RESUMO
A substantial amount of information on the biosynthesis of tylosin has been obtained over the past ten years. Physiological studies and experiments with tylosin-blocked (tyl) mutants have suggested the probable pathway by which tylactone is converted to tylosin. The development of recombinant DNA methodology for streptomycetes in general, and for Streptomyces fradiae in particular, has allowed us to apply gene cloning techniques in further studies of tylosin biosynthesis in S. fradiae. The macrocin O-methyltransferase (MOMT), which catalyzes the last step in tylosin biosynthesis, was purified, and the sequence of the 35 amino acids at its amino-terminus was determined. A synthetic 44 base oligonucleotide probe was constructed on the basis of the amino acid sequence. The probe was used to identify sequences containing the MOMT structural gene in bacteriophage and cosmid libraries of S. fradiae DNA. Complementation of tyl mutants with the cloned DNA sequences identified nine tyl biosynthetic genes (tylC, D, E, F, H, J, K, L, and M) in a 42 kb stretch of DNA. Genes complementing four mutant classes, tylA, B, G, and I were not found. A tylosin-resistance gene, tlrB, was located just left of the tyl gene cluster. Tylosin-sensitive mutants of S. fradiae, which were isolated from regenerated protoplasts and which have pleiotropic deficiencies in tylosin biosynthesis, contained deletions which included at least some of the identified tyl loci and one or both of two tylosin-resistance genes, tlrB and tlrC. Possible schemes for the functional organization of the tyl region of the S. fradiae genome are discussed.
Assuntos
DNA Recombinante , Leucomicinas/biossíntese , Streptomyces/metabolismo , DNA Bacteriano/biossíntese , Resistência Microbiana a Medicamentos/genética , Leucomicinas/farmacologia , Fenótipo , Plasmídeos , Streptomyces/efeitos dos fármacos , Streptomyces/genéticaRESUMO
Mesenchyme (UGM) and epithelium (UGE) isolated from the urogenital sinuses (UGS) of 17-day male and female rat embryos were separated by using a trypsinization procedure, grown on soft agar, transplanted into syngeneic pubertal male hosts as subcapsular renal grafts, and then collected after 29-30 days. Neither UGM nor UGE underwent prostatic morphogenesis when grown under these conditions. However, tissue recombinants composed of UGM + UGE grew and produced prostatic glands with acinar secretory material. Further, UGM + UGE recombinants were made by varying the proportions of mesenchymal and epithelial tissues. The size of the implants was a function of the absolute amount of mesenchyme; increasing the absolute amount of UGM produced larger specimens whereas varying the UGE had no effect. The UGM was also found to be essential for supporting the growth of small glandular elements derived from the ventral prostate of pubescent rats. Segments isolated from the terminal vesicles (TIPs) and from prostatic tissue adjacent to the urethra (PDCT) regressed when implanted alone under the kidney capsule. However, combination of the prostatic segments with UGM produced prostatic glands with relative wet weight and DNA content responses of the following order: UGM + TIP greater than UGM + PDCT = UGM + UGE. Two-dimensional gel electrophoretic protein patterns from UGM + PDCT and UGM + TIP specimens had differential expression of three protein regions unique to the ventral prostate Quantitative and qualitative responses of the TIP and PDCT segments to UGM inductive influences indicate that differences exist between the epithelia of the TIP and PDCT regions of the ventral lobes of the rat prostate.
Assuntos
Genitália Masculina/embriologia , Animais , Células Cultivadas , DNA/análise , Células Epiteliais , Epitélio/transplante , Feminino , Genitália Feminina/citologia , Genitália Feminina/embriologia , Genitália Masculina/citologia , Genitália Masculina/transplante , Masculino , Ratos , Transplante IsogênicoRESUMO
Immunocytochemical techniques were used to identify human proinsulin chimeric protein in cytoplasmic inclusion bodies of genetically modified Escherichia coli. Antibodies to proinsulin chimeric protein (human proinsulin coupled at its amino-terminus to a portion of the E. coli tryptophan E gene product) were localized in E. coli using post-embedding staining with protein A-peroxidase labelling for transmission electron microscopy. The observable distribution of the labelled antibody was limited to that portion of the E. coli cytoplasm occupied by inclusion bodies. The localization of human peptides as insoluble masses within the bacterial cytoplasm has important implications in relation to the synthesis, recovery and purification of pharmacologically useful substances produced through the application of recombinant DNA technology.
Assuntos
Proteínas de Bactérias/análise , Escherichia coli/ultraestrutura , Corpos de Inclusão/análise , Proinsulina/análise , Proteínas Recombinantes de Fusão , Escherichia coli/metabolismo , Histocitoquímica , Humanos , Técnicas Imunoenzimáticas , Microscopia EletrônicaRESUMO
Escherichia coli that has been genetically manipulated by recombinant DNA technology to synthesize human insulin polypeptides (A chain, B chain, or proinsulin) contains prominent cytoplasmic inclusion bodies. The amount of inclusion product within the cells corresponds to the quantity of chimeric protein formed by the bacteria. At peak production, the inclusion bodies may occupy as much as 20 percent of the Escherichia coli cellular volume.
Assuntos
Escherichia coli/ultraestrutura , Insulina/genética , Clonagem Molecular/métodos , Grânulos Citoplasmáticos/ultraestrutura , DNA Recombinante , Escherichia coli/metabolismo , Humanos , Microscopia Eletrônica , PlasmídeosAssuntos
Fibrina/metabolismo , Fibrinogênio/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Animais , Cromatografia em Gel , Feminino , Fibrinogênio/farmacologia , Iodoacetamida/farmacologia , Lisossomos/metabolismo , Macrófagos/enzimologia , Masculino , Plasminogênio , Coelhos , Ácido Tricloroacético/farmacologiaAssuntos
Osso e Ossos/metabolismo , AMP Cíclico/metabolismo , Osteoblastos/metabolismo , Animais , Osso e Ossos/ultraestrutura , Calcitonina/farmacologia , Células Cultivadas , Técnicas de Cultura/métodos , Masculino , Microscopia Eletrônica , Osteoblastos/ultraestrutura , Hormônio Paratireóideo/farmacologia , Ratos , Fatores de TempoAssuntos
2-Naftilamina , Naftalenos , Peptídeo Hidrolases/análise , 2-Naftilamina/análogos & derivados , Amidoidrolases/análise , Aminoácidos , Aminopeptidases/análise , Animais , Catepsinas/análise , Quelantes , Fenômenos Químicos , Química , Colorimetria , Corantes , Compostos de Diazônio , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Inibidores Enzimáticos , Complexo de Golgi/enzimologia , Histocitoquímica , Lisossomos/enzimologia , Microscopia Eletrônica , Naftalenos/análogos & derivados , Peptídeo Hidrolases/metabolismo , Resinas Vegetais , Espectrometria de FluorescênciaAssuntos
Penicillium , Vírus de Plantas , Vírus de RNA , Isótopos de Carbono , Centrifugação com Gradiente de Concentração , Centrifugação Zonal , Dicroísmo Circular , RNA Polimerases Dirigidas por DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Temperatura Alta , Microscopia Eletrônica , Peso Molecular , Desnaturação de Ácido Nucleico , Nucleoproteínas/análise , Penicillium chrysogenum , Vírus de Plantas/análise , Vírus de Plantas/enzimologia , Vírus de Plantas/isolamento & purificação , RNA Viral/análise , RNA Viral/isolamento & purificação , Espectrofotometria , Proteínas Virais/análiseAssuntos
Encéfalo/efeitos dos fármacos , Morfina/farmacologia , Acetilcolinesterase/análise , Fosfatase Ácida/análise , Animais , Pressão Atmosférica , Encéfalo/enzimologia , Química Encefálica/efeitos dos fármacos , Fracionamento Celular , Centrifugação Zonal , Masculino , Mesencéfalo/análise , Proteínas do Tecido Nervoso/análise , Tamanho do Órgão , Ratos , Frações Subcelulares/efeitos dos fármacosRESUMO
Rabies virus produced in duck embryo cell culture was concentrated from volumes of 14 to 30 liters to 400 to 800 ml by zonal centrifugation. Virus titers of peak fractions were from 100- to 1,000-fold greater than those of the starting material. Vaccines were prepared by combining fractions with peak virus titers and diluting back to 10 times concentration. The resulting beta-propiolactone-inactivated vaccines, when prepared as lyophilized vaccines with AlPO(4) adjuvant diluents, were low in protein nitrogen (0.01 mg/ml), and three of four lots passed the National Institutes of Health potency test when tested as equivalent to a standard 10% suspension of duck embryo or mouse brain tissue vaccine. These vaccines also induced good sero-conversion in adult rabbits after a single 1-ml dose of vaccine. Guinea pigs sensitized with zonal-centrifuged purified duck embryo vaccine (with AlPO(4) adjuvant) did not exhibit anaphylactic shock reactions when challenged with homologous vaccine. Also, no anaphylactic shock reactions were observed when guinea pigs were sensitized with either a 10% experimental duck embryo vaccine or cell culture vaccine and then challenged with the zonal-purified vaccine. However, guinea pigs sensitized with cell culture or zonal-purified vaccine and then challenged with the 10% experimental vaccine did show slight transitory congestion. The 10% experimental whole duck embryo vaccine was responsible for all observed anaphylactic shock reactions whether homologous or heterologous.
Assuntos
Centrifugação Zonal , Vacina Antirrábica/normas , Vírus da Raiva/isolamento & purificação , Raiva/prevenção & controle , Adjuvantes Imunológicos , Alumínio , Anafilaxia , Animais , Formação de Anticorpos , Técnicas de Cultura , Patos , Liofilização , Cobaias , Compostos Heterocíclicos , Humanos , Imunoquímica , Lactonas , Camundongos , Fosfatos , Propionatos , Coelhos , Vacinação , Cultura de VírusAssuntos
Penicillium , Vírus , Álcoois , Animais , Bioensaio , Centrifugação Zonal , Precipitação Química , Cromatografia por Troca Iônica , Meios de Cultura , Formaldeído , Temperatura Alta , Interferons/análise , Interferons/biossíntese , Células L , Métodos , Camundongos , Microscopia Eletrônica , Desnaturação de Ácido Nucleico , Penicillium/crescimento & desenvolvimento , Fenóis , RNA Viral/análise , RNA Viral/isolamento & purificação , RNA Viral/farmacologia , Ribonucleases , Espectrofotometria , Raios Ultravioleta , Vírus da Estomatite Vesicular Indiana , Vírus/isolamento & purificaçãoAssuntos
Metilcolantreno/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Fenobarbital/farmacologia , Amidas/farmacologia , Animais , Centrifugação Zonal , Citocromos/biossíntese , Densitometria , Indução Enzimática , Etionina/farmacologia , Masculino , Microssomos Hepáticos/metabolismo , RatosRESUMO
New zonal centrifuges can conveniently process as much as five orders of magnitude (10(5)) greater sample volumes than conventional swinging-bucket rotors. The continuous-sample-flow-with-banding versions may be used in series with ancillary purification procedures. Here we have studied the combined process: absorption and elution of influenza virus with barium sulfate followed by concentration and isopycnic banding of the virus in a buffered sucrose gradient. Kilogram quantities of impurity have been rapidly separated from grams of purified virus, which have been conveniently concentrated several hundred-fold by the purification process. Experimental vaccines made by these procedures are being evaluated.