RESUMO
Mouse liver DNA was cut out with BamHI and cloned into YIp5, which contained the URA3 gene of Saccharomyces cerevisiae in pBR322. Of the several plasmids isolated, two plasmids, pMU65 and pMU111, could transform S. cerevisiae from the URA- to the URA+ phenotype and could replicate autonomously within the transformant, indicating that mouse DNA fragments present in pMU65 or pMU111 contain autonomously replicating sequences (ARS) for replication in S. cerevisiae. Furthermore, to determine the correlation between ARS function in yeast cells and that in much higher organisms, we tried to challenge these plasmids with the simian virus 40 (SV40) DNA replication system. Of the two plasmids tested, the EcoRI-BglII region of pMU65 could be hybridized with a chemically synthesized 13-nucleotide fragment corresponding to the origin region of SV40 DNA. Both pMU65 (the EcoRI-BglII region cloned in pBR322) and its subclone pMU65EB could replicate semiconservatively, and initiation of DNA replication started from the EcoRI-BglII region when the replicating activity of these plasmids was tested in the in vitro SV40 DNA replication system we have established before. Furthermore, pMU65 and pMU65EB could replicate autonomously within monkey Cos cells which produce SV40 T antigen constitutively. These results show that a 2.5-kilobase fragment of the EcoRI-BglII region in pMU65 contains the ARS needed for replication in the SV40 DNA replication system.
Assuntos
Antígenos Virais de Tumores/fisiologia , Replicação do DNA , DNA Recombinante/metabolismo , Vírus 40 dos Símios/fisiologia , Proteínas Virais/fisiologia , Animais , Antígenos Transformantes de Poliomavirus , Linhagem Celular , DNA Viral/biossíntese , Vetores Genéticos , Haplorrinos , Humanos , Camundongos , Camundongos Endogâmicos C3H/genética , Replicon , Saccharomyces cerevisiae/genéticaRESUMO
Circulating interleukin-6 (IL-6) levels are directly correlated to fatal outcome in both patients and animal models with endotoxin shock. However, whether IL-6 is deleterious or protective in regard to survival is obscure. We investigated the action of IL-6 in the pathogenic progress of endotoxin shock. C3H/HeN mice received 10 micrograms of natural human IL-6 (Hu-IL-6) s.c. at various times before or after challenge with Escherichia coli lipopolysaccharide (LPS) at a lethal dose. Pretreatment with Hu-IL-6 0, 1, and 4 h before LPS administration improved the survival rate of the mice. However, no protection was observed when Hu-IL-6 was administered 24 h before or 1 h after the LPS injection. The protective mechanism of Hu-IL-6 pretreatment was not explained on the changes in the circulating levels of tumor necrosis factor and endogenous murine IL-6 (Mu-IL-6). The induction of fibrinogen and immunosuppressive acidic protein, a type of acute-phase proteins, may have contributed to the protection. The results show that the order and the time interval in which the administered Hu-IL-6 and the Mu-IL-6 induced by LPS act are the key to the determination of fatal outcome.
Assuntos
Interleucina-6/uso terapêutico , Choque Séptico/tratamento farmacológico , Proteínas de Fase Aguda/biossíntese , Animais , Esquema de Medicação , Avaliação Pré-Clínica de Medicamentos , Humanos , Injeções Subcutâneas , Interleucina-6/efeitos adversos , Interleucina-6/sangue , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Choque Séptico/sangue , Choque Séptico/induzido quimicamente , Especificidade da Espécie , Taxa de Sobrevida , Resultado do TratamentoRESUMO
The growth of inoculated colon 26 adenocarcinoma (colon 26) in mice gradually increased the platelet count owing to murine IL-6 secreted from the tumor, while Lewis lung carcinoma (LLC) decreased the platelet count in the hosts, depending on the tumor growth. Natural human IL-6 injections (hIL-6), 280 micrograms/kg/day, stimulated the platelet production in both types of carcinoma-bearing mice. When the administration of mitomycin C or cisplatin decreased the platelet number as a side reaction with a concomitant of suppressing the growth of colon 26 and LLC, respectively, hIL-6 could also increase the platelet count without the augmentation of tumor growth. However, loss of carcass weight was observed in colon 26-bearing mice treated with hIL-6, suggesting the development of cachexia is associated with hIL-6 administration. Despite the possibility of inducing cachexia in some types of tumors, our results suggest that IL-6 could be a useful means of restoring the decreased platelet number in cancer patients after intensive chemotherapy.
Assuntos
Adenocarcinoma/patologia , Neoplasias do Colo/patologia , Hematopoese/efeitos dos fármacos , Interleucina-6/farmacologia , Contagem de Plaquetas/efeitos dos fármacos , Adenocarcinoma/sangue , Animais , Divisão Celular/efeitos dos fármacos , Neoplasias do Colo/sangue , Humanos , Interleucina-6/biossíntese , Interleucina-6/isolamento & purificação , Cinética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fatores de TempoRESUMO
Of the several DNA fragments present in the human lung cancer gene, 1.1 and 2.0 kilobase (kb) fragments corresponding to the intron of this gene were hybridized to a half part of the 27 nucleotides perfect palindrome present in the initiation part of replication in simian virus 40 (SV40) DNA. These two fragments cloned in pBR322 had good template activity, and the initiation of DNA replication started from the region of these fragments in an in vitro system, in which the initiation of DNA replication occurs on cloned DNA containing SV40 origin of DNA replication as described previously. Furthermore, these two clones could replicate autonomously in nuclei of SV40 transformed Cos cells, producing SV40 T antigen constitutively when the clones were transfected into Cos cells. These results show that functional SV40 origin-like sequences are present in human genomes, and they can replicate autonomously within the cells which are producing SV40 T antigen.
Assuntos
Antígenos Virais de Tumores/genética , Replicação do DNA , Proteínas Oncogênicas Virais/genética , Oncogenes , Vírus 40 dos Símios/genética , Antígenos Transformantes de Poliomavirus , Sequência de Bases , Clonagem Molecular , Enzimas de Restrição do DNA , Humanos , Neoplasias Pulmonares/genética , Plasmídeos , Moldes GenéticosRESUMO
Medullasin, an inflammatory serine protease in bone marrow cells, modifies the functions of natural killer cells, monocytes, and granulocytes. We have cloned a medullasin cDNA from a human acute promyelocytic cell (ML3) cDNA library using oligonucleotide probes synthesized from the information of N-terminal amino acid sequence of natural medullasin. The cDNA contained a long open reading frame encoding 237 amino acid residues beginning from the second amino acid of natural meduallasin. The deduced amino acid sequence of medullasin shows a typical serine protease structure, with 41% homology with pig elastase 1.
Assuntos
Medula Óssea/enzimologia , Clonagem Molecular , DNA/metabolismo , Serina Endopeptidases/genética , Sequência de Aminoácidos , Sequência de Bases , Enzimas de Restrição do DNA , Humanos , Dados de Sequência MolecularRESUMO
To identify the primary structure of CD59 antigen and to elucidate its function, a full-length cDNA clone of CD59 was isolated. The cDNA sequence contained an open reading frame that encodes an 128-amino-acid peptide. The amino-terminal 25 amino acids represented a typical signal peptide sequence and the carboxy-terminal hydrophobic amino acids were characteristic for phosphatidylinositol-anchored proteins. The predicted mature protein sequence showed 35% homology with murine Ly-6C.1 and 31% with Ly-6A.2. The number and the distribution of cysteine residues were conserved, implying that the CD59 represented a human homologue of murine Ly-6. RNA blot hybridization analysis revealed the expression of CD59 mRNA in placental, lung, and pancreatic tissues. The mRNA was not only expressed in T-cell lines but in some of monocytic, myeloid, and B-cell lines. In all of these tissues and cell lines, at least four mRNA species were detected. DNA blot hybridization analysis revealed a rather simple genomic structure, which suggested a single gene as compared with the complex multigene family of murine Ly-6.
Assuntos
Antígenos CD/genética , Antígenos de Diferenciação/genética , Glicoproteínas de Membrana/genética , Sequência de Aminoácidos , Animais , Antígenos CD/biossíntese , Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos Ly/genética , Sequência de Bases , Northern Blotting , Southern Blotting , Antígenos CD59 , Células Cultivadas , Clonagem Molecular , Biblioteca Gênica , Humanos , Glicoproteínas de Membrana/biossíntese , Camundongos , Dados de Sequência Molecular , Monócitos , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Homologia de Sequência do Ácido Nucleico , Transfecção , Membro 7 da Superfamília de Receptores de Fatores de Necrose TumoralRESUMO
We investigated mRNA expression for medullasin (an inflammatory serine protease in bone marrow cells) in peripheral blood mononuclear cells (PBMC) obtained from 36 patients with primary IgA nephropathy (IgAN), 30 patients with other types of primary glomerular disease, 18 patients with secondary IgA nephritis including lupus nephritis and hepatic glomerulosclerosis and 24 healthy age-matched controls. The majority of patients with IgAN (86%) showed elevated medullasin expression in PBMC, while no medullasin mRNA expression was detected in PBMC obtained from patients with other types of primary glomerular disease, secondary IgA nephritis or normal healthy controls. A positive correlation was noted between mRNA levels and urinary protein excretion. The medullasin mRNA expression in PBMC also correlated with the severity of the histopathologic changes in renal tissue obtained from patients with IgAN. All the patients with severe proteinuria (more than 3.0 g/day) showed strong [more than (++)] medullasin mRNA expression in their PBMC. In addition, all the patients with more than (++) medullasin mRNA expression are grade III or IV histopathological findings. These studies suggest that abnormally regulated medullasin gene expression in PBMC may be associated with the progression of primary IgAN.
Assuntos
Regulação Enzimológica da Expressão Gênica , Glomerulonefrite por IGA/genética , Leucócitos Mononucleares/enzimologia , RNA Mensageiro/genética , Serina Endopeptidases/genética , Adulto , Northern Blotting , Feminino , Glomerulonefrite/enzimologia , Glomerulonefrite/genética , Glomerulonefrite por IGA/enzimologia , Humanos , Nefrite Lúpica/enzimologia , Nefrite Lúpica/genética , Masculino , Hibridização de Ácido Nucleico , Serina Endopeptidases/biossínteseAssuntos
Antígenos CD/imunologia , Proteínas do Sistema Complemento/imunologia , Glicoproteínas de Membrana/imunologia , Transplante Heterólogo/imunologia , Animais , Antígenos CD/genética , Sequência de Bases , Southern Blotting , Antígenos CD59 , Cães , Haplorrinos , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Especificidade da Espécie , SuínosAssuntos
Antígenos CD/imunologia , Proteínas do Sistema Complemento/imunologia , Citotoxicidade Imunológica , Glicoproteínas de Membrana/imunologia , Transplante Heterólogo/imunologia , Células 3T3 , Animais , Antígenos CD/genética , Antígenos CD59 , Células Clonais , Clonagem Molecular , Vetores Genéticos , Humanos , Linfócitos/imunologia , Glicoproteínas de Membrana/genética , Camundongos , Mapeamento por Restrição , TransfecçãoAssuntos
Antígenos CD/metabolismo , Proteínas Inativadoras do Complemento/metabolismo , Proteínas do Sistema Complemento/fisiologia , Glicoproteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD/biossíntese , Antígenos CD/isolamento & purificação , Sequência de Bases , Sangue , Western Blotting , Antígenos CD59 , Linhagem Celular , Primers do DNA , Glicosilação , Haplorrinos , Humanos , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/isolamento & purificação , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Suínos , Transfecção , Transplante Heterólogo/imunologiaAssuntos
Anemia Diseritropoética Congênita/tratamento farmacológico , Anemia Hemolítica Congênita/tratamento farmacológico , Desferroxamina/administração & dosagem , Adulto , Anemia Diseritropoética Congênita/sangue , Desferroxamina/uso terapêutico , Avaliação de Medicamentos , Ferritinas/sangue , Humanos , MasculinoRESUMO
A small reaction vessel, composed of a balled sintered glass filter and a test tube with a stopcock, was designed for the solid phase phosphotriester synthesis of oligodeoxyribonucleotide. Operation of small scale synthesis (nucleoside on resin: 3-6 mumol, activated diester of dimer: 20-30 mumol) was performed under the atmosphere of argon. The yield of each coupling reaction was 60-100%. Twelve short oligonucleotides (6-16mer) were obtained in 19-78% overall yield by this method. These oligomers were prepared as a part of the control region of gene to increase the translation efficiency.
Assuntos
Oligodesoxirribonucleotídeos/síntese química , Oligonucleotídeos/síntese química , Sequência de Bases , Indicadores e Reagentes , Métodos , Microquímica , Relação Estrutura-AtividadeRESUMO
A plasmid was constructed to express a mouse beta-interferon (IFN-beta) in Escherichia coli under the control of the modified tryptophan (trp) promoter. E. coli carrying the plasmid were cultivated in a minijar fermentor and synthesized up to 2.7 X 10(6) IU/ml of antiviral activity. At the end of the cultivation the cells became elongated and curved.
Assuntos
Interferon Tipo I/biossíntese , Proteínas Recombinantes/biossíntese , Animais , Clonagem Molecular/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Sintéticos , Interferon Tipo I/genética , Camundongos , Plasmídeos , Regiões Promotoras Genéticas , Proteínas Recombinantes/genéticaRESUMO
We have cloned a full length cDNA for human leukocyte elastase (HLE, EC 3.4.21.37)/medullasin from the cDNA library of human leukemic cell line, ML3. Recombinant plasmid for the expression of HLE cDNA in eukaryotic cells was constructed in which HLE cDNA was fused in a frame to a leader sequence of human interleukin-2 (IL-2). COS-1 cells, transfected with the plasmid, secreted fusion protein consists of N-terminal 8 amino acid (aa) residues of human IL-2 and 238 aa residues of HLE. As the fusion protein was designed to be connected through lysine residue, elastase activity was generated after digestion of the fusion protein with lysyl-endopeptidase.
Assuntos
DNA/genética , Leucócitos/enzimologia , Elastase Pancreática/genética , Serina Endopeptidases/genética , Transfecção , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular/métodos , Expressão Gênica , Vetores Genéticos , Humanos , Interleucina-2/genética , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Plasmídeos , Mapeamento por RestriçãoRESUMO
NF-IL6, a member of the basic leucine zipper (bZIP) family transcription factors, is involved in expression of inducible genes involved in immune and inflammatory responses. We observed that coexpression of oncogenic p21ras stimulated the transactivating activity of NF-IL6 and induced phosphorylation of Thr-235 located just N-terminal to the DNA binding domain of NF-IL6. Recently, mitogen-activated protein (MAP) kinases have been shown to be implicated in the cellular response to activated ras. Purified MAP kinases specifically phosphorylated Thr-235 of NF-IL6 in vitro. Mutation of Thr-235 abolished the ras-dependent activation of NF-IL6. From these results, we conclude that NF-IL6 is regulated through phosphorylation by MAP kinases in response to activated ras.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Genes ras , Proteínas Nucleares/metabolismo , Proteínas Quinases/metabolismo , Treonina , Fatores de Transcrição/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas Estimuladoras de Ligação a CCAAT , Proteínas Quinases Dependentes de Cálcio-Calmodulina , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Zíper de Leucina/genética , Luciferases/genética , Luciferases/metabolismo , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Nucleares/genética , Mapeamento de Peptídeos , Peptídeos/síntese química , Fosforilação , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/metabolismo , TransfecçãoRESUMO
Two expression plasmids (pSVIFN gamma/BPV97 and pSVIFN gamma/AdDHFR) for constitutive production of human interferon-gamma (HuIFN-gamma) were constructed and introduced into the two different mammalian cell lines, mouse C127 cells and Chinese hamster ovary (CHO) cells. Genetically engineered C127 and CHO cells grew on microcarriers having high productivity of HuIFN-gammas for at least six months. Isoelectric focusing patterns and molecular weight analyses suggest that C127- and CHO-HuIFN-gammas are glycoproteins and that both HuIFN-gammas have different molecular structures. The development of a microcarrier culture system for genetically engineered mammalian cells has enabled us to prepare glycosylated HuIFN-gammas on a large scale.
Assuntos
Interferon gama/biossíntese , Animais , Linhagem Celular Transformada , Cricetinae , Humanos , Interferon gama/análise , Camundongos , Peso Molecular , Plasmídeos , Proteínas Recombinantes/biossínteseRESUMO
In order to evaluate the structural identification of various recombinant human interferon-beta 1s, the recombinant proteins were produced in four different mammalian cells (human PC12 and PC8 lung adenocarcinoma cells, Chinese hamster ovary cells and mouse C127 cells) and characterized. Each mammalian-cell-derived recombinant human interferon-beta 1 represented a single band of 23 kDa on sodium dodecyl sulphate/polyacrylamide gel electrophoresis, the same molecular mass as fibroblast-derived natural human interferon-beta 1. Specific activities, amino acid compositions, amino-terminal sequences, peptide maps on C18 reversed-phase high-performance liquid chromatography and circular dichroic spectra of recombinant proteins were in good agreement with natural ones. On the other hand, the patterns of isoelectric focusing were different between mammalian-cell-derived recombinant human interferon-beta 1s and natural human interferon-beta 1. Sugar composition analysis revealed that the recombinant protein from Chinese hamster ovary cells has a similar sugar composition to that of natural protein and the other recombinant proteins have increased amounts of galactose and glucosamine in comparison to the natural protein. Furthermore, there is no galactosamine in the natural protein, while small amounts of galactosamine were detected in the oligosaccharides released from PC8- and C127-derived recombinant proteins by N-glycanase. These results indicate that mammalian-cell-derived recombinant human interferon-beta 1s have identical polypeptides to those of natural human interferon-beta 1 but their carbohydrate moieties, including unusual N-linked oligosaccharides, are individually different from natural ones and depend on the host cell.
Assuntos
Carboidratos/análise , Interferon Tipo I/biossíntese , Peptídeos/análise , Proteínas Recombinantes/biossíntese , Adenocarcinoma , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Carboidratos/genética , Linhagem Celular , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Cricetinae , Cricetulus , Feminino , Humanos , Interferon Tipo I/análise , Interferon Tipo I/genética , Focalização Isoelétrica , Neoplasias Pulmonares , Dados de Sequência Molecular , Ovário , Mapeamento de Peptídeos , Peptídeos/genética , Proteínas Recombinantes/análise , Proteínas Recombinantes/genéticaRESUMO
A multiple growth factor-producing tumor cell line (NIM-1) was newly established from a patient with thyroid cancer and remarkable neutrophilia. NIM-1 cells also caused severe neutrophilia in nude mice bearing tumors. NIM-1-conditioned medium (NIM-1CM) contained activities that supported not only granulocyte, macrophage and eosinophil colony formation of human bone marrow cells but also the growth of colony-stimulating factor (CSF)-dependent cell lines, NFS60-KX and TF-1. Northern blot hybridization analysis revealed the constitutive expression of granulocyte-CSF (G-CSF), granulocyte/macrophage-CSF (GM-CSF) and interleukin(IL)-6 mRNAs in NIM-1 cells. Enzyme-linked immunosorbent assays (ELISA) using NIM-1CM also confirmed the production of IL-1 alpha and a small amount of IL-1 beta besides G-CSF, GM-CSF and IL-6 in NIM-1 cells. In addition, unexpected production of IL-11 in NIM-1 cells was detected by northern blot hybridization analysis and by bioassay using an IL-11-dependent cell line. Therefore, NIM-1 cell line is shown to produce multiple cytokines including potentially megakaryopoietic growth factors such as GM-CSF, IL-6 and IL-11.
Assuntos
Adenocarcinoma Papilar/metabolismo , Fator Estimulador de Colônias de Granulócitos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Interleucinas/biossíntese , Neoplasias da Glândula Tireoide/metabolismo , Animais , Feminino , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Interleucinas/genética , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , RNA Mensageiro/análise , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
The possibility of early detection of chronic obstructive pulmonary disease (COPD) was investigated among subjects whose lung function could be followed for more than ten years. Ten subjects aged 60 or more were selected from among about 1500 cases using the following diagnostic criteria; (1) subjects had complained of exertional dyspnea (2) chest X-ray abnormalities were suggestive of COPD (3) FEV1.0% was under 55%. Data on FVC/height, FEV1.0/height and FEV1.0% of these subjects were available for the previous ten years. Individual linear regression equations were estimated. Average annual changes of cases of obstruction were compared with those of normal cases, and individual deviations from the normal standard range were observed. Declines of the regression lines were as follows; FEV1.0/height: normal case--0.016 +/- 0.017, obstruction cases--0.038 +/- 0.018 (L/m/yr), FEV1.0%: normal cases 0.164 +/- 0.627, obstruction cases--1.522 +/- 0.638 (%/yr). Significant differences were observed between the normal and the obstruction cases (p < 0.01). FEV1.0% of all obstruction cases was under--2 SD of that of normal subjects. All individual FEV1.0% regression lines of the obstruction cases were outside +/- 2 RSD of the normal standard range more than 5 years before the onset of symptoms. Following individual annual changes in lung function was considered to be useful for the early detection of COPD.
Assuntos
Pneumopatias Obstrutivas/fisiopatologia , Pulmão/fisiopatologia , Adulto , Idoso , Seguimentos , Volume Expiratório Forçado , Humanos , Pneumopatias Obstrutivas/diagnóstico , Masculino , Pessoa de Meia-Idade , Triagem Multifásica , Análise de Regressão , Fatores de TempoRESUMO
Acute-phase response factor (APRF) is a transcription factor that binds to the interleukin-6 (IL-6)-responsive elements identified in the promoters of various acute-phase protein genes. We report here the purification and cloning of APRF. APRF exhibits a 52.5% overall homology at the amino acid level with p91, a component of the interferon (IFN)-stimulated gene factor 3 complexes. The cloned APRF protein is tyrosine phosphorylated and translocated into the nucleus in response to IL-6, but not in response to IFN-gamma. Tyrosine phosphorylation was also observed in response to other cytokines, such as leukemia inhibitory factor, oncostatin M, and ciliary neurotrophic factor, whose receptors share the IL-6 receptor signal transducer gp130. In contrast, we observed that p91 is not tyrosine phosphorylated in response to IL-6. These results suggest that this novel p91-related protein may play a major role in the gp130-mediated signaling pathway and that selective activation of p91-related factors may explain the diversity of cellular responses to different cytokines.