CYTOKINE INDUCTION BY A 48 KD MYCOPLASMA GENE PRODUCT

48 KD 支原体基因产物诱导细胞因子

• 批准号: 2895749
• 负责人: ROBERT E HALL
• 金额: $ 10.91万
• 依托单位: UNIVERSITY OF TENNESSEE KNOXVILLE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2002-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2895749
• 关键词: Baculoviridae; CHO cells; Mycoplasma; affinity chromatography; bacterial proteins; cell differentiation; cell growth regulation; cytokine; flow cytometry; gene expression; hematopoietic stem cells; human tissue; immunofluorescence technique; immunomodulators; laboratory mouse; lipopolysaccharides; monocyte; posttranslational modifications; protein purification; recombinant proteins; tumor necrosis factor alpha

Hematopoietic/lymphoid cell differentiation is an area of intense investigation, and a number of cytokines and other factors have been identified which modulate growth, differentiation, and functional activity of hematopoietic and lymphoid cells. These include a number of microbial products such as LPS, TSST-1, erythrogenic toxins A and C, and several Mycoplasma products. In the process of examining tumor cell line conditioned media for cytokines and factors with differentiation activity, several years ago we identified, purified, and began characterizing a 48 kd factor termed P48. The biochemical characteristics and biologic activities of this factor include induction of monocytic differentiation and cytolytic activity in the HL-60 (human promyelocytic) cell line, growth inhibition (but not nonspecific toxicity) toward a large panel of other human and mouse cell lines, induction of secretion of IL-1 and TNF-alpha by human peripheral blood monocytes, and induction of accumulation of mRNAs for IL1- alpha, IL1-beta, lL6, and TNF-alpha in monocytes and monocyte-like cell lines. Over the past three years, we have further characterized this factor including cDNA and genomic cloning, sequencing, expression of the gene in E. coli, and purification of the biologically active recombinant protein. We have found that P48 represents a Mycoplasma fermentans gene product, and sequence analysis revealed a number of characteristics of a lipoprotein suggesting that P48 may be functionally similar to bacterial LPS. This revised grant proposal is designed to further characterize the biochemistry, molecular biology, and biology of P48 in detail including (I) characterization of the surface or cytoplasmic receptor for P48 on monocytes and monocyte-like cell lines and possible involvement of serum binding proteins; (2) examination of the biochemistry and functional significance of post-translational modification (e.g. lipid and/or carbohydrate) of the molecule; (3) examination of the mechanism of P48 induction of inflammatory cytokines with comparison to LPS; and (4) expression in eukaryotic systems including Baculovirus/insect cells and CHO cells P48 is an important immunomodulatory molecule which may be important in the pathophysiology of Mycoplasma infections and may be useful in dissecting the mechanisms involved in mammalian hematopoietic cell differentiation and cytokine biosynthesis.

造血/淋巴细胞分化是一个强烈的区域， 调查，以及一些细胞因子和其他因素， 已经鉴定出调节生长、分化和 造血和淋巴细胞的功能活动。这些 包括许多微生物产物，例如LPS，TSST-1， 红细胞生成毒素A和C以及几种支原体产物。 在 检查肿瘤细胞系条件培养基的过程 具有分化活性的细胞因子和因子，多年 之前，我们鉴定，纯化，并开始表征一个48 kd的 称为P48的因子。其生化特性和生物学特性 该因子的活性包括诱导单核细胞 在HL-60（人）中的分化和细胞溶解活性 早幼粒细胞）细胞系，生长抑制（但非非特异性 毒性）对一大组其他人类和小鼠细胞系， 人外周血淋巴细胞诱导IL-1和TNF-α分泌 血液单核细胞，并诱导IL 1 - 1 mRNA的积累， 单核细胞和单核细胞样单核细胞中的IL-1-β、IL-6和TNF-α 细胞系过去三年，我们进一步 包括cDNA和基因组克隆， 测序、在E.大肠杆菌，并纯化 具有生物活性的重组蛋白。我们发现P48 代表发酵支原体基因产物， 分析揭示了脂蛋白的一些特征， 提示P48在功能上可能与细菌LPS相似。 这一修订后的赠款建议旨在进一步特点， 生物化学、分子生物学和P48的生物学 包括（I）表征表面或细胞质 单核细胞和单核细胞样细胞系上的P48受体， 可能涉及血清结合蛋白;（2）检查 翻译后的生物化学和功能意义 分子的修饰（例如脂质和/或碳水化合物）;（3） P48诱导炎症反应机制的研究 与LPS相比的细胞因子;和（4）在真核细胞中的表达 包括杆状病毒/昆虫细胞和CHO细胞的系统，P48是一种 重要的免疫调节分子，其在免疫调节中可能是重要的。 支原体感染的病理生理学，并可用于 剖析哺乳动物造血细胞 分化和细胞因子生物合成。