Differential expression of binding sites for chemokine RANTES on human T lymphocytes

The C-C chemokine RANTES, a T lymphocyte chemoattractant, is considered an important mediator of inflammation, allergy, and host defense against HIV-1 infection. In this study, we investigated the modulation of binding of RANTES to T lymphocytes. Human peripheral blood CD3+ T cells, when freshly isolated from buffy-coat blood, expressed a considerable number of high-affinity binding sites for RANTES. These cells also showed significant chemotactic migration in response to RANTES in vitro. After 6–15 h incubation at 37°C, the binding of RANTES, but not of macrophage inflammatory protein-1α (MIP-1α) or of monocyte chemotactic protein-3 (MCP-3), consistently increased. Scatchard analyses indicated that the number of binding sites for RANTES increased about threefold by 15 h without any change in the affinity. The increase in RANTES binding was no longer detected by 24 h. This increase in the specific binding was mainly attributable to CD4⁺ T cells and was not associated with increased chemotactic activity of these cells in response to RANTES. Incubation with anti-CD3 antibody for 15 h markedly reduced the binding capability of T cells for RANTES and was associated with decreased chemotactic activity. On the other hand, when T cells were incubated with interleukin-2 (IL-2) for 1 week, the specific binding for all three C-C chemokines, RANTES, MIP-1α, and MCP-3 was markedly increased in comparison to cells cultured in the absence of IL-2. These results suggest that the expression of binding sites on T cells for RANTES is differentially modulated, indicating the existence of novel receptors for RANTES that do not bind MIP-1α.     

Mouse macrophage development in the absence of the common γ chain: defining receptor complexes responsible for IL-4 and IL-13 signaling

The common γ chain (γ_c) forms a critical component of the receptors for interleukins (IL)-2, IL-4, IL-7, IL-9, and IL-15. We analyzed γ_c-deficient mice to define a role for γ_c signaling in the development and function of the macrophage lineage. No major differences in absolute cell numbers, cell surface phenotype, or in vitro function of γ^?_c compared to γ⁺_c macrophages were observed. We therefore conclude that signaling through the γ_c chain is not essential for the differentiation of mouse macrophages. Although B and T cells require γ_c for IL-4 responses, IL-4 up-regulated major histocompatibility class II molecules and inhibited nitric oxide production from γ^?_c macrophages following stimulation with lipopolysaccharide and interferon-γ. γ^?_c macrophages could also respond to IL-13, consistent with the model of a type II IL-4 receptor α/IL-13R which can function in the absence of γ_c. Both IL-4 and IL-13 responses could be completely inhibited with the mouse IL-4 antagonist QY, suggesting that all of the observed IL-13 responses pass through the type II receptor, making it the primary signaling receptor complex for IL-13 in mouse macrophages.     

利用国产~(125)碘源制备~(125)I-心得静及实验考核

用国产Na~(125)I源、氯胺-T法、制备(-)~(125)I心得静标记配体【(-)~(125)I-IPIN】,放射色谱法分离复合物,测得(-)_(125)I-IPIN的Rf值为0.35,比活度为46.12～55.32TBq/mmol,标记率为37％,放射化学纯度达95％以上,该标记配体与小鼠脑细胞膜β受体的结合用受体的放射配体结合分析法(RBA)考核,呈典型的饱和曲线,Scatchard分析呈一直线,平衡解离常数(Kd)为0.041±0.001nM,Hill系数接近于1(0.99)。     

Galanin receptors in the post-mortem human brain. Regional distribution of I-galanin binding sites using the method of in vitro receptor autoradiography

The distribution of putative receptors for the peptide galanin was studied in the normal post-mortem human brain by using ¹²⁵I-galanin (0.5 nM) in combination with in vitro receptor autoradiography. Specific binding of ¹²⁵I-galanin was found in a large number of brain areas throughout the neuraxis. Highest binding densities occurred in the basal forebrain and hypothalamus, while the basal ganglia, major parts of the thalamus and the tectum were found to be poor in binding sites. All cortical areas harboured ¹²⁵I-galanin binding, and in the visual cortex a laminated pattern was present. In the hippocampus, ¹²⁵I-galanin binding occurred in layer 2 of the entorhinal cortex, in the uncus and in the hippocampal-amygdala area. In the brain-stem, ¹²⁵I-galanin binding was found in serotoninergic and noradrenergic cellgroups as well as in the reticular formation and in the parabrachial nuclei. Galanin receptors may, thus, mediate the response of galanin in numerous structures in the human brain.     

MAPK 通路抑制剂对低氧高二氧化碳性肺动脉收缩的影响

目的： 观察肺主动脉环、二级肺动脉环在急性低氧高二氧化碳介质中张力的变化；探讨 MAPK 信号通路抑制剂 U0126、SB203580 对低氧高二氧化碳性肺血管收缩的影响。方法: 制备离体 SD 大鼠肺主动脉环、二级肺动脉环。分别观察肺主动脉环、二级肺动脉环在常氧及急性低氧高二氧化碳介质中的张力变化；在急性低氧高二氧化碳条件下分别用 U0126、SB203580 孵育二级肺动脉，观察各自对低氧高二氧化碳性肺动脉收缩的影响。结果: 在常氧条件下,肺主动脉、二级肺动脉张力均无明显变化。急性低氧高二氧化碳条件下二级肺动脉发生双向性收缩反应，肺主动脉只在低氧高二氧化碳早期出现较明显的收缩峰，后期则变化不明显。二级肺动脉分别经ERK1/2上游激酶抑制剂 U0126、p38 MAPK 通路抑制剂 SB203580 孵育后，Ⅱ期持续收缩幅度明显下降（P<0.05)，Ⅰ期快速收缩峰、Ⅰ期舒张均没有明显变化。结论: 在离体条件下，急性低氧高二氧化碳（PO₂ = 30-35 mmHg，PCO₂=55-60 mmHg)可使肺主动脉出现早期快速收缩，并可使二级肺动脉环发生双向性收缩反应；急性低氧高二氧化碳条件下，U0126、SB203580 均能减弱二级肺动脉环的Ⅱ期持续收缩反应。这为临床治疗缺氧和高碳酸血症引起的肺血管收缩及肺动脉高压提供了理论依据。     

N-乙酰半胱氨酸对脂多糖诱导的小鼠肝MAPK磷酸化的影响

目的： 探讨N-乙酰半胱氨酸（NAC）对脂多糖（LPS）诱导的肝MAPK磷酸化的影响。方法: 雄性昆明种小鼠54只随机分为对照组(n=6)：0.9 % NaCl 0.2 mL ip；LPS组（n=24）：LPS 5 mg ip；NAC+LPS组（n=24）：NAC 150 mg·kg-1·d-1ip，连续3 d；第3 d NAC灌胃后1 h时，LPS 5 mg ip。将小鼠分别在注射LPS或生理盐水后0.5 h、1 h、2 h和6 h时，在戊巴比妥钠麻醉下开腹取肝，测定肝MDA和还原型谷胱甘肽（GSH）含量；Western blotting方法测定肝脏MEK1/2、ERK1/2、p38MAPK磷酸化水平，放免法测定肝TNF-α含量。结果: NAC预处理使肝MDA含量明显下降，使肝GSH含量升高。NAC预处理显著抑制了LPS所致的肝MEK1/2、ERK1/2、p38MAPK磷酸化，同时使肝TNF-α水平显著降低。结论: 在LPS诱导的急性肝损伤过程中，活性氧（ROS）在激活MAPK信号转导中起重要作用。NAC通过其抗氧化作用部分抑制了LPS诱导的MAPK磷酸化，使TNF-α生成减少，从而发挥抗损伤作用。     

人可溶性IL-6R基因在COS7细胞中的表达及活性分析

目的在ＣＯＳ７细胞中表达具有生物学功能的人可溶性ＩＬ－６Ｒ（ｓＩＬ－６Ｒ），作为研究ｓＩＬ－６Ｒ结构与功能关系的基础。方法首先利用ＰＣＲ技术扩增出人可溶性ＩＬ－６Ｒ（ｈｓＩＬ－６Ｒ）编码基因片段，并重组入克隆载体ｐＡＬＴＥＲ－１。通过基因序列分析确定了目的基因的核苷酸序列，并进一步构建了由ＳＶ４０晚期启动子和ＨＣＭＶ早期启动子控制的表达质粒ｐＳＶＬ６Ｒ和ｐＣＭＶ６Ｒ。用脂质体介导的方法将表达质粒转染ＣＯＳ７细胞，并分别在ｍＲＮＡ水平（斑点杂交）和蛋白水平（ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ－ｂｌｏｔ）检测ｓＩＬ－６Ｒ基因在ＣＯＳ７细胞中的表达。在７ＴＤ１，ＬＴ１２两种ＩＬ－６反应细胞系上检测转染细胞上清（含ｓＩＬ－６Ｒ）的生物学活性。结果在ｍＲＮＡ水平和蛋白水平分别检测到ｓＩＬ－６Ｒ基因在ＣＯＳ７细胞中的表达，表达产物分子量约为５００００。表达产物在７ＴＤ１，ＬＴ１２细胞系上检测到明显的生物学活性。结论天然ｓＩＬ－６Ｒ基因在ＣＯＳ７细胞中的成功表达为进一步制备ｓＩＬ－６Ｒ突变体及其结构与功能关系的研究奠定了基础     

三磷酸肌醇在IL—2诱导的T细胞增殖分化中的信息传递作用

用不同浓度的IL-2刺激静息T淋巴细胞,其细胞内IP_3无明显改变,但ConA刺激则使IP_3增加45%。在IL-2依赖性T细胞,IL-2R表达率达83%,IL-2刺激时IP_3的变化依浓度不同而异,10u—50u/ml的IL-2使IP_3增高,以50u/ml最为显著,增加60%,但100u/ml IL-2及ConA不改变胞内IP_3的浓度。IL-2R封闭后的T淋巴细胞在IL-2刺激时IP_3的增加明显减弱。这些结果提示:IP_3作为细胞内的第二信使介导了IL-2诱导的T细胞的增殖反应,这种作用与IL-2的剂量及IL-2R表达有密切的关系。     

Longer term progesterone treatment induces changes of GABAA receptor levels in forebrain sites in the female hamster: quantitative autoradiography study

Summary Quantitative receptor autoradiography was applied to evaluate the effects of one and three injections of 1 mg progesterone (P) on ³H muscimol binding levels in the different forebrain areas of the female hamster. The overall effect of P resulted in substantial increases in ³H muscimol binding in brain areas containing gonadal steroid receptors: medial preoptic area and ventromedial hypothalamic nucleus as well as in bed nucleus stria terminalis and subiculum. Similarly, the caudate putamen, a region where gonadal steroid receptors are not abundant, also showed substantial increases of ³H muscimol binding receptor levels. Moreover, female hamsters treated with P for 3 days presented altered ³H muscimol binding levels in the amygdala and thalamic nucleus that were, in some cases, not produced by one dose of P. P treatment also decreased GABA_A binding in two areas of the thalamus. These results are consistent with the proposal that P may alter GABAergic inhibitory activity via changes in the levels of GABA_A receptors in certain forebrain areas in the female hamster, changes which may be linked to the mediation of anxiolytic effects and to the inhibition of aggressive behavior. These data also suggest that P treatment increases the binding of high affinity GABA receptors in some forebrain sites and may be responsible for maintenance of the anxiolytic effects.     

PKA参与脊髓背角C-纤维诱发电位LTP的诱导和早期维持

目的: 探讨环一磷酸腺苷依赖的蛋白激酶 (PKA) 在脊髓背角C-纤维诱发电位长时程增强(LTP)的诱导和维持中的作用。方法:细胞外记录技术在脊髓腰膨大部记录背角浅层神经元C-纤维诱发电位。 结果:(1) 8-Br-cAMP诱发脊髓背角C-纤维诱发电位LTP,且8-Br-cAMP-诱导的 LTP 遮蔽强直刺激诱导的LTP。(2) PKA的选择性抑制剂Rp-CPT-cAMPS阻断C-纤维诱发电位LTP的诱导和时间依赖性翻转C-纤维诱发电位LTP。(3)蛋白质合成抑制剂茴香霉素彻底阻断8-Br-cAMP诱发的 LTP。(4)MAPK选择性抑制剂PD98059阻断8-Br-cAMP诱发的LTP。 结论:脊髓背角神经元存在PKA信号途径,并参与脊髓背角C-纤维诱发LTP的诱导和早期维持。