RESUMEN
A new method for an assay of human granulocyte colony-stimulating factor (hG-CSF) has been developed using human promyelocytic HL-60 cells. The proliferation of HL-60 cells had been suppressed by the addition of dimethyl sulfoxide (DMSO) or retinoic acid (RA). These differentiating agent-treated HL-60 cells exhibited an increase in their number in response to recombinant hG-CSF (rhG-CSF). Neither dibutyl-cAMP nor interferon-gamma (IFN-gamma)-treated HL-60 cells, however, showed an increase in their number in response to rhG-CSF. The proliferation rate of DMSO-pretreated HL-60 cells was linearly increased from 0.3 to 10 ng/ml of rhG-CSF. L-Value of HL-60 cells assay was 0.027 +/- 0.012. The activities of non-glycosylated rhG-CSF produced by Escherichia coli and glycosylated rhG-CSF produced by chinese hamster ovary (CHO) cells were compared using DMSO-treated HL-60 cells; no significant difference between them. DMSO-treated HL-60 cells also responded to interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF), but did not respond to erythropoietin or macrophage colony-stimulating factor, suggesting that the responsiveness of these cells to growth factor is restricted to myelogenic cytokines. In conclusion, DMSO- or RA-treated HL-60 cells are useful for the measurement of bioactivity of hG-CSF.
Asunto(s)
Dimetilsulfóxido/farmacología , Factor Estimulante de Colonias de Granulocitos/análisis , Leucemia Promielocítica Aguda/metabolismo , Neutrófilos/efectos de los fármacos , Tretinoina/farmacología , Animales , Bioensayo , Células CHO , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Cricetinae , Citocinas/farmacología , Glicosilación , Células HL-60 , Humanos , Proteínas RecombinantesRESUMEN
In an effort to analyze the genomic region of the distal half of human chromosome 4p, to where Huntington disease and other diseases have been mapped, we have isolated the cosmid clone (CRS447) that was likely to contain a region with specific repeat sequences. Clone CRS447 was subjected to detailed analysis, including chromosome mapping, restriction mapping, and DNA sequencing. Chromosome mapping by both a human-CHO hybrid cell panel and FISH revealed that CRS447 was predominantly located in the 4p15.1-15.3 region. CRS447 was shown to consist of tandem repeats of 4.7-kb units present on chromosome 4p. A single EcoRI unit was subcloned (pRS447), and the complete sequence was determined as 4752 nucleotides. When pRS447 was used as a probe, the number of copies of this repeat per haploid genome was estimated to be 50-70. Sequence analysis revealed that it contained two internal CA repeats and one putative ORF. Database search established that this sequence was unreported. However, two homologous STS markers were found in the database. We concluded that CRS447/pRS447 is a novel tandem repeat sequence that is mainly specific to human chromosome 4p.
Asunto(s)
Cromosomas Humanos Par 4/genética , Secuencias Repetitivas de Ácidos Nucleicos , Animales , Secuencia de Bases , Células CHO , Mapeo Cromosómico , Clonación Molecular , Cósmidos , Cricetinae , Cartilla de ADN/genética , Repeticiones de Dinucleótido , Marcadores Genéticos , Humanos , Enfermedad de Huntington/genética , Células Híbridas , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Plásmidos , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Lugares Marcados de SecuenciaRESUMEN
To investigate the role of phosphatase in O2- generation, the effects of the potent phosphoprotein phosphatase inhibitors, Calyculin A and FK506, were analyzed during phagocytosis using rat peritoneal macrophages. O2- generation was continuously measured after addition of opsonized zymosan (op. ZY) or IgG-coated zymosan (IgG-ZY). The rate of O2- generation was directly proportional to the number of macrophages, up to 1-2 x 10(6) cells/ml. It was found that the rate and duration of O2- generation were markedly inhibited by Calyculin A. The addition of 100 nM of Calyculin A reduced O2- generation to about one-tenth of the control value. In contrast, FK506 did not inhibit O2- generation, suggesting that calcium calmodulin phosphatase is not involved in the activation of NADPH oxidase. This result indicates that the process of dephosphorylation might involve activation of NADPH oxidase as a control mechanism in phagocytosis by rat peritoneal macrophages. Furthermore, since Calyculin A is an inhibitor of phosphatase 1 and 2A, it is suggested that dephosphorylation may be evoked by these phosphatases.
Asunto(s)
Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Oxazoles/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Superóxidos/metabolismo , Tacrolimus/farmacología , Animales , Macrófagos Peritoneales/enzimología , Toxinas Marinas , Proteína Fosfatasa 1 , RatasRESUMEN
The genes that cause a variety of neurologic and neuromuscular disorders have been mapped to the distal region of Xq. In an effort to isolate genes from this area, a regional genomic library of the distal 30% of Xq was constructed from a single metaphase spread by means of laser microdissection and single unique primer-polymerase chain reaction. Using pooled probes of 1000 clones from the genomic library, human brain cDNA libraries were screened for expressed sequences encoded by this region. From the 250,000 cDNA clones screened so far, 10 nonoverlapping sequences that mapped back to the target portion were isolated. The complete nucleotide sequences of these cDNA clones have been determined. Analysis of the sequences indicates that none has significant similarity to previously characterized primate genes. One sequence mapping to Xq27.3-qter contained an open reading frame of 281 amino acids and was expressed in every tissue tested. This gene, as well as others isolated in this manner, may prove to be a candidate gene for heritable disorders mapping to this region.
Asunto(s)
Hominidae/genética , Cromosoma X , Secuencia de Aminoácidos , Aneuploidia , Animales , Secuencia de Bases , Encéfalo/metabolismo , Bandeo Cromosómico , Mapeo Cromosómico , Clonación Molecular , Cartilla de ADN , Femenino , Feto , Humanos , Cariotipificación , Masculino , Datos de Secuencia Molecular , Enfermedades del Sistema Nervioso/genética , Enfermedades Neuromusculares/genética , Sondas de Oligonucleótidos , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa/métodosRESUMEN
We have developed an argon laser chromosome microdissection technique in conjunction with a polymerase chain reaction (PCR) approach to directly amplify microdissected chromosomes. The single 22-mer primer used in PCR, although unique in sequence (5'-TAGATCTGA-TATCTGAATTCCC-3'), randomly primed and amplified any target DNA. These methods were applied to the distal half of the short arm of human chromosome 4 containing the Huntington disease (HD) locus. Forty-four percent of representative clones from this library identify single-copy DNA sequences. This calculation suggests that the resulting chromosome-specific DNA library contains approximately 600 nonoverlapping sequences with an average size 350 bp at an average spacing of 30 kbp along chromosome 4. This microdissection and PCR cloning procedure is a simple and general approach for constructing a chromosome region-specific DNA library from a single metaphase spread.