RESUMO
The fibroblast growth factor receptor 1 (flg) contains eight acidic amino acids between the first and second immunoglobulin domain. This report examines the role of the acidic domain in the interaction of the flg receptor with its ligands. We observed a marked inhibition of binding of bFGF to the receptor when the acidic domain was completely deleted, but mutants with two and four amino acids deleted (flg delta A2 and flg delta A4, respectively) still bound the ligand. After addition of a bifunctional cross-linking reagent, cross-linked complexes (between bFGF and receptor) with the expected size were observed in cells expressing mutants lacking two or four acidic residues, but not in cells expressing mutants lacking six or eight acidic residues. Immunoprecipitation with anti-flg antibody followed by electrophoresis produced a band of 90 Kd in tunicamycin-treated cells expressing the mutant as well as the wild-type receptors, indicating that the inhibition of binding was not due to defective expression of the protein. The ability of flg delta A8 to mediate a mitogenic response to FGFs was also greatly reduced when this mutated receptor was expressed in receptor-negative cells. The effect of replacing the acidic amino acids with lysine residues was also studied. Binding of bFGF to cells transfected with a plasmid encoding a mutated protein with four amino acid substitutions was totally inhibited, but an eight amino acid substitution did not alter ligand binding to the receptor. In this case the mutation with four amino acids substitution caused a drastic impairment of protein expression. Thus the acidic domain of the FGFR-1 plays an essential role in receptor function, either because it is important for a stable protein configuration or for ligand-receptor interaction.
Assuntos
Aminoácidos/análise , Aminoácidos/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Receptores Proteína Tirosina Quinases , Receptores de Fatores de Crescimento de Fibroblastos/química , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Sequência de Aminoácidos , Aminoácidos/fisiologia , Animais , Células CHO , Cricetinae , DNA/genética , Eletroforese em Gel de Poliacrilamida , Ligantes , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Testes de Precipitina , Ligação Proteica , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/genética , TransfecçãoRESUMO
Ribonucleotide reductase activity is markedly elevated in cell lines selected for resistance to hydroxyurea, a cytotoxic drug known specifically to inhibit ribonucleotide reductase. From a cDNA library constructed from a highly hydroxyurea-resistant hamster lung cell line, 600H in which the activity is elevated more than 80-fold, we have isolated a full length cDNA for the small subunit of the reductase. The cDNA is 3.48 kb long with an open reading frame of 1158 nucleotides and a long 3' flanking region of 2169 nucleotides from the termination codon. The derived polypeptide sequence is closely similar to the small subunit of the mouse, differing from it in 20 amino acid positions. Most of these replacements occur in the N-terminal segment of the protein. The hamster subunit does not contain 4 amino acid residues found in the mouse small subunit near the C-terminal end. RNA blots probed with the cDNA show two poly(A)+ RNA species which are elevated in hydroxyurea-resistant cells.
Assuntos
DNA , Ribonucleotídeo Redutases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Cricetinae , Dados de Sequência Molecular , RNA/metabolismo , Regiões Terminadoras GenéticasRESUMO
Three cases of non-parasitic liver cysts are reported. Each of them presented with upper abdominal pain and enlarged liver, and a cyst of enormous size was detected by ultrasound and isotopic scan. Residual cavity following cyst surgery was obliterated by different surgical techniques.
Assuntos
Cistos/congênito , Hepatopatias/congênito , Dor Abdominal/etiologia , Adulto , Feminino , Hepatomegalia/etiologia , Humanos , Pessoa de Meia-IdadeRESUMO
Cell lines selected in multiple steps for increasing resistance to hydroxyurea have been shown to have corresponding increases in ribonucleotide reductase activity. We have isolated a number of cDNA clones from a cDNA library constructed from a highly hydroxyurea-resistant hamster cell line, 600H, in which the activity of ribonucleotide reductase is elevated more than 80-fold. These clones correspond to genomic DNA sequences amplified in the 600H cell line compared with the V79 parental line. One of these cDNA clones, termed P5, codes for a 50 kDa protein detected by in vitro translation of poly(A)+ RNA isolated by hybridization/selection. The cDNA sequence contains a single open reading frame of 1317 nucleotides which encodes a polypeptide of 439 amino acids. The amino acid sequence deduced from the cDNA insert contains two copies of the 11-amino-acid sequence Val-Glu-Phe-Tyr-Ala-Pro-Trp-Cys-Gly-His-Cys. Duplicate copies of this sequence also occur in the active site of rat and human protein disulphide isomerase (also known as the beta-subunit of human prolyl 4-hydroxylase, tri-iodothyronine-binding protein) and in Form I phosphoinositide-specific phospholipase C, indicating that P5 falls into this newly defined superfamily of proteins. Genomic sequences similar to the cDNA clone are amplified 10-20-fold in hamster cells selected for resistance to increasing concentrations of hydroxyurea, a phenomenon observed earlier with cDNA clones for the M2 subunit of ribonucleotide reductase and ornithine decarboxylase. RNA blots probed with P5 cDNA show two poly(A)+ RNA species which are elevated in hydroxyurea-resistant cells.
Assuntos
Amplificação de Genes , Hidroxiureia/farmacologia , Isomerases/genética , Diester Fosfórico Hidrolases/genética , Proteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Cricetinae , DNA/química , DNA/metabolismo , Desoxirribonuclease EcoRI , Resistência a Medicamentos , Isomerases/química , Dados de Sequência Molecular , Peso Molecular , Ornitina Descarboxilase/química , Ornitina Descarboxilase/genética , Ornitina Descarboxilase/metabolismo , Fosfatidilinositol Diacilglicerol-Liase , Diester Fosfórico Hidrolases/química , Biossíntese de Proteínas , Isomerases de Dissulfetos de Proteínas , Proteínas/química , Proteínas/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
An abscess forming in a giant cavernous hemangioma of the left lobe of the liver is reported. It was treated by lateral segmentectomy.
Assuntos
Hemangioma Cavernoso/patologia , Neoplasias Hepáticas/patologia , Adulto , Feminino , Hemangioma Cavernoso/complicações , Humanos , Abscesso Hepático/complicações , Abscesso Hepático/patologia , Neoplasias Hepáticas/complicaçõesRESUMO
Sarcoma-180 ascites tumour-bearing male mice were injected i.p. with single dose of 0.5, 2.5, 5 mg/kg body wt. of rifampicin. Cells were sampled for mitotic chromosome analysis 4, 16 or 24 h after treatment. The maximal yield of chromatid-type aberrations induced was found 24 h after treatment with 5 mg/kg of rifampicin. More than 60% of the cells carried at least one chromatid exchange. The majority of these were exchanges derived from breaks in the centromeric heterochromatin.