Characterization of a membrane-associated protein kinase of multidrug-resistant HL60 cells which phosphorylates P-glycoprotein

Cells containing increased levels of the membrane phosphoprotein P-glycoprotein exhibit a multidrug-resistant phenotype. In the present study we have analyzed protein kinases capable of phosphorylating P-glycoprotein in membranes of HL60 cells isolated for resistance to vincristine. Analysis of this system demonstrates that in isolated membranes the protein kinase inhibitor staurosporine greatly reduces P-glycoprotein phosphorylation. In contrast, the kinase inhibitor H-7 does not affect this reaction. Fractionation of solubilized membrane proteins from sensitive and resistant cells on DEAE-cellulose reveals a major protein kinase (PK-1) which exhibits optimal activity in the presence of Mn2+ and histone H1. This enzyme fraction does not contain detectable levels of protein kinase C or cAMP-dependent protein kinase. PK-1 phosphorylation of two endogenous proteins is, however, greatly enhanced in the presence of phosphatidylserine or phosphatidyl-inositol. In reaction mixtures containing Mg2+ or Mn2+ in the absence of phospholipid, PK-1 from resistant cells phosphorylates an endogenous protein of 180 kilodaltons (P180), which exhibits an electrophoretic mobility identical to P-glycoprotein. In parallel experiments with PK-1 from sensitive cells there is no detectable phosphorylation of a P180 protein. P180 phosphorylated by PK-1 from resistant cells is immunoprecipitated by antibody against P-glycoprotein. Additional studies demonstrate that PK-1 is capable of phosphorylating specific synthetic peptides which correspond to the sequence of P-glycoprotein. Peptide phosphorylation occurs at both serine and threonine residues. These studies thus identify a novel membrane-associated protein kinase in HL60 cells which is capable of phosphorylating P-glycoprotein. This enzyme may have an important role in regulating levels of multidrug resistance.     

Adriamycin resistance in HL60 cells in the absence of detectable P-glycoprotein

Previous studies have shown that the development of multi-drug resistance in cell lines treated with chemotherapeutic agents is closely associated with the overexpression of a 170-180 kilodalton surface membrane glycoprotein (P-glycoprotein). In the present study a monoclonal antibody against the P-glycoprotein was used to determine if this protein is overexpressed in multi-drug resistant HL60 cells. Using either indirect immunofluorescent staining or immunoblot analysis P-glycoprotein could not be detected in HL60 cells isolated for resistance to adriamycin. In contrast HL60 cells isolated for resistance to vincristine contain the P-glycoprotein and the amount of this material increases with increasing levels of resistance. These studies thus demonstrate adriamycin resistance in P-glycoprotein negative HL60 cells. Furthermore adriamycin and vincristine are found to have distinct effects in inducing overexpression of P-glycoprotein in the HL60 cell line. This information could be useful in the development of therapeutic strategies for the treatment of certain forms of cancer.     

Generation of, lipid neutrophil chemoattractant activity by histamine-stimulated cultured endothelial cells

Endothelial cell-neutrophil interactions are an important aspect of inflammatory responses. Because vascular endothelial cells respond to the inflammatory mediator histamine, these studies determined whether histamine could induce endothelial cells to release substances that affect human neutrophil migration. Cultured bovine and human endothelial cells incubated with histamine released neutrophil chemoattractant activity within 1 min; peak levels were noted in 45 min. Cimetidine, an H2 receptor antagonist, blocked chemoattractant production, whereas diphenhydramine, an H1 receptor antagonist, did not. Cycloheximide did not inhibit release of chemoattractant activity, suggesting de novo protein synthesis was not necessary for its appearance. Extraction with acidified diethyl ether partitioned all neutrophil chemoattractant activity into the organic phase. The lipoxygenase pathway inhibitors, diethylcarbamazine and 5,8,11,14 eicosatetraynoic acid, inhibited generation of this lipophilic chemoattractant activity, whereas indomethacin, a cyclo-oxygenase inhibitor, did not. Resolution of the histamine-induced endothelial cell-derived chemoattractant activity by reverse-phase high pressure liquid chromatography yielded several peaks of chemoattractant activity, none of which co-eluted with leukotriene B4, platelet-activating factor, or two mono-hydroxyeicostetraenoic acids. These findings suggest that endothelial cells release lipid neutrophil chemoattractant activity that may play a role in inflammatory responses associated with histamine.     

土壤中含EB病毒诱导物的检测

从广西壮族自治区梧州市、苍梧县、罗城县和北京市收集的土壤标本中发现有EB病毒诱导物。梧州市和苍梧县沿公路和江河两旁桐油树下的土壤标本,对EB病毒早期抗原诱导的阳性率为40～58％。在其他大戟科植物下的土壤标本中,也发现有EB病毒诱导物。对桐油树下土壤中EB病毒诱导物与鼻咽癌发生的可能关系进行了讨论。     

从广州市散发性脑炎病人血清中查出雪鞋野兔病毒抗体升高

近几年来夏季,在我国南方一些省市发现一些散发性病毒性脑炎病例,主要是儿童。他们的流行性乙型脑炎抗体阴性。病因不明。 为了研究本病的病因,于1983年4月至10月,我们在广州市儿童医院收集了34例散发性脑炎病人的双份血清,15例其它病种(如百日咳、心肌炎、钩端螺旋体脑炎、多发性神经根     

抗胃癌细胞系单克隆抗体PD4的初步研究

以胃癌细胞系MGC803免疫Balb/c小鼠,取其脾细胞与小鼠骨髓瘤细胞NS-1融合。经选择培养、筛选及克隆化,获得恒定地分泌抗胃癌细胞系单克隆抗体(MoAb)的杂交瘤细胞系PD4。MoAb PD4与3/4胃癌细胞系有强结合反应,与4/4肺癌细胞系有弱结合反应,但与淋巴细胞、ABO红细胞、骨髓细胞、二倍体成纤维细胞及经检测的其他肿瘤细胞均无反应。PD4抗原主要表达于靶细胞的膜上,不耐热,为分子量40kD的蛋白性抗原。该抗原与HLA抗原系统,血型抗原系统无关,亦不同于其他作者所报告的其他胃癌相关抗原。     

甲型肝炎病毒对理化因子的稳定性

用放射免疫成斑法研究了甲型肝炎病毒FM-175株的理化稳定性和灭活条件,证明甲型肝炎病毒理化稳定性与其它肠道病毒相似,具有广范围的pH(2～10)稳定性;Mg~(++)和Ca可增强其热稳定性,不能抵抗冷冻干燥,但对热的抵抗力明显高于普通肠道病毒,可被紫外线迅速杀灭,也可被多种消毒剂如3～8％的甲醛液,50～90％的乙醇,2％的石炭酸及400ppm的有效氯等杀灭,但可抵抗0.1％甲醛液,2～5％的来苏儿及200ppm的有效氯1小时以上,本研究工作为甲型肝炎病毒的理化性状、保存及消毒条件等提供了实验数据。     

黄素氧还蛋白参与的固氮链电子传递

棕色固氮菌中电子载体Fld直接向固氮酶铁蛋白传递电子。Fld_(ox)至Fld_R是双电子二步还原反应,极谱半波电位分别为-210、-550 mV。Fld_(ox)至Fld_(SR)的中点电位为-280 mV,Fld_(SR)至Fld_R为-500mV。铁蛋白中点电位为-256mV,加MgATP后为-390 mV。Fld_R与铁蛋白ox组成的电池电动势为244mV,电子传递可自发进行,反应的J△G~o为-23KJ/摩尔,铁蛋白被Fld_R还原的K_a=1.3×120~4,加入MgATP后△G~o为-10.6KJ/摩尔,K_a=72。因此,未加入MgATP时电子传递反应更易进行。