RESUMO
Monolayer and suspension cell cultures prepared from Hodgkin's disease tumors in the spleen were examined microscopically and by cytogenetics, tested for lymphocyte and monocyte cell surface properties, and assayed for enzymes by histochemical and spectrophotometric techniques. Hodgkin's disease monolayer cultures were composed of rapidly proliferating round and polygonal cells that were capable of propagation in vitro for an indefinite period of time. Abnormal aneuploid chromosomes were found in short-term Hodgkin's disease monolayers that had been passaged 16-20 times, and in established cell lines carried in culture longer than 3 yr and passaged more than 200 times. Cells fromHodgkin's disease monolayers contained lysozyme (muramidase), fluoride-resistant alpha naphthol acetate esterase, acid and alkaline phosphatase, and chymotrypsin-like activity. The monolayers did not exhibit specific cell surface markers or phagocytosis. Suspension cultures derived from Hodgkin's disease monolayers were composed of cells with aneuploid karyotypes and similar enzymes. The Hodgkin's disease suspension culture cells had surface receptors for complement and IgGFc, lacked surface or cytoplasmic immunoglobulin, and did not form Erosettes, react with an antithymocyte serum, nor exhibit phagocytosis. Normal monolayer culture cells, derived from adult spleen and human fetal spleen and thymus, were composed of spindle cells with a diploid number of chromosomes that could be carried for only a finite period of time in vitro. Normal cultured cells contained similar esterases and phosphatases, but were devoid of lysozyme and chymotrypsin-like activity. The morphologic, cytogenetic, cell surface, and enzymatic findings indicate that our Hodgkin's disease monolayer and suspension cultures are composed of cells with many properties suggesting an origin from monocytes (macrophages) rather than lymphocytes or fibroblasts. The presence of aneuploid karyotypes is consistent with a neoplastic origin and derivation from a malignant cell of Hodgkin's disease.
Assuntos
Doença de Hodgkin/patologia , Neoplasias Esplênicas/patologia , Fosfatase Alcalina/metabolismo , Membrana Celular/imunologia , Células Cultivadas , Técnicas de Cultura , Doença de Hodgkin/enzimologia , Humanos , Fragmentos Fc das Imunoglobulinas , Técnicas Imunológicas , Linfócitos/enzimologia , Linfócitos/patologia , Linfócitos/ultraestrutura , Monócitos/enzimologia , Monócitos/patologia , Monócitos/ultraestrutura , Naftol AS D Esterase/metabolismo , Espectrofotometria , Neoplasias Esplênicas/enzimologia , Coloração e RotulagemRESUMO
An antigen in tissue cultures derived from Hodgkin's disease tumors was investigated by polyacrylamide gel electrophoresis, column chromatography, and isotopic antibody techniques. Fourteen long-term, serially passaged monolayer cultures prepared from tumor nodules of Hodgkin's disease in the spleen were studied; 11 monolayers derived from normal adult spleen and human fetal spleen and thymus were used as controls. Cell-free medium from Hodgkin's disease and normal cultures were centrifuged, and the pellet fractions were sedimented in a discontinuous sucrose gradient, solubilized with dodium dodecyl sulfate, and labeled with radioiodine. Gel filtration and electrophoresis revealed a component in samples prepared from medium of Hodgkin's disease cultures that was not observed in medium from normal cultures. An antiserum made in rabbits against this component reacted by radioiodine-labeled antibody assay with an antigen on the surface on cells from Hodgkin's disease cultures that was present in very small amounts, or in a cryptic state, on normal cultured cells. This antigen, intimately associated with propagation of cells obtained from the tumor in vitro, was not demonstrable in noncultured Hodgkin's disease tissue...
Assuntos
Antígenos de Neoplasias/análise , Doença de Hodgkin/imunologia , Membrana Celular/imunologia , Cromatografia em Gel , Técnicas de Cultura , Eletroforese em Gel de Poliacrilamida , RadioimunoensaioRESUMO
Lysyl-tRNA synthetase was purified to 70-90% of homogeneity from Escherichia coli K-12. The enzyme was purified from wild-type cells grown in minimal medium, or minimal medium containing either 20 mM L-alanine or 3 mM glycly-L-leucine. The synthetase was similarly purified from a mutant strain grown in minimal medium plus 20 mM L-alanine. Results based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and trypsin inactivation studies indicate (A) that the presence of L-alanine of glycyl-L-leucine in the culture medium alters the properties of the wild-type enzyme; (B) that the alteration of the synthetase by l-alanine and glycyl-L-leucine is different; and (c) that the molecular weight of lysyl-tRNA synthetase is at least 135000--140000. The results suggest that most likely the metabolites modify the structure of lysyl-tRNA synthetase, but the possibility that the metabolites induce the synthesis of a new lysyl-tRNA synthetase cannot be completely eliminated.
Assuntos
Alanina/farmacologia , Aminoacil-tRNA Sintetases , Dipeptídeos/farmacologia , Escherichia coli/enzimologia , Lisina-tRNA Ligase , Aminoacil-tRNA Sintetases/metabolismo , Glicina , Leucina , Lisina-tRNA Ligase/metabolismo , Substâncias Macromoleculares , Peso Molecular , Mutação , Conformação Proteica , Especificidade da Espécie , TripsinaAssuntos
Biossíntese de Proteínas , Ribossomos/fisiologia , Animais , Fracionamento Celular , Código Genético , Morfogênese , Conformação de Ácido Nucleico , Precursores de Ácido Nucleico/metabolismo , RNA Mensageiro/metabolismo , RNA Ribossômico/metabolismo , RNA de Transferência/metabolismo , Proteínas Ribossômicas/fisiologia , Ribossomos/análise , Ribossomos/ultraestruturaRESUMO
Platelets from cat and cattle with Chediak-Higashi disease were found completely devoid of Ap4A as measured by high performance liquid chromatography. Using a very sensitive firefly biolumnescence method 6% of the normal content of Ap4A was, however, found in platelets from sick animals. A content of Ap A of 1.90 +/- 0.11 X 10 M (means +/- SEM, n = 10) was found in whole normal human blood as measured by firefly bioluminescense method in trichloroacetic acid extracts of the blood samples. This concentration corresponds to the contribution from the platelets, thus the contribution of Ap4A from erythrocytes and the "buffy-coat" is negligible. Using the same method an Ap4A contents in platelets of 0.063 and 0.021 nmol/mg of protein compared to the normal content of 0.42 nmol/mg of protein (1) was found in two patients with severe myeloproliferative disorder calculated in this way on basis of platelet counts and on the assumption that 10(11) platelets contain 189 mg of protein (2). Comparison of these figures with parallel HPLC analyses on acid extracts of platelets isolated from the same patient were in agreement. The storage pool deficiency of adenine nucleotides in this disease found by others on basis of release experiments (3) can thus be diagnosed by rapid and simple measurements of Ap4A in whole blood using the advantage of Ap4A being a specific components stored in dense granules.
Assuntos
Nucleotídeos de Adenina/sangue , Transtornos Plaquetários/sangue , Fosfatos de Dinucleosídeos , Deficiência do Pool Plaquetário/sangue , Animais , Plaquetas/metabolismo , Gatos , Bovinos , Síndrome de Chediak-Higashi/sangue , Grânulos Citoplasmáticos/metabolismo , Humanos , Medições Luminescentes , Deficiência do Pool Plaquetário/diagnósticoRESUMO
Biological science is a rapidly flowing experimental stream, at times encountering a dam that impedes further progress. At such a pomt, a single crack may induce a major breakthrough Discovery of the double helical structure of DNA in 1953 (1) caused such an event, with flooding of new information into the area now known as molecular biology.
Assuntos
Diferenciação Celular , Neoplasias/metabolismo , RNA de Transferência , Aminoácidos/metabolismo , Sequência de Bases , Carcinógenos , Genes Reguladores , Código Genético , Genética Médica , Genética Microbiana , Metilação , Metiltransferases/análise , Metiltransferases/metabolismo , Biologia Molecular , Mutação , RNA de Transferência/análise , RNA de Transferência/isolamento & purificação , RNA de Transferência/metabolismo , Supressão Genética , Transferases/metabolismoAssuntos
Cobre , RNA de Transferência , Acetatos , Aminas , Soluções Tampão , Isótopos de Carbono , Escherichia coli , Formiatos , Concentração de Íons de Hidrogênio , Cinética , ValinaAssuntos
Aminoacil-tRNA Sintetases/metabolismo , Cisteína , Escherichia coli/enzimologia , Etilaminas , Alanina/farmacologia , Aminoácidos , Arginina , Isótopos de Carbono , Meios de Cultura , Resistência Microbiana a Medicamentos , Estabilidade de Medicamentos , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/isolamento & purificação , Genótipo , Glutamatos , Cinética , Lisina , Mutagênicos/farmacologia , Mutação , Especificidade da Espécie , Relação Estrutura-Atividade , Ácidos Sulfônicos/farmacologia , Temperatura , Fatores de TempoAssuntos
Aminoácidos/metabolismo , Escherichia coli/enzimologia , Ligases/antagonistas & inibidores , RNA de Transferência/biossíntese , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Arginina/metabolismo , Isótopos de Carbono , Cianatos , Difosfatos/metabolismo , Esterases , Ligases/análise , Lisina/metabolismo , NitrobenzenosAssuntos
Escherichia coli/enzimologia , Ligases , Lisina , Nucleotídeos de Adenina , Trifosfato de Adenosina , Arginina , Difosfatos , Estabilidade de Medicamentos , Ativação Enzimática , Etanol , Cinética , Ligases/antagonistas & inibidores , Modelos Biológicos , Modelos Químicos , Isótopos de Fósforo , Cloreto de Potássio , RNA Bacteriano , RNA de Transferência , Cloreto de Sódio , Temperatura , Termodinâmica , TrometaminaAssuntos
Aminoacil-tRNA Sintetases , Cisteína , Escherichia coli/enzimologia , Etilaminas , Cromatografia em Gel , Resistência Microbiana a Medicamentos , Estabilidade de Medicamentos , Escherichia coli/efeitos dos fármacos , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Lisina , Substâncias Macromoleculares , Biologia Molecular , Peso Molecular , Mutação , Desnaturação Proteica , Especificidade da Espécie , UreiaAssuntos
HIV-1/efeitos dos fármacos , Oligorribonucleotídeos/farmacologia , Tionucleotídeos/síntese química , Sequência de Bases , Desenho de Fármacos , Estabilidade de Medicamentos , Genes tat/genética , HIV-1/genética , Dados de Sequência Molecular , Desnaturação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/farmacologia , Oligodesoxirribonucleotídeos/farmacologia , Oligorribonucleotídeos/síntese química , RNA de Cadeia Dupla/farmacologiaRESUMO
Diadenosine-5',5'''-P(1),P(4)-tetraphosphate (Ap(4)A) and its analog P(2),P(3)-monochloromethylene diadenosine-5',5'''-P(1),P(4)-tetraphosphate (AppCHClppA) are competitive inhibitors of adenosine diphosphate-induced platelet aggregation, which plays a central role in arterial thrombosis and plaque formation. In this study, we evaluate the imaging capabilities of positron-emission tomography (PET) with P(2),P(3)-[(18)F]monofluoromethylene diadenosine-5',5'''-P(1),P(4)-tetraphosphate ([(18)F]AppCHFppA) to detect atherosclerotic lesions in male New Zealand White rabbits. Three to six months after balloon injury to the aorta, the rabbits were injected with [(18)F]AppCHFppA, and microPET imaging showed rapid accumulation of this radiopharmaceutical in the atherosclerotic abdominal aorta, with lesions clearly visible 30 min after injection. Computed tomographic images were coregistered with PET images to improve delineation of aortoiliac tracer activity. Plaque macrophage density, quantified by immunostaining with RAM11 against rabbit macrophages, correlated with PET measurements of [(18)F]AppCHFppA uptake (r = 0.87, P < 0.0001), whereas smooth-muscle cell density, quantified by immunostaining with 1A4 against smooth muscle actin, did not. Biodistribution studies of [(18)F]AppCHFppA in normal rats indicated typical adenosine dinucleotide behavior with insignificant myocardial uptake and fast kidney clearance. The accumulation of [(18)F]AppCHFppA in macrophage-rich atherosclerotic plaques can be quantified noninvasively with PET. Hence, [(18)F]AppCHFppA holds promise for the noninvasive characterization of vascular inflammation.
Assuntos
Aterosclerose/diagnóstico , Fosfatos de Dinucleosídeos , Tomografia por Emissão de Pósitrons/métodos , Animais , Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/farmacocinética , Modelos Animais de Doenças , Masculino , Estrutura Molecular , Coelhos , RatosRESUMO
The synthesis of an oligonucleotide functionalized to attach two different reporter groups at specific internucleotide linkages is described. To incorporate the amine specific reporter group the internucleotide linkage is modified to phosphoramidate (N-1-aminoalkyl) and for a thiol specific reporter group the internucleotide linkage is modified to a phosphorothioate diester. The synthetic cycle for introducing the modified internucleotide linkages at specific sites can be carried out using an automated DNA synthesizer. Combination of reporter groups have been attached successfully.
Assuntos
Aminas , Oligodesoxirribonucleotídeos/química , Compostos de Sulfidrila , Amidas , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Dados de Sequência Molecular , Estrutura Molecular , Oligodesoxirribonucleotídeos/síntese química , Ácidos FosfóricosRESUMO
The tridecamer d(A-A-T-G-G-T-A-A-A-A-T-G-G), which is complementary to 13 nucleotides of the 3'- and 5'-reiterated terminal sequences of Rous sarcoma virus 35S RNA, was added to chick embryo fibroblast tissue cultures infected with Rous sarcoma virus. Inhibition of virus production resulted. The inference emerges that the tridecamer and its counterpart with blocked 3'- and 5'-hydroxyl termini enter the chick fibroblast cells, hybridize with the terminal reiterated sequences at the 3' and 5' ends of the 35S RNA, and interfere with one or more steps involved in viral production and cell transformation. Likely sites of action are (i) the circularization step of the proviral DNA intermediate, and (ii) the initiation of translation, the latter being described in the following communication [Stephenson, M. L. & Zamecnik, P. C. (1978) Proc. Natl. Acad. Sci. USA 75, 285--288].