Dendritic nucleic acid structures.

Dendritic molecules are highly-branched arborescent structures and have found applications as chemical reagents, lubricants, contrast media for magnetic resonance, and others. Dendritic nucleic acids could be extremely useful for the development of nucleic acid diagnostics as signal amplification tools and potentially as drug (antisense) delivery vehicles. Further, due to the relatively large size of nucleic acid molecules, nucleic acid dendrimers could be readily labeled with numerous fluorescent compounds and/or protein moieties with limited steric hindrance and/or quenching. Herein, we present a physical-mathematical model of a new class of dendrimers, constructed entirely from unique nucleic acid monomers that are designed such that sequential hybridization adds successive layers of monomer in a geometric expansion of both mass and free single-stranded sequences, called arms, at the surface. The specially designed monomer is a heterodimer of two single-stranded nucleic acid oligomers possessing a central double-stranded waist and four single-stranded arms for binding. Assembly of a dendrimer is initiated from a single monomer and proceeds in layers, the first comprising four monomers, which provides 12 single-stranded arms. Thus, the second layer adds 12 monomers resulting in 36 single-stranded arms. After addition of the 6th layer, the dendrimer is comprised of 1457 monomers, of which 972 reside in the 6th layer, which possesses 2916 single-stranded arms. The accompanying mathematical description of a dendrimer's growth is generic. A natural consequence and limiting condition of the growth process we describe is a saturated solution of nucleic acid, which is, in effect, a "nucleic acid membrane".

Activation of human monocyte cytotoxicity by natural and recombinant immune interferon.

Human T cell hybridomas were established by fusion of SH9 cells, the 6-thioguanine-resistant mutant line of human T lymphoma Hut 102-B2, with concanavalin A-stimulated human peripheral blood lymphocytes. Hybridoma line L38 produced a macrophage activating factor (MAF) with the ability to activate human peripheral blood monocytes to show enhanced cytotoxicity against human colon adenocarcinoma HT-29 cells in a 72-hr 125iododeoxyuridine-release assay. The L38 line was then cloned by the limiting dilution technique and two sublines, L38B and L38D, were found to produce high levels of MAF constitutively. Interferon activity was also detected in L38B and L38D supernatants. When interferon activity was neutralized with specific antiserum to purified human immune interferon (IFN-gamma), MAF activity was abrogated. To confirm that the MAF activity is indeed due to IFN-gamma, IFN-gamma was purified from the culture supernatant of another human T cell hybridoma, L265K2, a cell line known to produce high levels of IFN-gamma. Two highly purified IFN-gamma fractions with m.w. of 20,000 and 25,000, respectively, were obtained by NaDodSO4/polyacrylamide gel electrophoresis (SDS-PAGE). Similar fractions were obtained from IFN-gamma derived from human peripheral blood lymphocyte (PBL) cultures induced with 12-0-tetradecanoylphorbol-13-acetate (TPA) and phytohemagglutinin (PHA). In comparison, Escherichia coli-derived recombinant human IFN-gamma separated by SDS-PAGE yielded two major active fractions with m.w. of 17,000 and 34,000. With all three types of preparations, a close correlation was found between the presence of IFN-gamma activity demonstrable in an antiviral assay and MAF activity in individual fractions. Substantial quantitative differences were observed in the ability of various human IFN to activate monocytes. Although no MAF activity was detected with IFN-alpha and IFN-beta at concentrations up to 200 U/ml, both natural and recombinant IFN-gamma showed marked MAF activity at concentrations as low as 0.3 to 1 U/ml.

Lymphokine production by human T cell hybridomas.

Human T cell hybridomas were generated by hybridization of SH9 cells, the 6-thioguanine-resistant variant of human T lymphoma Hut102-B2, with concanavalin A-stimulated human peripheral blood lymphocytes. The hybrid nature of the established cell lines was documented by the difference in D14S1 restriction fragment length polymorphism between SH9 and the hybridoma cell DNA, and by the expression of OKT11 antigen on hybrid cells. T cell growth factor, macrophage growth factor (MGF), and interferon (IFN) activities have been demonstrated in the supernatants of different hybrid cultures, but not in SH9 cell cultures. Substantial quantities of MGF were secreted by several hybrids including the L23 line. MGF activity was dose-dependent, heat-labile, and synergistic with indomethacin. High titers of IFN activity were found in the cultures of hybridoma L415 and its subclones. Neutralization with specific antisera showed the IFN synthesized by L415 clones was immune interferon (IFN-gamma). Like the parental SH9 line, all of the hybridomas producing these lymphokines exhibited a cell surface phenotype typical for helper T cells. The hybridoma system therefore shows potential for the study of various lymphokines produced by human helper T lymphocytes.

Detection of a feline X-linked antigen in somatic cell hybrids. Single-cell analysis using monoclonal antibodies.

We tested the premise that monoclonal antibodies to either intracellular or membrane antigens can greatly facilitate the construction of linkage maps of mammals whose chromosomes can be introduced into rodent cells. Monoclonal antibodies against antigenic determinants of cat lymphocytes and fibroblasts were used to analyze feline antigen expression in cat-mouse somatic cell hybrid populations selected to contain the X-linked feline HPRT locus. The frequency of antigen expression as measured by fixed cell immunofluorescence (IF) assays, varied greatly within hybrid populations for all but the antigen designated as VP382. Its frequent presence in hybrid cells led to the prediction, confirmed by 8-azaguanine selection experiments, that its expression was controlled by a gene, or genes, on the feline X chromosome. The antigens identified by the rest of the antibodies segregated independently of each other in cat-mouse somatic cell hybrids and their expression appeared to be controlled by autosomal genes of the cat.

Feline oncornavirus-associated cell membrane antigen: a viral and not a cellularly coded transformation-specific antigen of cat lymphomas.

The feline oncornavirus-associated cell membrane antigen (FOCMA) was defined as a tumor antigen common to cat lymphomas and fibrosarcomas induced by feline leukemia virus (FeLV) and feline sarcoma virus (FeSV), respectively. The antigen was recognized by sera from cats thought to be resistant to leukemogenesis. We report here that a common denominator in the activity of naturally occurring viremic cat antisera to FOCMA is, in fact, their reactivity to FeLV C antigenic determinants. The cat antisera, monoclonal antibodies to FOCMA, and monoclonal antibodies to FeLV C, all reacted in immunofluorescence assays with FeLV C-infected cells and immunoprecipitated a molecule electrophoretically indistinguishable from envelope glycoprotein of FeLV. Viremic cat antisera to FOCMA bound to budding virus particles of FeLV C-infected cells, even though some of them could not be absorbed by mature virion proteins. Thus, the unusual feature of cat antibodies to FOCMA is their binding to nascent but not to mature virus particles. FOCMA-positive cat lymphomas expressed antigenic determinants of FeLV-C gp70, with or without productive infection. FeLV-negative tumors not expressing FeLV C gp70 were also FOCMA negative. Furthermore, most of the viremic cat sera and the monoclonal antibodies to FOCMA did not react with FeSV-transformed nonproducer cells. The absence of FOCMA from these cells and from FeLV-negative lymphoid tumors and its presence in FeLV-C infected fibroblasts indicated that this antigen is virus encoded and not a cellular tumor-specific antigen.

Human T cell hybridomas secreting immune interferon.

Human T-cell hybridomas were established by hybridization of concanavalin A-stimulated human peripheral blood lymphocytes with a 6-thioguanine-resistant mutant cell line, designated SH9, derived by irradiation from a cloned human cutaneous T lymphoma line, Hut102-B2. High levels of interferon (IFN) were demonstrated in the supernatants of hybridoma L265 and its subclones. Whereas no IFN was detected in SH9 cell cultures, up to 1,330 units of IFN per ml were produced spontaneously by the hybrids. On induction with 12-omicron-tetradecanoylphorbol 13-acetate, IFN synthesis in hybridoma cultures was enhanced 8- to 16-fold. Neutralization with specific antisera and determination of antiviral activities in human and bovine cells showed that the IFN secreted by the hybridomas was immune IFN (IFN-gamma). Analysis of DNA content, karyotype, and cell surface phenotype, including T cell specific antigens and receptors, confirmed the T cell hybrid nature of L265 clones. No correlation was found in the hybridomas between IFN production and the expression of HTLV, a retrovirus released by Hut102-B2 and SH9 cells.

Marked increase in ribosomal RNA gene multiplicity in a rat hepatoma cell line.

An H4-IIE-C3 hepatoma cell line derived from an ACI rat has been shown to have differentially stained regions attached to the short arms of chromosomes 3, 11 and 13 and the long arm of an unidentified small chromosome. There is cell to cell variability in the number and size of the differentially stained regions, which contain, on the average, about 5% of the total DNA. A series of secondary constrictions occur at intervals along the length of each differentially stained region. These stain with silver by the Ag-AS method, indicating that the differentially stained regions contain sites of active 45S ribosomal precursor RNA transcription. In situ hybridization to metaphase chromosomes shows that the hepatoma cells have a 10 fold increase in DNA coding for 18S and 28S ribosomal RNA, 90% of it located in the differentially stained regions, and no change in the number of genes coding for 5S RNA. These results have been confirmed by filter disc hybridization.

Variability in the amount of beta-globin mRNA in beta0 thalassemia.

Globin mRNA isolated from a number of beta0 thalassemia patients of different ethnic origins was analyzed by RNA-cDNA hybridization and, in two cases, by fingerprint analysis of 125I-labeled mRNA. Quantitation of the relative amounts of alpha- and beta-mRNA by hybridization to purified alpha-and beta-cDNA revealed that in approximately half the cases, there was less than 1% as much beta-mRNA as alpha-mRNA. In the rest of the cases, low levels of beta-like mRNA were detected in amounts 4-12% as abundant as alpha-mRNA. There was variability in the yield of beta-like mRNA in patients of the same racial group, in the same patient at different times and in similarly affected siblings: beta-mRNA was virtually absent in some samples, whereas low but significant levels were found in other samples. In one patient, beta-like mRNA was not detected in peripheral blood RNA, but was present in the RNA of bone marrow cells. In one case, the thermal stability of the beta0 thalassemia mRNA-beta-cDNA hybrid was measured and found to be slightly lower than that of the authentic beta-mRNA-beta-cDNA hybrid. In none of the cases tested was there synthesis of beta-globin chains directed by beta0 thalassemia mRNA in a cell-free protein-synthesizing system, even when beta-like mRNA was detected in the sample by hybridization assays. mRNA from two patients was labeled in vitro with 125I, digested with T1 RNAase and fractionated in two dimensions. Analysis of the resulting fingerprints revealed the presence of prominent alpha chain-specific oligonucleotides without detectable beta chain-specific oligonucleotides, and thereby confirmed the results of hybridization assays showing absent or very low levels of beta-mRNA in the same RNA samples. Our results support the concept that beta0 thalassemia is heterogeneous in its molecular basis even within the same racial group: in some patients, it is associated with absent beta globin mRNA, whereas in other patients, it is associated with low but significant levels of nonfunctional beta or beta-like globin mRNA. The variable amounts of beta-like mRNA detected in different samples from the same patient, and in patients with the same genotype, indicate that as yet undefined factors can influence the yield of beta-like mRNA observed in beta0 thalassemia.

Chromosome damage in Chinese hamster cells produced by 125I-UdR at the site of its incorporation.

Metaphase chromosomal aberrations were produced by 125I-labeled iododeoxyuridine (125I-UdR) incorporated into Chinese hamster Don cells at the end of the S-period of the cell cycle. Chromosome damage and the number of autoradiographic silver grains were recorded for whole cells, for chromosome pairs No. 4 and No. 5, and for the X and the Y chromosomes. The X and the Y chromosomes, which label late in S, were at least twice as heavily labeled as chromosome pairs No. 4 and No. 5--two readily recognizable autosomes of similar size. The incidence of chromosome damage was at least six times that which would have been expected from equivalent doses of X-rays and the incidence of damage was directly related to the number of silver grains over each chromosome. We estimate that it takes four to ten disintegrations to produce a visible chromosome aberration. The finding that chromosome damage is localized at the site of the 125I decay is most readily explained by the high flux of low energy Auger electrons occurring at the site of the decay of the incorporated 125I atom.

Blood thymidine level and iododeoxyuridine incorporation and reutilization in DNA in mice given long-acting thymidine pellets.

A long-acting thymidine pellet consisting of 190 mg of cholesterol and 60 mg of thymidine has been developed for the study of thymidine metabolism and reutilization in vivo. Implantation of such a pellet s.c. in adult mice will maintain the blood plasma concentration of thymidine at levels between 40 and 8 X 10(-6) M, which are from 36 to 7 times those of normal mice, for periods up to 48 hr. During this period, in vivo uptake and reutilization of [125I]iododeoxyuridine, a thymidine analog, into intestinal and tumor DNA were almost completely suppressed. While iododeoxyuridine reutilization is not large in normal proliferative tissue even in the absence of pellet implants, reutilization of over 30% was measured in large, rapidly growing ascites tumors. The inhibition of iododeoxyuridine incorporation by elevated thymidine blood levels is directly proportional to serum concentration. This appears to be due to a thymidine pool in rapid equilibrium with blood thymidine. This pool is at least 10 times larger than the 4-nmole pool of extracellular thymidine.