RESUMO
The production of recombinant human proteins in the milk of transgenic dairy animals offers a safe, renewable source of commercially important proteins that cannot be produced as efficiently in adequate quantities by other methods. A decade of success in expressing a variety of proteins in livestock has brought three human recombinant proteins to human clinical trials. Recent progress has drawn on molecular biology and reproductive physiology to improve the efficiency of producing and reproducing useful transgenic founder animals, and to improve the expression of heterologous proteins in their milk.
Assuntos
Animais Domésticos/genética , Animais Geneticamente Modificados/genética , Produtos Biológicos/biossíntese , Biotecnologia/métodos , Proteínas Recombinantes/biossíntese , Animais , Animais Domésticos/metabolismo , Animais Domésticos/fisiologia , Animais Geneticamente Modificados/metabolismo , Animais Geneticamente Modificados/fisiologia , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Leite , Técnicas de Transferência Nuclear , Técnicas Reprodutivas , Células-Tronco/citologia , Células-Tronco/metabolismo , Transgenes/genéticaRESUMO
Flow cytometric analysis of 5-bromodeoxyuridine (BrdU) incorporation during DNA synthesis was used to characterize the effects of X-rays on cell-cycle kinetics in the DNA-repair deficiency disease ataxia telangiectasia (AT). Cultured fibroblasts from homozygotes (at/at), heterozygotes (at/+) and normal controls (+/+) were either: (1) irradiated, cultured, then pulsed with BrdU and harvested, or (2) pulsed with BrdU, irradiated, cultured and then harvested. Cells were then fixed and stained with both a fluoresceinated monoclonal antibody against BrdU to identify S-phase cells and with propidium diiodide to measure total DNA content. Irradiation of +/+ and at/+ cells induced a similar, transient G2/M arrest detectable within 8 h, which subsequently delayed by 6-8 h the passage of cells into G1 and depleted early S phase. In contrast, at/at cells failed to arrest in G2/M phase and entered the next cell cycle without pausing to repair radiation-induced damage. X-Rays also blocked entry of +/+ G1 cells into S phase, subsequently reducing the total S-phase population. This effect was not observed in at/at cells. These cell-cycle responses to radiation may be of diagnostic use and ultimately may help explain the basic defect in AT.
Assuntos
Ataxia Telangiectasia/genética , Ciclo Celular/efeitos da radiação , Citometria de Fluxo , Adolescente , Adulto , Bromodesoxiuridina , Divisão Celular/efeitos da radiação , Criança , Pré-Escolar , DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Feminino , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Genótipo/efeitos da radiação , Humanos , Recém-Nascido , Interfase/efeitos da radiação , Masculino , Pessoa de Meia-Idade , Mitose/efeitos da radiaçãoRESUMO
Flow cytometry was used to identify heterozygotes for the autosomal recessive DNA-repair deficiency disease ataxia telangiectasia (AT). Confluent G0/G1 fibroblasts from 4 homozygotes (at/at), 5 obligate heterozygotes (at/+) and 7 presumed normal controls (+/+) were X-irradiated with 200 Rad and subcultured immediately in medium containing 5-bromodeoxyuridine (BrdU). Cells were harvested 72 h later and stained with fluoresceinated anti-BrdU antibody to identify cells that had entered S phase. They were counterstained with propidium iodide to measure total DNA content. On the basis of relative release from G0/G1, the at/+ strains as a group (33 +/- 3% release) were distinguished from both the presumed +/+ strains (60 +/- 3%) and at/at strains (85 +/- 3%), although the individual values for some strains did show overlap between genotypes. When 10 cell strains were coded and analyzed in 'blind' experiments, all 4 heterozygotes were correctly assigned, although one poorly growing presumed normal line was incorrectly assigned as a heterozygote. By a similar assay in which exponentially growing cultures were pulsed briefly with BrdU 8 h after irradiation with 400 Rad and then harvested immediately, presumed +/+ cells as a group could be distinguished from at/at cells but not from at/- cells. This combination of assays assists in the identification of all 3 AT genotypes. This should be of both basic and diagnostic use, particularly in families known to segregate AT.
Assuntos
Ataxia Telangiectasia/genética , Reparo do DNA/efeitos da radiação , Citometria de Fluxo , Triagem de Portadores Genéticos , Adolescente , Adulto , Ataxia Telangiectasia/patologia , Células Cultivadas , Criança , Pré-Escolar , Feminino , Fibroblastos/efeitos da radiação , Genótipo/efeitos da radiação , Humanos , Recém-Nascido , Interfase/efeitos da radiação , Masculino , Pessoa de Meia-IdadeRESUMO
Linkage disequilibrium between the T/t complex and Pgk-2 - two loci flanking the murine major histocompatibility complex on chromosome 17 - has been found in a population of wild mice. By subjecting 78 wild males to progeny testing, we extracted 15 recessive t haplotypes of the T/t complex and assigned 12 of them to the tw5 complementation group. We also determined the allelic phase of T/t, Pgk-2, and Ce-2 on both chromosomes from each wild male. The allele Pgk-2c was always associated with a t haplotype, although the converse was not true.
Assuntos
Ligação Genética , Camundongos/genética , Alelos , Animais , Animais Selvagens , Catalase/genética , Genes Letais , Haploidia , Complexo Principal de Histocompatibilidade , Fosfoglicerato Quinase/genéticaRESUMO
The activities of phosphoglycerate kinase B and lactate dehydrogenase C4 were correlated with the state of spermatogenesis in testes of sex-reversed (Sxr) mice. Both isoenzymes were present in Sxr/+,XO testes, which contained late spermatids. LDH-C4 was detected in testes from 5 of 9 Sxr/+,XX mice, but PGK-B was present in testes of only 1 of the 5 mice. Pachytene spermatocytes were the predominant cell types in those Sxr/+,XX testes that contained spermatogenic cells. These observations confirm that PGK-B is restricted to spermatogenic cells and is not present in somatic testicular cells. Furthermore, they suggest that PGK-B synthesis may be initiated later than pachytene, when synthesis of the LDH-C polypeptide is known to commence.
Assuntos
Isoenzimas/metabolismo , L-Lactato Desidrogenase/metabolismo , Fosfoglicerato Quinase/metabolismo , Espermatogênese , Animais , Masculino , Camundongos , Camundongos Endogâmicos , Espermatozoides/enzimologia , Testículo/patologiaRESUMO
K562 cells are human leukemia cells inducible for hemoglobin synthesis by a variety of agents. This report demonstrates that hypoxanthine, which alone has no inductive effect, enhances induction by thymidine, resulting in a greater absolute, as well as relative, percentage of benzidine positive cells. This effect is seen over a 20-fold concentration range for both thymidine and hypoxanthine. This enhancement involves commitment, i.e., a process in which the induction of hemoglobin synthesis is coupled to a limitation in the number of subsequent cell divisions. Although thymidine alone increases the percentage of cells in S phase, hypoxanthine does not augment this. Purines other than hypoxanthine also enhance induction by thymidine. This enhancement by hypoxanthine of thymidine induction is inhibited by pyrimidine nucleosides. Mycophenolic acid, an inhibitor of IMP dehydrogenase, itself an effective K562 inducer, is not additive to thymidine and hypoxanthine, suggesting that hypoxanthine may act by reducing the supply of guanosine nucleosides.
Assuntos
Hipoxantinas/farmacologia , Leucemia/metabolismo , Timidina/farmacologia , Adenina/farmacologia , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Sinergismo Farmacológico , Guanina/farmacologia , Hemoglobinas/biossíntese , Humanos , Hipoxantina , Ácido Micofenólico/farmacologia , Xantina , Xantinas/farmacologiaRESUMO
It is generally assumed that cellular and humoral immunity decline in aging humans. Although there have been reports that the naturally-occurring ABO antibodies also decline with age, some of the data are incomplete and others contradictory. Our study involved only healthy women of various ages and included assays of anti-A and anti-B titers in A, B, and O subjects. Statistical analyses of the data showed that while the antibody titers decreased with age, the amount of change was very small relative to the amount of variability among individuals and the variability attributable to blood type and season of the year. Comparisons with a previous study (1) from our laboratory showed that the changes due to aging were also small relative to the variability in titer scores within an individual over the course of a year. Based on this study, we conclude that the decrease in antibody titers with age is of doubtful clinical significance.
Assuntos
Sistema ABO de Grupos Sanguíneos/imunologia , Envelhecimento/imunologia , Isoanticorpos/sangue , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Análise de RegressãoRESUMO
Multiparameter single-cell analysis by flow cytometry was used to distinguish between size-related changes in K562 cell transferrin receptor (TfR) expression and changes in membrane receptor density throughout the cell cycle and over time in culture. Light-scatter pulse-width time-of-flight, a direct and readily calibrated measure of cell diameter, was used to calculate receptor density as the average number of receptors per unit cell surface area. Cell surface TfRs were unimodally distributed over the cell population and were present throughout the cell cycle. The number of receptors increased as cells progressed through the cell cycle, but cell cycle phase was also correlated with cell volume. However, when size heterogeneity was factored out by reanalysis of listmode data, there was a clear cell-cycle effect: among cells of the same size, both the number of receptors per cell and the receptor density increased from G1 to S to G2/M. TfR expression was also followed over time in culture after dilution into fresh medium. A decrease in growth rate after four days was preceded by one to two days by a decrease in both number of TfRs per cell and mean receptor density, indicating that decreased TfR expression represented true "down-regulation" and not just decreased cell size or an increase in the proportion of smaller G1 cells. This type of analysis is generally applicable for resolving the effects of cell size heterogeneity and cell cycle on membrane protein distribution and for other studies of ligand-receptor interaction.
Assuntos
Ciclo Celular , Divisão Celular , Receptores de Superfície Celular/metabolismo , Células Cultivadas , Citometria de Fluxo , Humanos , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/patologia , Receptores da Transferrina , Fatores de TempoRESUMO
Single-cell analysis of K562 human erythroleukemia cells by flow cytometry was used to demonstrate the specific role of iron in regulating transferrin receptors (TfRs) and to establish that TfR expression does not necessarily correlate with growth rate. Exogenous iron concentration in culture was manipulated by supplementing the medium with sera having different iron concentrations over the range 0.6 to 5.4 micrograms/ml, by the addition of iron in the form of FeCl3, iron-saturated serum, or diferric transferrin, and by the addition of the iron chelator Desferal (desferrioxamine). TfR expression was negatively correlated with exogenous iron content: any treatment that reduced exogenous iron supply by at least 15% resulted in as much as a 1.8-fold increase in external receptors, detected as binding by both transferrin and monoclonal anti-TfR antibodies, and a 1.5-fold increase in the pool of internal receptors, as detected by anti-TfR antibody binding. None of these treatments altered growth rate, total cellular protein content, protein synthetic rate, cell cycle distribution or cell size. The rapid (12 hr) and reversible induction of internal and external receptors by Desferal was inhibited by cycloheximide and therefore may have resulted from de novo synthesis and not just mobilization of internal receptor pool to the cell surface. The correlation between growth rate and TfR expression previously observed in these and other cells must be secondary to cellular mechanisms that maintain intracellular iron pools by regulating synthesis, recycling, and cell surface expression of TfRs.
Assuntos
Ferro/farmacologia , Leucemia Eritroblástica Aguda/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Sangue , Bovinos , Linhagem Celular , Humanos , Ferro/sangue , Leucemia Eritroblástica Aguda/patologia , Receptores da Transferrina , Transferrina/sangueRESUMO
K562 human erythroleukemia cells are an established cell line derived from an adult with chronic myelogenous leukemia. Hemin stimulates their synthesis of embryonic and fetal hemoglobins. We have found that their globin synthetic pattern depends on the concentration of added hemin. Clone RA6 was cultured with 0--100 microM hemin and the globin synthetic pattern determined by 3H-leucine incorporation and analysis of 3H-globins by polyacrylamide gel electrophoresis in Triton X acid urea followed by fluorography and densitometry. The higher the hemin concentration, the greater the synthetic rate of each type of globin. However, the relative increase was greatest for alpha-globin. We propose that the differential dependence of alpha synthesis on added hemin is a reflection of translational inefficiency of alpha messenger RNA and that this property is exposed when the translational capacity of the cell is limited by hemin deficiency. We suggest that the differential dependence of alpha-chain synthesis on added hemin in clone RA6 is evidence of an intrinsic deficiency in heme synthesis.