白细胞介素－2镇痛功能位点的研究

白细胞介素-2(IL-2)是重要的免疫调节因子,近来发现还有中枢镇痛作用,用不同IL-2突变体测定其对大鼠痛阈的影响,发现完全丧失免疫刺激作用的20Leu-IL-2(20Asp→Leu)仍能显著提高大鼠的痛阈,其作用强度与天然IL-2无显著差异,而另一突变体45Val-IL-2(45Tyr→Val)虽保留免疫学活性却不能提高大鼠的痛阈.这些结果证明IL-2分子中具有镇痛作用与具有免疫作用的功能位点是相互独立的;IL-2分子中第45位Tyr对IL-2镇痛作用的发挥起重要作用.     

定点突变法对人白细胞介素—2C末端a螺旋的结构和功能研究

用寡核苷酸诱导的基因定位突变法,将人白细胞介素－２Ｃ末端两亲性ａ螺旋中１２５位和１２７位基分别或同时突变为Ｐｒｏ,并测定其生物活性和空间结构。结果发现所得突变体的生物学活性均急下降,同时ａ螺旋含量以及Ｃ端螺旋中残基的空间位置也发生了不同程度的变化。结果提示,Ｃ端ａ螺旋尤其是其疏水面的完整性对维持白介素－２的生物活性有重要作用。     

白细胞介素－2第17和20位残基在结构功能中的重要性研究

用基因定位突变法,将白细胞介素－２（ＩＬ－２）分子中１７Ｌｅｕ和２０Ａｓｐ进行一系列突变,并测定各突变体生物活性与空间结构的变化。分析结果表明１７Ｌｅｕ突变为Ａｓｐ时,ＩＬ－２的空间结构无明显变化。生物活性却显著下降;２０Ａｓｐ突变为Ｌｅｕ,以及１７Ｌｅｕ与２０Ａｓｐ对调后,均导致ＩＬ－２的空间结构发生变化,并严重影响其生物活性。上述结果说明１７Ｌｅｕ突变为Ａｓｐ后对活性的影响并非由空间结构变化所引起,而与残基本身性质有关：１７Ｌｅｕ与２０Ａｓｐ这两个重要的残基,必须位于各自特定的空间位置,才能发挥其生物作用。     

人白细胞介素—2第20位残基局部空间结构稳定性对活性的影响

用寡核苷酸诱导的基因定位突变法,将人白细胞介素－２（ＩＬ－２）第２０位Ａｓｐ分别突变为Ａｒｇ,Ｌｙｓ和Ａｓｎ,比较第２０位残基碱性基团对ＩＬ－２活力的影响,结果＾２０Ａｓｐ突变为碱性残基时,ＩＬ－２活性急剧下降,但突变为Ａｒｇ时所导致的活性下降较突变为Ｌｙｓ时所导致的活性下降较突变为Ｌｙｓ严重３０００倍以上。从空间结构变化上对这２个碱性残基造成的如此大的活性差异进行了分析,发现＾２０Ａｒｇ突变后对     

白细胞介素2新的功能位点及其中枢镇痛作用

白细胞介素２（ＩＬ－２）,不仅是重要的免疫调节因子,而且具有重要的中枢调节作用。本工作表明：（１）ＩＬ－２具有中枢镇痛作用;（２）ＩＬ－２除具有免疫调节作用功能位点外,还存在着另一新的与之相互独立的镇痛功能位点;（３）ＩＬ－２的中枢镇痛作用,主要是由ＩＬ－２第４５位Ｔｙｒ残基以及空间结构上邻近的４４、１１７位Ｐｈｅ等残基共同构成的镇痛功能位点与阿片受体直接结合所介导。本工作提示,细胞因子的多功能性,可能是其相互独立的功能位点作用于不同的受体或受体亚型所致。     

白细胞介素－2微柱剂型

白细胞介素－２微柱剂型张世联（河北省医学科学院生化研究室,石家庄市０５００２１）关键词白细胞介素－２,微柱剂型白细胞介素－２（ＩＬ－２）是一种免疫反应调节因子,它的生物活性与它在体内存留时间及其浓度有关。增加ＩＬ－２在体内存留时间或延缓清除时间的办法...     

白细胞介素—2的中枢镇痛作用

本实验采用侧脑室给药,以钾离子透入法引起大鼠甩尾反应为指标,测定动物的痛阈,发现白细胞介素－２具有显著提高大鼠痛阈的作用,此作用能被抗ＩＬ－２单克隆抗体所阻断。纳洛酮能反转ＩＬ－２的镇痛作用,表明其作用机理与阿片受体有关。     

新型基因重组白细胞介素-2的研究开发

本文根据IL- 2基因定点突变研究所提供的有关IL- 2结构和功能重要信息 ,特别是某些关键氨基酸残基的改变对IL- 2生物活性影响 ,探讨了IL- 2和IL- 2受体相互作用关系、新型IL- 2的研制方法和变异IL -2作用机制 ,并提出设计新型天然IL -2单位点到多位点突变的策略 ,从而指导研制更有效或毒副作用降低的新型IL- 2类似物。     

人白细胞介素－2的两个部分拮抗剂

通过定点诱变技术得到6个生物活性剧烈下降的人白细胞介素-2(IL-2)突变体,其中两个突变体即15Val-IL-2和126Asp-IL-2可以在一定浓度范围内使IL-2的生物效应降低.在对高亲和力IL-2受体(IL-2R)的竞争抑制实验中,15Val-IL-2和126Asp-IL-2又表现了一定的竞争能力.这些结果表明15Val-IL-2和126Asp-IL-2的部分拮抗天然IL-2的作用.结合IL-2二级结构分析及对IL-2与IL-2R相互作用的已有认识,可认为15Val-IL-2和126Asp-IL-2的部分拮抗作用产生的原因在于替换残基在空间上对IL-2与IL-2R βγ亚基结合微环境的轻微扰动,干扰了IL-2有关残基与IL-2R βγ亚基的结合,但尚不能完全阻止其与IL-2R βγ亚基的结合.     

Probing the catalytic roles of n2-site glutamate residues in Escherichia coli glutamine synthetase by mutagenesis.

The contribution of metal ion ligand type and charge to catalysis and regulation at the lower affinity metal ion site (n2 site) of Escherichia coli glutamine synthetase (GS) was tested by mutagenesis and kinetic analysis. The 2 glutamate residues at the n2 site, E129 and E357, were changed to E129D, E129H, E357H, E357Q, and E357D, representing conservative and nonconservative alterations. Unadenylylated and fully adenylylated enzyme forms were studied. The Mn(2+)-KD values, UV-cis and fluorescence emission properties were similar for all mutants versus WTGS, except E129H. For kinetic determinations with both Mn2+ and Mg2+, nonconservative mutants (E357H, E129H, E357Q) showed lower biosynthetic activities than conservative mutants (E129D, E357D). Relative to WTGS, all the unadenylylated Mn(2+)-activated enzymes showed reduced kcat/Km values for ATP (> 7-fold) and for glutamate (> 10-fold). Of the unadenylylated Mg(2+)-activated enzymes, only E129D showed kinetic parameters competitive with WTGS, and adenylylated E129D was a 20-fold better catalyst than WTGS. We propose the n2-site metal ion activates ADP for departure in the phosphorylation of glutamate by ATP to generate gamma-glutamyl phosphate. Alteration of the charge density at this metal ion alters the transition-state energy for phosphoryl group transfer and may affect ATP binding and/or ADP release. Thus, the steady-state kinetic data suggest that modifying the charge density increases the transition-state energies for chemical steps. Importantly, the data demonstrate that each ligand position has a specialized spatial environment and the charge of the ligand modulates the catalytic steps occurring at the metal ion. The data are discussed in the context of the known X-ray structures of GS.     

中枢白细胞介素-1在应激反应中的作用

白细胞介素-1（interleukin-1,IL-1）系统分子广泛分布于中枢神经系统。中枢IL-1具有极其丰富的生物学功能,作为经典炎性细胞因子,在多种生理、病理生理过程中起重要作用。近几年来,中枢IL-1的应激介质作用备受关注。本文综述了中枢IL-1在应激反应中作用的最新进展,包括应激对中枢IL-1系统的影响,中枢IL-1对应激反应的启动和介导作用;参与中枢IL-1-应激介导作用的神经环路和细胞信号转导通路,以及中枢IL-1对应激时脑高级功能和行为反应的影响。     

突变型人白细胞介素-2基因在巴斯德毕赤酵母中的表达

为了提高人重组白细胞介素 2的稳定性和活性以及减少毒副作用 ,有必要定向改造rhIL 2的分子结构 .用PCR法从白细胞介素 2 (IL 2 )cDNA全序列中扩增成熟的肽基因片段 ,并利用定点突变技术将人重组白细胞介素 2第 12 5位游离的半胱氨酸编码序列突变为丙氨酸序列 .编码 18位亮氨酸的序列突变为蛋氨酸序列 ;编码 19位亮氨酸的序列突变为丝氨酸序列 .突变型人白细胞介素 2 (MvIL 2 )基因与表达载体pPIC9K重组 ,酶切线性化后用Invitrogen转化毕赤酵母试剂盒导入酵母细胞进行整合 ,经筛选得到一高表达白介素 2的克隆 .SDS PAGE显示 ,表达量约占总量的4 5 7% .经Western印迹验证 ,重组人白介素 2有免疫活性 ;与野生型IL 2相比 ,所获得的突变型IL 2纯品的比活性为 4 0× 10 7IU mg蛋白 ,比天然型IL 2高 4～ 5倍     

人白介素6受体结合功能域的构效关系研究

以分子对接（docking）方法研究人白介素6受体胞外区配基结合功能域“WSXWS”区氨基酸残基定点突变对受体与配基人白介素6结合时的相互作用能量、分子间相互作用的影响,从分子力学、分子动态学分析了人白介素6受体胞外区功能域关键氨基酸残基在受体与配基结合中的构象变化以及与人白介素6间的相互作用.     

Levamisole and Interleukin-2 for advanced malignancy

Therapy for cancer patients with biologically active immune modulators is attractive but has met with limited clinical success. Interleukin-2 (IL2) stimulates T-cells and natural killer (NK) cells to kill tumor cells and levamisole (LMS) is an immunostimulant which has been shown to increase NK cells and activated T-cells in patients receiving this adjuvantly along with 5FU for Stage III colon cancer. This study was designed to evaluate whether treatment with LMS prior to IL2 would provide synergistic activity and improve response rates. Four patients with advanced malignancies were treated with LMS at 50mg po TID for 3 days followed on day 4 with 600,000 units/kg IL2 as a single iv bolus. This treatment was repeated weekly until progression. Serum soluble IL2 receptor (sIL2R) and interferon- levels were monitored throughout the treatment course as markers of immune activation. All patients had eventual progression of disease. Toxicity was minimal with Grade II orthostatic hypotension the major consequence of therapy. The pattern of sIL2R levels in 3/4 patients revealed a steady increase over the several weeks of therapy, indicating ongoing immunostimulation (r =0.53 , p= 0.001). Short-term treatment with LMS, however, resulted in a significant and consistent decreases in sIL2R levels (2198 U/ml vs. 1969 U/ml, p=0.001) in all patients. In conclusion, LMS/IL2 in the dose and schedule utilized here was not clinically effective. However, LMS reduced sIL2R levels immediately following a three-day course. This reduction in sIL2R by LMS may improve the possibility of response to IL2 by facilitating a decrease in inhibitory sIL2R. Combinations of these two agents should continue to be investigated as potential synergistic anti-tumor agents.     

Substrate specificities in class A beta-lactamases: preference for penams vs. cephems. The role of residue 237

Site saturation mutagenesis has been carried out at Ala-237 in RTEM-1 beta-lactamase to assess the role of this site in modulating differences in specificity of beta-lactamases for penams vs. cephems as substrates. (An Ala-237 Thr mutation had previously been shown to increase activity on cephems by about 30-80%.) Screening of all 19 possible mutants on penams and cephems revealed the even more active Ala-237 Asn mutant. Detailed kinetic analysis shows that this mutant has about four times the activity toward cephalothin and cephalosporin C as the wild-type enzyme. Both mutations reduce the activity toward penams to about 10% that of RTEM-1 beta-lactamase and lower by about 5 degrees C the temperature at which the enzyme denatures. Functional properties of the other mutants have also been surveyed. The most interesting aspect of these results is that two quite disparate amino acids, threonine and asparagine, when introduced for Ala-237, cause such similar changes in enzyme specificity while more similar residues do not alter the catalytic properties of the enzyme to such a significant degree.     

林麝IL-2细胞因子的克隆、表达和纯化

用林麝（Moschus berezovskii）外周血单核细胞（peripheral blood mononuclear cells,PBMC）提取总RNA,RT-PCR扩增IL-2（interleukin-2,IL-2）,连接pMD18-T载体后转化大肠杆菌,筛选阳性克隆并测序。然后将IL-2成熟肽编码序列连接到pGEX4T-1原核表达载体进行表达、纯化。结果表明林麝IL-2开放阅读框全长468bp,编码155个氨基酸。序列分析表明,林麝IL-2及其受体结合域与水牛的高度保守,而在翻译后修饰的预测活性位点上和水牛的有所不同。IFFG诱导林麝IL-2蛋白表达,得到约41kD的目标带;优化表达后,林麝IL-2表达含量达到了总蛋白量的25％。经GST亲和柱分离纯化,得到了约41kD的单一目标带,这说明林麝IL-2基因的原核表达成功。