammographically benign, should raise suspicion of a metastasis. Pathological features include a histologic pattern similar to the primary neoplasm and an absence of in situ carcinoma which characterizes primary breast cancer. Surgical excision for local control and systematic therapy is the most appropriate treatment.
TL;DR: The progressive increase in the size of the core-associated, BrUMP-containing transcripts indicated that they were still engaged by elongating RNA polymerase, consistent with a predominant pathway of mRNA 3'-end formation by virions that involves VTF-dependent transcription termination.
TL;DR: It is found that TGF-beta 1 could induce hemoglobin accumulation in K-562 cells, a human multipotential hematopoietic cell line, and showed the same electrophoretic characteristics as the hemoglobin induced by hemin.
TL;DR: The results indicate that each ligand either has different binding site(s) in chromatin or modulates chromatin structure in a specific way by changing the affinity of different sets of proteins for their respective binding sites, resulting in their selective extraction or entrapment.
Abstract: The nuclear chromatin binding sites of the antitumor drugs mitoxantrone, ametantrone, doxorubicin, mithramycin, and actinomycin D and the intercalating ligand ethidium were studied by polyacrylamide gel electrophoresis of the proteins released from rat liver nuclei in the presence and absence of these drugs in buffer of low ionic strength (10 mM NaCl). At 25-50 microM free ligand concentration, each drug produced a specific and reproducible pattern of extractable proteins of different molecular weight by (i) releasing new proteins, (ii) altering the quantity of particular extracted proteins, and/or (iii) selectively entrapping other proteins in the nuclei. Ethidium, up to 100 microM, did not affect release of proteins from the nuclei. These results indicate that each ligand either has different binding site(s) in chromatin or modulates chromatin structure in a specific way by changing the affinity of different sets of proteins for their respective binding sites, resulting in their selective extraction or entrapment. The lack of effect of ethidium indicates that intercalation of the ligand to DNA, per se, does not alter the release of nuclear proteins. If patterns of nuclear proteins selectively released or retained by antitumor drugs are found to correlate with biological activity, this type of analysis may be helpful in new drug design and screening.
TL;DR: Limited proteolysis with trypsin was shown to convert the vaccinia capping enzyme into a form capable of generating a Mr = 59,000 enzyme-GMP complex, and a model for the organization of functional domains within the capping enzymes is suggested.
Journal Article•
TL;DR: The results indicate that the in vivo administration of IL-1 results in neutrophilia and generation of myelopoietic suppressive effects, mediated by cyclo-oxygenase pathway products.
Abstract: Injection of human rIL-1 alpha in intact normal mice has positive and negative effects on myelopoiesis. Within 6 h postinjection, peripheral neutrophilia can be demonstrated. However, bone marrow and spleen cells capable of inhibiting CFU-granulocyte macrophage proliferation are detected between 6 and 48 h postinjection. These myelopoietic suppressor cells belong to the monocytic lineage and are identical to inhibitory cells induced by PGE2. Treatment of mice with indomethacin, a PG synthesis inhibitor, completely blocked the generation of IL-1-alpha-induced myelopoietic suppressor cells, and significantly enhanced femoral and splenic CFU-GM proliferation after a single injection of 0.4 microgram/mouse IL-1. The peripheral blood neutrophilia observed within 6 h after IL-1 injection was delayed to 18 to 24 h postinjection in indomethacin-pretreated mice. In mice treated with four consecutive daily injections of 0.4 microgram IL-1, a sustained peripheral neutrophilia was observed. IL-1 had little effect on femoral CFU-GM in these animals, however, splenic CFU-GM was increased 7- to 10-fold by 4 to 7 days postinjection. In IL-1 plus indomethacin-treated mice, sustained peripheral neutrophilia was observed although to a lesser degree than with IL-1 alone. Marrow CFU-GM were relatively unaffected, however, splenic CFU-GM were increased by 27-fold. These results indicate that the in vivo administration of IL-1 results in neutrophilia and generation of myelopoietic suppressive effects, mediated by cyclo-oxygenase pathway products. Blockade of PG synthesis by using the cyclo-oxygenase inhibitor indomethacin abrogates the myelopoietic suppressive effects associated with IL-1 administration and optimizes its myelopoietic stimulatory capacity. The inclusion of a cyclo-oxygenase inhibitor may have significant relevance to the clinical use of IL-1.
TL;DR: Carpal tunnel syndrome is much commoner in those with limited joint mobility of the hands than in those without this complication of insulin-dependent diabetes mellitus.
Abstract: Carpal tunnel syndrome is much commoner in those with limited joint mobility of the hands than in those without this complication of insulin-dependent diabetes mellitus. The implications of this are discussed.
Patent•
30 Jun 1989TL;DR: In this article, a class of compounds having two or more nonpolar components connected by a polar group and having polar groups on the termini of the compound is presented. And the authors provide a method of treating a patient having a tumor characterized by proliferation of neoplastic cells which comprises administering to the patient an amount of compound effective to selectively induce terminal differentiation of such cells, thereby inhibiting their proliferation and suppressing oncogenicity.
Abstract: The invention provides a class of compounds having two or more nonpolar components connected by a polar group and having polar groups on the termini of the compound. The invention also concerns a method of selectively inducing terminal differentiation of neoplastic cells and thereby inhibiting proliferation of such cells which comprises contacting the cells under suitable condition with an amount of the compound effect to selectively induce terminal differentiation. Moreover, the invention provides a method of treating a patient having a tumor characterized by proliferation of neoplastic cells which comprises administering to the patient an amount of the compound effective to selectively induce terminal differentiation of such neoplastic cells, thereby inhibiting their proliferation and suppressing oncogenicity. Lastly, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the compound in an amount effective less than an amount which would cause toxicity in the patient.
TL;DR: An improved high-performance liquid-chromatographic method was developed for estimation of polyamines in crude plant extracts that requires preparation and analysis time similar to other current methods but provides greater sensitivity and selectivity.
Abstract: An improved high-performance liquid-chromatographic method was developed for estimation of polyamines in crude plant extracts. Polyamines were derivatized with o-phthalaldehyde and mercaptoethanol (OPT). The fluorescent derivatives were eluted from a C18 column with the dimethylcyclohexylamine-phosphate buffer derived by T. Skaaden and T. Greibrokk ([1982] J Chromatogr 247: 111-122) after treatment to remove impurities in the buffer. The method had a sensitivity of 1-2 picomoles and completely resolved nine polyamines (agmatine, spermine, nor-spermidine, spermidine, 3,5-homospermidine, 4,4-homospermidine, 1,3-diaminopropane, putrescine, and cadaverine) in 12 to 14 minutes. An optional ion-exchange step was used to remove less basic amines (including amino acids) and to concentrate the crude extracts. This method was compared with benzoyl chloride derivatization. Use of the benzoyl chloride method vastly under-estimated the amount of polyamine in some plant extracts, a problem not encountered with the OPT procedure. Additionally, the OPT procedure resolved two isomers of homospermidine found in Azolla caroliniana. These two isomers were not resolved with the benzoylation method. Overall, the OPT method described here requires preparation and analysis time similar to other current methods but provides greater sensitivity and selectivity.
TL;DR: Results suggest that RNA II-p BR322 DNA hybrid formation is essential to permit preprimosome assembly during pBR322 DNA replication under conditions where both RNase H and DNA polymerase I are absent.
TL;DR: It is suggested that down-regulation of the CD8 antigen may require its expression in a T- cell environment and/or the association of CD8 with the T-cell receptor or other T cell-specific molecules.
Abstract: The CD4 and CD8 molecules are rapidly phosphorylated following exposure of CD4+ or CD8+ human cytotoxic T lymphocytes (CTL) clones to B-lymphoblastoid cell lines bearing the relevant target alloantigens. Treatment of CD4+ or CD8+ CTL clones with phorbol myristate acetate (PMA), phytohemagglutinin, or mitogenic combinations of CD2-specific antibodies also resulted in CD4 or CD8 phosphorylation. Down-regulation of the surface expression of these molecules could be demonstrated in both CD4+ and CD8+ clones following exposure to the relevant alloantigen or PMA. Parallel experiments were conducted using mouse L cells in which the human CD4 or CD8 antigens were stably expressed. Exposure of these transfectants to PMA induced rapid phosphorylation of the CD4 and CD8 molecules. As in CD4+ CTL clones, rapid modulation of the CD4 antigen could be demonstrated in L cells following PMA treatment. In contrast, there was no demonstrable down-regulation of the CD8 antigen in PMA-treated CD8+ L cell transfectants. These studies demonstrate a significant differential property of the CD4 and CD8 antigens and suggest that down-regulation of the CD8 antigen may require its expression in a T-cell environment and/or the association of CD8 with the T-cell receptor or other T cell-specific molecules.
TL;DR: The progress of 108 children identified by the vision screening programme in school as having defective vision was reviewed and treatment resulted in improvement in visual acuity of the worst eye for 16 children, but the need to treat children with lesser degrees of amblyopia is questioned.
Abstract: The progress of 108 children who were identified by the vision screening programme in school as having defective vision (excluding those with puberty onset myopia) was reviewed. Treatment of these children resulted in improvement in visual acuity of the worst eye (two lines or better) for 16 children. Eighteen children had severe amblyopia (6/24 or worse). Among these the vision of only five was improved by treatment. Two thirds of the children had refractive errors in the better eye which required correction. It seems sensible to identify and treat children with bilateral refractive errors, but the need to treat children with lesser degrees of amblyopia is questioned.