RESUMO
Biological activities of human tumor necrosis factor (TNF) and its derivatives were compared. In cytotoxicity assay with L929 cells, one derivative, designated as TNF(Asn), showed significantly lower activity than any other TNF examined. In binding assay, this derivative was also shown to have lower affinity for TNF receptors on L929 cells, suggesting that the cytotoxic activity of TNFs on L929 cells correlates with their affinity for receptors. We also found that the cytotoxic activity of TNF on A673 cells and its inhibitory effect on lipoprotein lipase were parallel with the cytotoxic activity on L929 cells, but the growth-enhancing activity on FS-4 cells and the cytotoxic activity on endothelial cells were not. It was also shown that TNF(Asn) had lower affinity than any other TNF for receptors on these target cells tested. These results suggested that there might be at least two types of cellular responses to TNF; one might correlate with the receptor-binding affinity of TNFs and the other not.
Assuntos
Glicoproteínas/fisiologia , Animais , Divisão Celular , Sobrevivência Celular , Células Cultivadas , Humanos , Lipase Lipoproteica/antagonistas & inibidores , Camundongos , Receptores de Superfície Celular/fisiologia , Receptores do Fator de Necrose Tumoral , Proteínas Recombinantes , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfaRESUMO
Diuretic and smooth muscle-relaxing peptides, designated atrial natriuretic peptides (ANPs), have been identified in human and rat atrial tissues and implicated in the control of fluid volume and vascular function. Recently, cDNAs encoding the human and rat ANP precursors have been sequenced. We previously isolated from human tissue a natriuretic peptide of molecular weight (MW) 13,000 (gamma-hANP) comprising 126 amino acid residues, the largest natriuretic peptide so far identified, and showed that it is directly derived from the 151-residue human ANP precursor by the removal of a signal peptide. We now report the isolation and sequence analysis of a novel rat atrial natriuretic peptide (gamma-rANP) of MW 13,000, which derives from the rat ANP precursor. We also report the molecular cloning and nucleotide sequence analysis of the cDNA of the 152-residue rat ANP precursor, which is remarkably similar to the human 151-residue precursor (pre-hANP) except at the C-terminus. Differences in the rat and human precursor nucleotide sequences around the termination codons lead to a difference in processing pattern.
Assuntos
DNA/metabolismo , Proteínas Musculares/genética , Sequência de Aminoácidos , Animais , Fator Natriurético Atrial , Composição de Bases , Sequência de Bases , Galinhas , Clonagem Molecular , Proteínas Musculares/isolamento & purificação , Proteínas Musculares/farmacologia , Relaxamento Muscular/efeitos dos fármacos , Fragmentos de Peptídeos/análise , Ratos , Reto/fisiologia , TripsinaRESUMO
A gene coding for human growth hormone, which consists of 192 amino acids, was chemically synthesized. The synthesis entailed ligating 78 deoxyribooligonucleotides, which had been synthesized on polymer supports by the phosphotriester method with frequently occurring amino acid codons of Escherichia coli. The chemically synthesized gene was inserted into an E. coli plasmid downstream from the E. coli trp promoter, with a modified ribosome-binding region carried on pBR322. E. coli cells transformed with this recombinant plasmid synthesized 2.9 X 10(6) molecules per cell of human growth hormone upon induction. The induced polypeptide was identical with natural human growth hormone in size and in immunological properties, as well as in biological activity as examined by the tibial test with hypophysectomized rats.
Assuntos
Clonagem Molecular , Escherichia coli/genética , Genes Sintéticos , Genes , Hormônio do Crescimento/genética , Oligodesoxirribonucleotídeos/síntese química , Oligonucleotídeos/síntese química , Sequência de Aminoácidos , Sequência de Bases , Enzimas de Restrição do DNA , Escherichia coli/ultraestrutura , Humanos , Indicadores e Reagentes , Microscopia Eletrônica , Óperon , Biossíntese de Proteínas , Transcrição GênicaRESUMO
Two genes for human immune interferon (hIFN-gamma), synthetic (GIFs) and cDNA prepared from mRNA (GIFm), have been expressed in E. coli under the control of the trp promoter. The expression level of GIFs was 100-fold lower than that of GIFm. Secondary structure analysis of two genes' mRNA surrounding Shine-Dalgarno (SD) sequence showed the presence of a 8 base-pair stable stem structure preceding SD sequence in GIFs mRNA, but not in GIFm mRNA. Expression of a GIFs gene, in which the stem structure was rendered unstable by changing two bases, became 100 times greater than that of the original GIFs.
Assuntos
Interferon gama/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Sequência de Bases , Quimera , DNA/metabolismo , Escherichia coli/genética , Genes , Genes Sintéticos , Humanos , Conformação de Ácido Nucleico , ÓperonRESUMO
Human growth hormone gene with 584 base pairs has been synthesized by joining with DNA ligase of chemically synthesized deoxyoligonucleotides with the chain length of 15-25. The phosphotriester polymer support synthesis was employed to obtain 80 oligonucleotides.
