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1.
Radiat Res ; 165(6): 626-35, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16802862

RESUMO

In vitro experiments with C3H 10T(1/2) mouse cells were performed to determine whether Frequency Division Multiple Access (FDMA) or Code Division Multiple Access (CDMA) modulated radiofrequency (RF) radiations induce changes in gene expression. After the cells were exposed to either modulation for 24 h at a specific absorption rate (SAR) of 5 W/ kg, RNA was extracted from both exposed and sham-exposed cells for gene expression analysis. As a positive control, cells were exposed to 0.68 Gy of X rays and gene expression was evaluated 4 h after exposure. Gene expression was evaluated using the Affymetrix U74Av2 GeneChip to detect changes in mRNA levels. Each exposure condition was repeated three times. The GeneChip data were analyzed using a two-tailed t test, and the expected number of false positives was estimated from t tests on 20 permutations of the six sham RF-field-exposed samples. For the X-ray-treated samples, there were more than 90 probe sets with expression changes greater than 1.3-fold beyond the number of expected false positives. Approximately one-third of these genes had previously been reported in the literature as being responsive to radiation. In contrast, for both CDMA and FDMA radiation, the number of probe sets with an expression change greater than 1.3-fold was less than or equal to the expected number of false positives. Thus the 24-h exposures to FDMA or CDMA RF radiation at 5 W/kg had no statistically significant effect on gene expression.


Assuntos
Telefone Celular , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Expressão Gênica/fisiologia , Expressão Gênica/efeitos da radiação , Micro-Ondas , Proteoma/metabolismo , Animais , Linhagem Celular , Relação Dose-Resposta à Radiação , Perfilação da Expressão Gênica , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Doses de Radiação
2.
Genomics ; 77(3): 189-99, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11597144

RESUMO

The tilted (tlt) mouse carries a recessive mutation causing vestibular dysfunction. The defect in tlt homozygous mice is limited to the utricle and saccule of the inner ear, which completely lack otoconia. Genetic mapping of tlt placed it in a region orthologous with human 4p16.3-p15 that contains two loci, DFNA6 and DFNA14, responsible for autosomal dominant, nonsyndromic hereditary hearing impairment. To identify a possible relationship between tlt in mice and DFNA6 and DFNA14 in humans, we have refined the mouse genetic map, assembled a BAC contig spanning the tlt locus, and developed a comprehensive comparative map between mouse and human. We have determined the position of tlt relative to 17 mouse chromosome 5 genes with orthologous loci in the human 4p16.3-p15 region. This analysis identified an inversion between the mouse and human genomes that places tlt and DFNA6/14 in close proximity.


Assuntos
Surdez/genética , Membrana dos Otólitos/anormalidades , Mapeamento Físico do Cromossomo , Vestíbulo do Labirinto/fisiologia , Animais , Mapeamento Cromossômico , Cromossomos Humanos Par 4/genética , Mapeamento de Sequências Contíguas , Etiquetas de Sequências Expressas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Repetições de Microssatélites , Dados de Sequência Molecular , Mutação , Vestíbulo do Labirinto/anormalidades
3.
Shock ; 15(3): 165-70, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11236897

RESUMO

The traditional approach to the study of biology employs small-scale experimentation that results in the description of a molecular sequence of known function or relevance. In the era of the genome the reverse is true, as large-scale cloning and gene sequencing come first, followed by the use of computational methods to systematically determine gene function and regulation. The overarching goal of this new approach is to translate the knowledge learned from a systematic, global analysis of genomic data into a complete understanding of biology. For investigators who study shock, the specific goal is to increase understanding of the adaptive response to injury at the level of the entire genome. This review describes our initial experience using DNA microarrays to profile stress-induced changes in gene expression. We conclude that efforts to apply genomics to the study of injury are best coordinated by multi-disciplinary groups, because of the extensive expertise required.


Assuntos
Genômica/tendências , Pesquisa/tendências , Ferimentos e Lesões/fisiopatologia , Previsões , Técnicas Genéticas , Genoma Fúngico , Genômica/métodos , Humanos , Insuficiência de Múltiplos Órgãos/genética , Insuficiência de Múltiplos Órgãos/imunologia , Insuficiência de Múltiplos Órgãos/patologia , Projetos de Pesquisa , Saccharomyces cerevisiae/fisiologia , Baço/imunologia , Baço/lesões , Baço/fisiopatologia , Ferimentos e Lesões/genética
4.
Mol Microbiol ; 39(1): 26-36, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11123685

RESUMO

Plasmodium falciparum is a protozoan parasite responsible for the most severe forms of human malaria. All the clinical symptoms and pathological changes seen during human infection are caused by the asexual blood stages of Plasmodium. Within host red blood cells, the parasite undergoes enormous developmental changes during its maturation. In order to analyse the expression of genes during intraerythrocytic development, DNA microarrays were constructed and probed with stage-specific cDNA. Developmental upregulation of specific mRNAs was found to cluster into functional groups and revealed a co-ordinated programme of gene expression. Those involved in protein synthesis (ribosomal proteins, translation factors) peaked early in development, followed by those involved in metabolism, most dramatically glycolysis genes. Adhesion/invasion genes were turned on later in the maturation process. At the end of intraerythrocytic development (late schizogony), there was a general shut-off of gene expression, although a small set of genes, including a number of protein kinases, were turned on at this stage. Nearly all genes showed some regulation over the course of development. A handful of genes remained constant and should be useful for normalizing mRNA levels between stages. These data will facilitate functional analysis of the P. falciparum genome and will help to identify genes with a critical role in parasite progression and multiplication in the human host.


Assuntos
Eritrócitos/parasitologia , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Plasmodium falciparum/genética , Animais , Moléculas de Adesão Celular/genética , Análise por Conglomerados , Citoesqueleto/genética , Glicólise/genética , Humanos , Plasmodium falciparum/patogenicidade , Biossíntese de Proteínas/genética
5.
Curr Protoc Mol Biol ; Chapter 6: Unit6.10, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-18265255

RESUMO

This unit provides a series of protocols describing the analysis and manipulation of an isolated YAC clone. The procedures are based upon the use of the YAC vector pYAC4. Once an isolated YAC clone has been obtained from a core laboratory, the clone can be analyzed as described herein. Methods for analysis involve growing and storing YAC-containing yeast strains and purifying YAC DNA in a form suitable for assessing the size of the artificial chromosome and for conventional Southern blotting. Preparation of yeast chromosomes in agarose plugs for subsequent analysis by pulsed-field gel electrophoresis is also described. Additional protocols are provided for recovering DNA fragments from the ends of a YAC genomic insert to be used as probes for detecting chimerism and for chromosome walking. Finally, preparation of high-molecular-weight YAC DNA is described and a general method for subcloning YAC inserts into cosmid or lambda vectors for higher-resolution analysis is provided.


Assuntos
Passeio de Cromossomo , Cromossomos Artificiais de Levedura , DNA Fúngico/genética , Southern Blotting , Cromossomos Artificiais , Cromossomos Artificiais de Levedura/genética , Cromossomos Fúngicos , Clonagem Molecular , DNA Fúngico/isolamento & purificação , Eletroforese em Gel de Ágar , Biblioteca Genômica , Dados de Sequência Molecular , Mapeamento por Restrição
6.
Curr Protoc Mol Biol ; Chapter 6: Unit6.9, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-18265264

RESUMO

This unit provides an introduction to the use of yeast artificial chromosome-bearing yeast clones (hereafter referred to as YAC clones) in genome analysis. It describes criteria for designing a polymerase chain reaction (PCR) assay to be used in screening a YAC core library and discusses the rationale for verification and characterization of YAC clones obtained from these core laboratories. Protocols for maintaining YAC clones, analyzing YAC insert structure, preparing YAC DNA, and subcloning YAC inserts into other vectors are presented elsewhere in this volume.


Assuntos
Cromossomos Artificiais de Levedura/genética , Biblioteca Gênica , Genoma Humano , Reação em Cadeia da Polimerase/métodos , Clonagem Molecular , Primers do DNA , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Humanos , Peso Molecular
7.
Cytogenet Cell Genet ; 69(1-2): 101-7, 1995.
Artigo em Inglês | MEDLINE | ID: mdl-7835075

RESUMO

We screened two human yeast artificial chromosome (YAC) libraries by polymerase chain reaction (PCR) with oligonucleotides specific to the BCL1 major translocation breakpoint cluster region at 11q13. Five YACs were isolated. Two of them were chimeric. One of these and remaining three YACs were characterized by hybridization with various known 11q13 probes, Alu-PCR fingerprinting, in situ hybridization, and isolation of YAC ends. A map of this ca 700-kb YAC contig was obtained. This map was consistent with maps established from total human genomic DNA. Every YAC in this region was found unstable and gave rise to reproducibly deleted lineages. Analysis in detail of these deletions over many generations showed that more than a single sequence might be involved. The availability of cloned material will facilitate the search for the still elusive genetic elements responsible for amplifications, deletions and translocations observed at 11q13 in malignancies.


Assuntos
Cromossomos Artificiais de Levedura , Cromossomos Humanos Par 11 , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Translocação Genética , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Ciclina D1 , DNA/química , DNA/genética , Primers do DNA , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Peso Molecular , Família Multigênica , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Deleção de Sequência
8.
Biotechnology (N Y) ; 11(8): 911-4, 1993 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-7763914

RESUMO

We have previously described a strategy for integrating selectable marker genes into yeast artificial chromosomes (YACs) to facilitate their transfer into embryonic stem (ES) cells. Here we apply this technology to create mice carrying the core region of the human immunoglobulin (Ig) kappa light chain locus. A YAC was isolated which contains a 300 kb insert spanning three V kappa segments, the J kappa cluster, the C kappa region and extending downstream of the Kde element. After modification of this YAC to integrate the selectable neo marker gene, the YAC was introduced into ES cells by protoplast fusion. Several ES cell clones were obtained which appeared to harbor one complete copy of the YAC while retaining little or no other yeast DNA. The ES cells were injected into blastocysts and the chimaeric mice were shown to rearrange the introduced human light chain genes with the resultant production of antibodies containing human kappa light chains in the serum.


Assuntos
Cromossomos Fúngicos , Genoma Humano , Cadeias kappa de Imunoglobulina/genética , Animais , Sequência de Bases , Blastocisto , Quimera , Biblioteca Gênica , Rearranjo Gênico , Marcadores Genéticos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Protoplastos , Transfecção
9.
Hum Genet ; 89(5): 531-8, 1992 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-1353054

RESUMO

Friedreich ataxia (FA) is a severe autosomal recessive neurodegenerative disease. The defective gene has been previously assigned to chromosome 9q13-q21 by demonstration of tight linkage to the two independent loci D9S15 and D9S5. Linkage data indicate that FRDA is at less than 1 cM from both markers. Previous physical mapping has shown that probes defining D9S15 (MCT112) and D9S5 (26P) are less than 260 kb apart and are surrounded by at least six CpG clusters within 450 kb, which might indicate the presence of "candidate" genes for FA. We isolated and characterized a 530 kb YAC (yeast artificial chromosome) contig that contains five of the CpG clusters. The YACs were used to search for new polymorphic markers needed to map FRDA precisely with respect to the cloned segment. In particular, we found a (CA)n microsatellite polymorphism, GS4, that detects 13 alleles with a PIC value of 0.83 and allows the definition of haplotypes extending over 310 kb when used in combination with polymorphic markers at D9S5 and D9S15.


Assuntos
Cromossomos Fúngicos , Cromossomos Humanos Par 9 , DNA Satélite/genética , Ataxia de Friedreich/genética , Polimorfismo de Fragmento de Restrição , Sequência de Bases , Aberrações Cromossômicas , Clonagem Molecular , Cosmídeos , Nucleotídeos de Citosina/análise , DNA/análise , Eletroforese em Gel de Campo Pulsado , Nucleotídeos de Guanina/análise , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Linhagem , Reação em Cadeia da Polimerase , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição
10.
Genomics ; 13(3): 672-80, 1992 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-1639394

RESUMO

The yeast artificial chromosome (YAC) system (Burke et al., 1987, Science 236: 806-812) allows the direct cloning of large regions of the genome. A YAC contig map of approximately 700 kb encompassing the region surrounding the type 1 neurofibromatosis (NF1) locus on 17q11.2 has been constructed. A single YAC containing the entire NF1 locus has been constructed by homologous recombination in yeast. In the process of contig construction a novel method of YAC end rescue has been developed by YAC circularization in yeast and plasmid rescue in bacteria. YACs containing homology to the NF1 region but mapping to another chromosome have also been discovered. Sequences of portions of the homologous locus indicate that this other locus is a nonprocessed pseudogene.


Assuntos
Genes da Neurofibromatose 1 , Sequência de Bases , Cromossomos Fúngicos , Clonagem Molecular , DNA/genética , Biblioteca Gênica , Genoma Humano , Humanos , Dados de Sequência Molecular , Plasmídeos , Pseudogenes , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico
11.
Proc Natl Acad Sci U S A ; 89(12): 5457-61, 1992 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-1608955

RESUMO

The genetic defects in Prader-Willi syndrome (PWS) and Angelman syndrome (AS) map to 15q11-13. Using microdissection, we have recently isolated several DNA probes for the critical region. Here we report that microclone MN7 detects multiple loci in 15q11-13 and 16p11.2. Eight yeast artificial chromosome (YAC) clones, two genomic phage clones, and two placenta cDNA clones were isolated to analyze these loci in detail. Two of the YAC clones map to 16p. Six YAC clones and two genomic phage clones contain a total of four or five different MN7 copies, which are spread over a large distance within 15q11-13. One cDNA clone is from chromosome 15 and one is from chromosome 16. The chromosome 15 cDNA detects transcripts of 14 and 8 kilobases in various human tissues. The presence of multiple copies of the MN7 gene family in proximal 15q may conceivably be related to the instability of this region and thus to the etiology of associated disorders.


Assuntos
Cromossomos Humanos Par 15 , Cromossomos Humanos Par 16 , Deficiência Intelectual/genética , Família Multigênica , Síndrome de Prader-Willi/genética , Sequência de Bases , Southern Blotting , Bandeamento Cromossômico , Mapeamento Cromossômico , Clonagem Molecular , DNA/genética , DNA/isolamento & purificação , Sondas de DNA , Humanos , Riso , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Síndrome
12.
Genomics ; 12(1): 7-12, 1992 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-1733866

RESUMO

The region surrounding the steroid sulfatase (STS) locus on Xp22.3 is of particular interest since it represents a deletion hot spot, shares homology with the proximal long arm of the Y chromosome (Yq11.2), and contains genes for several well-described X-linked disorders. Here we describe yeast artificial chromosomes (YACs) covering 450 kb around the STS gene. Eight YAC clones were isolated from a human YAC library. Their STS exon content was determined and the overlap of the clones characterized. Two of the YAC clones were found to contain the entire STS gene. The most proximal and the most distal ends of the YAC contig were cloned but neither of them crossed the breakpoints in any of the previously described patients with entire STS gene deletions. This is consistent with deletions larger than 500 kb in all these patients. One of the YAC clones was found to contain sequences from the STS pseudogene on Yq11.2. Two anonymous DNA sequences, GMGXY19 and GMGXY3, previously mapped in the vicinity of the STS locus, were found within the YAC contig and their assignment with respect to the STS locus was thus possible. This contig is useful for the overlap cloning of the Xp22.3 region and for reverse genetic strategies for the isolation of disease genes in the region. Furthermore, it may provide insight into the molecular mechanisms of deletion and translocation events on Xp22.3 and in the evolution of sex chromosomes.


Assuntos
Arilsulfatases/genética , Cromossomo X , Arilsulfatases/metabolismo , Sequência de Bases , Southern Blotting , Mapeamento Cromossômico , Cromossomos Fúngicos , Clonagem Molecular , DNA , Genoma Humano , Humanos , Dados de Sequência Molecular , Esteril-Sulfatase
13.
Cytogenet Cell Genet ; 61(4): 263-5, 1992.
Artigo em Inglês | MEDLINE | ID: mdl-1486800

RESUMO

To understand better the organization and linkage of the interleukin genes, IL4 and IL5, we prepared long-range restriction maps of five yeast artificial chromosomes (YACs) containing IL5. We determined that IL4 and IL5 are within 100-170 kb, and that the regions surrounding these genes contain several GC-rich areas. Fluorescence in situ chromosomal analysis demonstrated that three of the five YAC clones contain non-contiguous genomic sequences originating from multiple human chromosomes.


Assuntos
Cromossomos Humanos Par 5 , Biblioteca Genômica , Interleucina-4/genética , Interleucina-5/genética , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Fúngicos , DNA de Cadeia Simples , Eletroforese em Gel de Campo Pulsado , Ligação Genética , Genoma Humano , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Mapeamento por Restrição
14.
Genomics ; 10(4): 976-84, 1991 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-1916829

RESUMO

The recently developed technique for cloning genomic DNA fragments of several hundred kilobases or more into yeast artificial chromosomes (YACs) makes it possible to isolate gene families while preserving their structural integrity. We have analyzed five independent yeast clones identified by PCR screening using oligonucleotides derived from the adult human beta-globin gene. Analysis of the five clones containing YACs by conventional and pulsed-field gel electrophoresis revealed that all of the clones include a YAC with sequences from the adult beta-globin gene as expected. One of the clones contains multiple, unstable YACs. Two other clones carry single YACs in which there are at least two unrelated human genomic inserts. The remaining two clones contain single YACs, 150 and 220 kb in size, that contain the entire beta-globin gene family and flanking regions in a single, structurally intact genomic fragment. These should prove useful in future studies of the regulation of expression of genes in the beta-globin gene cluster.


Assuntos
Cromossomos Fúngicos , Clonagem Molecular/métodos , Genes , Globinas/genética , Saccharomyces cerevisiae/genética , Linhagem Celular , Mapeamento Cromossômico , DNA/genética , DNA/isolamento & purificação , Impressões Digitais de DNA , Sondas de DNA , Humanos , Mapeamento por Restrição , Talassemia/genética
15.
Genomics ; 10(3): 756-64, 1991 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-1832411

RESUMO

The leukocyte-common antigen (CD45) is a transmembrane protein tyrosine phosphatase expressed uniquely by cells of hematopoietic origin. There are multiple isoforms of CD45 that are generated by the variable use of three exons (exons 4-6). The use of the variable exons results in changes near the amino-terminus of the mature glycoprotein. The gene is located on chromosome 1 for both human and mouse in a region that is homologous between these two species. This conserved linkage group contains a number of genes of immunological interest, such as the genes for complement regulatory proteins and the FCG2 receptor. Yeast artificial chromosomes provide a vector system in which large fragments of foreign DNA can be isolated and are suited to long-range physical mapping. To this end, three yeast artificial chromosomes containing the human CD45 gene have been isolated and characterized. They overlap to span 475 kb, establishing the largest physical map for DNA within the conserved linkage group. The CD45 gene is entirely encoded within one yeast artificial chromosome clone as determined by mapping with cDNA probes. A mouse B cell line transfected with this YAC clone expressed the low-molecular-weight isoform of the protein into the cell surface. The size of the human CD45 gene was determined to be approximately 120 +/- 10 kb.


Assuntos
Antígenos CD/biossíntese , Antígenos de Histocompatibilidade/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Animais , Antígenos CD/genética , Linfócitos B/metabolismo , Cromossomos Fúngicos , Éxons , Expressão Gênica , Vetores Genéticos , Antígenos de Histocompatibilidade/genética , Humanos , Antígenos Comuns de Leucócito , Camundongos , Reação em Cadeia da Polimerase , Splicing de RNA , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae , Transcrição Gênica , Transfecção
16.
Genomics ; 10(3): 661-5, 1991 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-1889812

RESUMO

A new method for screening of YAC libraries is described. Individual YACs were pooled into groups of 384 clones and prepared as samples suitable for pulsed-field gel electrophoresis. A five hit human YAC library (Brownstein et al., 1989) containing approximately 60,000 clones was condensed into 150 such pools and chromosomal DNAs in each sample were separated on three pulsed field gels containing 50 samples each. Southern blots prepared from these gels were hybridized with probes of interest to identify pools containing homologous YACs. Further purification was performed using standard colony hybridization procedures. Twenty-one probes used thus far have identified 47 positive pools and corresponding YACs have been purified from 28 of these. Some significant advantages of this method include avoidance of DNA sequence analysis and primer generation prior to YAC screening and the ability to handle the entire library on three filters. The screening approach described here permits rapid isolation of YACs corresponding to unsequenced loci and will accelerate establishment of YAC contigs for large chromosomal segments.


Assuntos
Southern Blotting/métodos , Cromossomos Fúngicos , DNA Recombinante/genética , Vetores Genéticos , Genoma Humano , Biblioteca Genômica , Saccharomyces cerevisiae/genética , Sondas de DNA , Eletroforese em Gel de Ágar/métodos , Humanos
17.
Science ; 250(4983): 994-7, 1990 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-2173146

RESUMO

Wilms tumor is an embryonal kidney tumor involving complex pathology and genetics. The Wilms tumor locus on chromosome 11p13 is defined by the region of overlap of constitutional and tumor-associated deletions. Chromosome walking and yeast artificial chromosome (YAC) cloning were used to clone and map 850 kilobases of DNA. Nine CpG islands, constituting a "CpG island archipelago," were identified, including three islands that were not apparent by conventional pulsed-field mapping, and thus were at least partially methylated. Three distinct transcriptional units were found closely associated with a CpG island within the boundaries of a homozygous DNA deletion in a Wilms tumor.


Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 11 , Fosfatos de Dinucleosídeos , Genes do Tumor de Wilms/genética , Tumor de Wilms/genética , Passeio de Cromossomo , Sondas de DNA , Humanos , Transcrição Gênica
18.
Cancer Res ; 50(13): 4111-20, 1990 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-2354460

RESUMO

alpha-Interferon (IFN-alpha) induced unique ultrastructural alterations in peripheral blood and splenic hairy cell leukemia (HCL) cells (14 of 20 cases) treated in vitro. To further investigate the effects of B-cell growth factor (BCGF) and IFN-alpha on target hairy cells (HCs), we utilized immunogold labeling in conjunction with scanning electron microscopy. This methodology, in contrast to other immunological methods, facilitated direct view of the expression, density, and rearrangement of selected antigens/receptors on individual cells before and after BCGF or IFN-alpha treatment. In addition to inducing proliferation of HCL cells, BCGF enhanced the expression of interleukin 2 receptors (CD25; T-activated cell antigen) with no change in the expression of class I and class II human leukocyte antigen. On the other hand, IFN-alpha did not exert a noticeable proliferative effect on HCL cells but rather inhibited the proliferation of BCGF-treated cells. In addition, IFN-alpha treatment revealed an enhanced expression of class I (4 of 9) and class II (12 of 15) human leukocyte antigen on target HCs. Two-day exposure of HCs to IFN-alpha resulted in enhanced expression of CD25 (11 of 14), whereas a decrease in CD25 expression was recorded in 4 of 5 cases treated with IFN-alpha for 3 days. Also, no significant change in the expression of two other HCL-related surface antigens, CD22 (S-HCL-1; Leu-14) and CD11c(S-HCL-3; Leu-M5), was recorded following up to 3 days of IFN-alpha or BCGF treatment. However, a 5-day exposure to IFN-alpha resulted in a significant decrease in expression of CD11c on treated HCs. Finally, the IFN-alpha-induced immunoultrastructural changes in target HCs were primarily encountered in cells from HCL cases classified as responders to in vivo IFN-alpha therapy. Our data add support to the concept that the effect of IFN-alpha in HCL is mediated by impairment of the response to B-cell growth factors and induction of further differentiation of the target cells.


Assuntos
Antígenos de Neoplasias/metabolismo , Antígenos HLA/metabolismo , Interferon Tipo I/farmacologia , Interleucina-4/farmacologia , Leucemia de Células Pilosas/patologia , Receptores de Interleucina-2/metabolismo , Idoso , Antígenos de Superfície/metabolismo , Divisão Celular/efeitos dos fármacos , Humanos , Interferon Tipo I/uso terapêutico , Interleucina-4/antagonistas & inibidores , Leucemia de Células Pilosas/imunologia , Leucemia de Células Pilosas/metabolismo , Leucemia de Células Pilosas/terapia , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade
19.
Science ; 248(4956): 732-5, 1990 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-2139735

RESUMO

Receptors for immunoglobulin G immune complexes (Fc gamma RII and Fc gamma RIII) are expressed on most hematopoietic cells and show much structural and functional diversity. In order to determine the genetic basis for this diversity, a family of genes encoding the human and mouse receptors was isolated and characterized. Humans have five distinct genes for low-affinity Fc gamma Rs, in contrast to two in the mouse. With the use of yeast artificial chromosomes, the genes encoding the human receptors were oriented and linked, which established the structure of this complex locus. Comparison of the human and mouse genes generated a model for the evolutionary amplification of this locus.


Assuntos
Antígenos de Diferenciação/genética , Família Multigênica , Receptores Fc/genética , Animais , Antígenos de Diferenciação/metabolismo , Sequência de Bases , Southern Blotting , Éxons , Genoma Humano , Humanos , Imunoglobulina G/metabolismo , Íntrons , Camundongos , Dados de Sequência Molecular , Mutação , Receptores Fc/metabolismo , Receptores de IgG , Recombinação Genética , Mapeamento por Restrição , Baço/imunologia
20.
Leuk Res ; 14(3): 263-71, 1990.
Artigo em Inglês | MEDLINE | ID: mdl-2319807

RESUMO

The nature of the interleukin 2 (IL-2) receptor on purified human B lymphocytes was examined. Both normal and malignant cells showed evidence of a 70-75,000 mol. wt (p75) IL-2 binding molecule as assessed by 125I-labeled IL-2 binding and receptor cross-linking. On normal, Tac-negative B lymphocytes the estimated number of p75 binding sites was 1100 per cell and the dissociation constant (Kd) was 1.7 nM. Consistent with this, cross-linking experiments demonstrated the presence of an IL-2 binding molecule of 70-75,000 mol. wt. Purified B cells from patients with hairy cell leukemia and chronic lymphocytic leukemia (CLL) also expressed the p75 IL-2 binding molecule. In the HCL samples, a small number of high-affinity IL-2 binding sites were detected (27-90) while the majority of binding sites (2100-10,800) were typical of low-affinity p55 Tac binding. IL-2 added to the purified normal and CLL B lymphocytes led to the induction of p55 Tac expression and the generation of high-affinity IL-2 receptors. This response to IL-2 was equivalent to the response observed when normal B lymphocytes were stimulated by Staphylococcus aureus Cowan I.


Assuntos
Linfócitos B/imunologia , Linfoma de Burkitt/imunologia , Interleucina-2/metabolismo , Receptores de Interleucina-2/metabolismo , Antígenos CD/análise , Linhagem Celular , Reagentes de Ligações Cruzadas/farmacologia , Humanos , Cinética , Peso Molecular , Receptores de Interleucina-2/isolamento & purificação , Succinimidas/farmacologia
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