8mol／L脲中的rhI—3含量的测定

为解决８ｍｏｌ／Ｌ脲中ｒｈＩＬ－３的定量问题,以卵清蛋白作内标,进行常规ＳＤＳ＿ＰＡＧＥ后,作激光灰度扫描,并计算ｒｈＩＬ－３和卵清蛋白的峰面积,发现两种蛋白峰面积的比值与ｒｈＩＬ－３浓度在０．２－１．０ｍｇ／ｍｌ间呈良好线性关系。查标准曲线可以计算出８ｍｏｌ／Ｌ脲中ｒｈＩＬ－３的含量。重复性测定表明该方法具有较好的重现性。     

H+-Lsfet型青霉素酶传感器

将sos型H^＋－ISFET与戊二醚交联的牛血清蛋白-青霉素酶膜组合成单管输出式青霉素酶－H＋－ISFET传感器的探头(以下简称青霉素－酶FET),并用于测定溶液中的青霉素含量。青霉素－酶FET在0．005mol/L、O．Olmol／L、0．02mol／L磷酸缓冲液中的响应灵敏度分别为11—12mV／m mol／L、7．5—8．0mV／m mol／L以及3．7—4．OmV／m m01／L;响应时间为30s,在0．02mol／L磷酸缓冲液中的标定曲线线性范围为O．5—25mmo】／L’相关系数为O．9976。该青霉素·酶FET在青霉索浓度为10mmol／L的0．01mo1／L磷酸缓冲液中重复测定8次的标准偏差sD为1．67mv,变异系数cv为2．1％;贮存寿命大于4个月,使用寿命在1个月以上(每天测试一次)。     

嗜酸乳杆菌表层蛋白提取方法及影响因素的研究

对嗜酸乳杆菌S层蛋白的提取方法进行了改进．用酸性5mol／L LiCl法,中性5mol／L LiCl法和8mol／L尿素法提取嗜酸乳杆菌的表层蛋白,考马斯亮兰法测蛋白浓度,进行常规SDS-PAGE,用Gel—Pro软件进行数据分析,得出其中酸性5mol／L LiCl法提取表层蛋白百分含量最多．分析影响提取条件的因素,设置0、4、室温（25℃）3个温度梯度和10、15、20、25、30min 5个时间梯度,进行常规SDS-PAGE,检测提取表层蛋白的效果．Sephadex G-100柱分离中性5mol／L LiCl法和8mol／L尿素法提取嗜酸乳杆菌的表层蛋白获得SA蛋白．酸性5mol／L LiCl法可用于分析各种表层蛋白;中性5mol／L LiCl法和8mol／L尿素法用于提取43kDa的SA蛋白．25℃处理15min得到最大量的表层蛋白．     

流感疫苗中卵清蛋白检定方法精确度的探讨

目的：探讨流感疫苗中卵清蛋白检定法的精确度。方法：将卵清蛋白标准抗原及待检流感疫苗适当地倍比稀释后进行寸流免疫电泳。经与标准抗原的沉淀线对比分析后,计算待检样品中的卵清蛋白含量。结果：流感疫苗中的卵清蛋白测定值可青确到0．1μg／ml。结论：对流免疫电泳法易于操作,精确度较高,比较适合大规模流感疫苗生产的检定需要。     

重组人白介素-10抑制晚期糖基化终产物诱导的大鼠血管平滑肌细胞和血管新生内膜的增殖

研究观察了重组人白介素10(rhIL-l0)对晚期糖基化终产物(AGE)刺激下离体大鼠胸主动脉血管平滑肌细胞增殖及对SD大鼠血管损伤后新生内膜增殖的影响。体外培养大鼠主动脉血管平滑肌细胞,采用MTS／PES法确定血管平滑肌细胞的增殖状态;应用流式细胞术测定细胞周期;利用p44／42磷酸化抗MAPK抗体的蛋白免疫印迹法测定p44／42 MAPK磷酸化蛋白表达。利用大鼠颈动脉血管损伤模型,观察rhIL—10对新生内膜增殖的影响。结果显示：(1)AGE处理组与对照组相比,AGE对血管平滑肌细胞增殖具有明显的刺激作用(P＜0．05)。rhIL-l0单独应用对血管平滑肌细胞生长没有影响(P＞0．05)。在AGE刺激下,低至100ng／ml的rhIL-l0可抑制血管平滑肌细胞的生长(P＜0．05)。(2)流式细胞术测定的结果显示,rhIL—10可以使AGE作用下的VSMC大部分处于Go／G1期,与对照组相比有明显差异(P＜0．01)。(3)AGE对p44／p42 MAPK磷酸化蛋白表达有显著的增强作用,此作用可被rhIL—10抑制(P＜0．001)。(4)大鼠颈动脉损伤后,rhIL—10治疗组的动脉血管新生内膜/中层面积比低于对照组约45％(P＜0．01)。表明抗炎细胞因子rhIL—10可抑制AGE诱导的大鼠血管平滑肌细胞增殖和血管新生内膜的增殖。     

维生素B2在玻碳电极上的伏安行为及痕量检测

玻碳电极在活化后,在pH=5．6的柠檬酸-磷酸二氢钠缓冲体系中,在-0．8—0．1V范围内进行循环伏安扫描,可观察到明显的准可逆氧化还原峰,峰电流与扫速成正比。实验表明,VB2在玻碳电极表面有弱吸附现象,VB2浓度与峰电流在1．0×10^-5～2．0×10^-4moL／L之间线性关系较好,最低检测隈为5．0×10^-7mol／L。     

8-羟基喹琳和对苯二酚对几种常用植物材料根尖细胞有丝分裂中期指数的影响

为了了解8-羟基喹啉（8-Hydroxyquinoline）和对苯二酚（P-hydroquinone）对蚕豆、洋葱和大蒜几种实验室常用植物有丝分裂材料根尖细胞中期分裂指数的效应．应用不同浓度的8-羟基喹啉和对苯二酚（0.002mol／L、0.004mol／L、0.006mol／L、0.008mol／L）分别处理3种植物根尖细胞．结果表明,8-羟基喹啉在0.004mol／L时处理洋葱和大蒜根尖,中期分裂指数较高：对苯二酚在0．004mol／L时处理蚕豆根尖,中期分裂指数较高;而对苯二酚处理洋葱和大蒜根尖时最佳浓度是0.008mol／L．该浓度根尖细胞有丝分裂指数达到最大值。     

G蛋白,蛋白激酶C和Na^＋—H^＋交换在内皮素—1诱导培养心肌细胞肥大反应中的

内皮系-1（ET-1）是一种强的生长因子,并诱导心肌细胞肥大反应。在本实验中,我们探讨了G蛋白、蛋白激酶C（PKC）和Na＋－H＋交换在ET-1诱导的培养新生大鼠心肌细胞肥大反应中的作用。ET-1（10－10～10－7mol／L）促进3H－亮氨酸掺入,增加细胞蛋白质的含量和心肌细胞的表面积,且呈剂量依赖性,它们的EC50分别为5．2×10-10,5．2×10－10和7．3×10－10mol／L。用蛋白激酶C（PKC）抑制剂,Staurosporin（2nmol／L）预处理心肌细胞,可完全阻断ET-1诱导的心肌细胞的这些肥大反应,而蛋白激酶C激动剂,佛波酸酯（PMA）（10－8～10－6mol／L）呈剂量依赖性促进心肌细胞的肥大反应。用Na＋－H＋交换抑制剂,氨氯毗咪（10-4mol／L）预处理心肌细胞,可抑制ET－1诱导的心肌细胞肥大反应,但不影响PMA诱导的心肌细胞肥大反应。百日咳毒素（150ng／ml）预处理心肌细胞,可明显抑制ET－1诱导的心肌细胞肥大反应。这些结果提示,ET-1诱导的培养新生大鼠心肌细胞肥大反应是与百日咳毒素敏感的G蛋白相耦联,蛋白激酶C和Na＋．H＋交换可能在ET-1诱导的心肌细胞肥大反应中是重要的细胞内信使转导途径。     

厚叶景天组织传感器的研究

利用厚叶景天的叶和茎组织作为生物催化材料,分别同二氧化碳气敏电极和氨气敏电极组合,研制了L－精氨酸传感器及,L－赖氨酸传感器。两种传感器的线性范围分别为1.0×10^-4 1.O×10^-3mol/L和8．0×10^-5—3.0×10^-3mol/L．检测下限分别为3．2×10^-5mol／L和2.2×10^-5mol/L,响应斜率分别为42.2mV／dec和41．4mv／dec。考察了两种传感器的回收率．结果表明,L－精氨酸传感器和L－赖氨酸传感器的回收率平均值分别为98．6％和101.6％,标准偏差分别为4.6％和4.0％。     

CYP3A4基因原核表达质粒构建与诱导表达

以质粒pCWNF14为模板,采用PCR技术扩增出CYP3A4基因,并将其接入pET-22b（＋）、pET-28b（＋）和pET-32a（＋）载体中,经PCR测序鉴定后,转化Rosetta（DE3）2pLysS细菌,并用IPTG诱导表达．采用考马斯亮蓝染色的方法检测重组蛋白表达,3个表达重组质粒中,只有pET-32a（＋）-CYP3A4可以表达目的蛋白;在低浓度IPTG（50μmol／L）诱导6h,所表达的CYP3A4蛋白约占细菌总蛋白的40％。且该蛋白不溶于8mol／L的尿素,而溶于去污剂10mmol／L 3-（（3-Cholamidopropyl）dimethvlammonio propanesulfonic acid（CHAPS））和0．3％ lauroyl sarcosine（SKL）．成功地使CYP3A4基因在原核系统中高效表达．     

Production and purification of refolded recombinant human IL-7 from inclusion bodies

A recombinant form of human rhIL-7 was overexpressed in Escherichia coli HMS174 (DE3) pLysS under the control of a T7 promoter. The resulting insoluble inclusion bodies were separated from cellular debris by cross-flow filtration and solubilized by homogenization with 6 M guanidine HCl. Attempts at refolding rhIL-7 from solubilized inclusion bodies without prior purification of monomeric, denatured rhIL-7 were not successful. Denatured, monomeric rhIL-7 was therefore initially purified by size-exclusion chromatography using Prep-Grade Pharmacia Superdex 200. Correctly folded rhIL-7 monomer was generated by statically refolding the denatured protein at a final protein concentration of 80-100 microg/ml in 100 mM Tris, 2mM EDTA, 500 mM L-arginine, pH 9.0, buffer with 0.55 g/l oxidized glutathione at 2-8 degrees C for at least 48 h. The refolded rhIL-7 was subsequently purified by low-pressure liquid chromatography, using a combination of hydrophobic interaction, cation-exchange, and size-exclusion chromatography. The purified final product was >95% pure by SDS-PAGE stained with Coomassie brilliant blue, high-pressure size-exclusion chromatography (SEC-HPLC), and reverse-phase HPLC. The endotoxin level was <0.05 EU/mg. The final purified product was biologically active in a validated IL-7 dependent pre-B-cell bioassay. In anticipation of human clinical trials, this material is currently being evaluated for safety and efficacy in non-human primate toxicology studies.     

Reduction of the acetylcholine-induced K+ current in identifiedAplysia neurons by human interleukin-1 and interleukin-2

1. Effects of bath-applied recombinant human interleukin-1 (rhIL-1) and interleukin-2 (rhIL-2) on the acetylcholine (ACh)-induced K+ current recorded from identified neurons (R9 and R10) of Aplysia kurodai were investigated with voltage-clamp and pressure ejection techniques. 2. Bath-applied rhIL-1 and rhIL-2 (10-40 U/ml) reduced the ACh-induced current in the neurons without affecting the resting membrane conductance and holding current. 3. The suppressing effects of these cytokines on the current were completely reversible. 4. Heat-inactivated rhIL-1 and rhIL-2 were without effect. 5. These results suggest that the immunomodulators, IL-1 and IL-2, can modulate the ACh-induced response in the nervous system.     

IL-3 promotes basophilic differentiation of KU812 cells through high affinity binding sites

The myeloid precursor cell line KU812 exhibits a constitutive potential to differentiate into basophilic cells. In the present study, the influence of recombinant human (rh)IL-2, rhIL-3, and recombinant human granulocyte-macrophage-CSF on basophilic differentiation of KU812-F cells was studied. Of all cytokines tested, rhIL-3 induced a significant increase in formation of metachromatically granulated cells (from 10% in control cultures up to 30% in cultures supplemented with 100 U/ml of rhIL-3) as well as dose-dependent (1.5- to 3 fold) increase in cellular histamine in KU812-F cell cultures. In addition, KU812-F cells exposed to rhIL-3 bound more IgE antibody than cells cultured in control medium with up to 3.3-fold increases in the mean fluorescence intensity on days 2 and/or 5 compared with control (p less than 0.001). RhIL-3 failed to induce significant changes in expression of the Tac-reactive subunit of the IL-2R (CD25), surface aminopeptidase N (CD13), ICAM-1 Ag (CD54), or CD40 Ag on KU812-F cells. To investigate the mechanism of IL-3 action on KU812-F cells, receptor analyses were performed by using 125I-radiolabeled rhIL-3. Quantitative binding studies and Scatchard plot analyses revealed the presence of a single class of 1910 to 2460 high affinity IL-3-binding sites per KU812-F cell with an apparent dissociation constant of 1.22 to 2.35 x 10(-9) M. Together, these results show that rhIL-3 promotes basophilic differentiation of KU812-F cells through a specific receptor.     

Recombinant human IL-1 alpha and -1 beta potentiate IgE-mediated histamine release from human basophils

In this study, we have explored the relationship between interleukins and human basophil activation. Previous studies by ourselves and others have found that recombinant human (rh) IL-3 causes histamine release. The ability to release histamine has also been claimed for IL-1 but we cannot confirm this. In experiments with the basophils of 29 donors (excluding one D2O responder), histamine release with 100 ng/ml rhIL-1 alpha was 1.3 +/- 1% (SEM), whereas with rhIL-1 beta, it was 0.8 +/- 1%. Both IL-1 alpha and -1 beta were also used at concentrations of 0.01 to 1000 ng/ml without causing release. Neither increasing the Ca2+ concentration nor adding D2O or cytochalasin B caused IL-1 alpha and -1 beta to become secretagogues. rhIL-1, however, did augment IgE-dependent histamine release. The enhancement was similar with both rhIL-1 alpha and -1 beta, i.e. they were dose-dependent between 0.1 and 3 ng/ml and reached a plateau from 3 to 100 ng/ml. At submaximal histamine release (less than 10%), there was enhancement of three IgE-dependent secretagogues: 125% with goat anti-human IgE (n = 7), 215% with Ag E (n = 10), and 260% with a histamine releasing factor (n = 7). Non-IgE-dependent stimuli (formyl-methionine-leucine-phenylalanine and the ionophore A23187, n = 10) were enhanced less than 5%. rhIL-1-enhancement persisted after cell washing (n = 10). rhIL-1 was active in preparations of 50 to 75% pure basophils in which mononuclear cells were reduced by greater than 95% (n = 4), and mAbH34 to IL-1 beta blocked the enhancement caused by that molecule. We postulate that basophils have an IL-1 receptor which, when occupied, upregulates the response to IgE-related signals. Thus, this work characterizes a second interaction between interleukins and the cells central to the allergic response.     

Dose-effect of interleukin-10 and its immunoregulatory role in Crohn's disease

BACKGROUND: Interleukin-10 (IL-10) is currently being extensively studied in clinical trials for the treatment of Crohn's disease (CD). Only marginal effects have, however, been reported, and the dose-response curve was bell-shaped contrasting with the reported data from in vitro experiments. AIM: To use another in vitro model to analyze the effect of rhIL-10 and rhIL-4 on the spontaneous mucosal TNF-alpha secretion in patients with CD, and to characterize the phenotype of the cells targeted by rhIL-10. METHODS: Non-inflamed colon biopsies from CD patients were cultured for 16 hours in presence of different concentrations of rhIL-10 or rhIL-4. The numbers of TNF-alpha-secreting cells among isolated lamina propria mononuclear cells (LPMNC) were estimated by Elispot. RESULTS: Both rhIL-10 and rhIL-4 down-regulate TNF-alpha secretion by LPMNC from CD patients, with a more pronounced effect with rhIL-10. These effects were closely linked to the cytokine concentrations used, with a bell-shaped dose-response curve. Residual TNF-alpha secretion, in the presence of optimal rhIL-10 concentration was mainly attributable to CD3+ T cells. In contrast, at higher rhIL-10 concentrations, CD3- cells contributed significantly to the TNF-alpha secretion. CONCLUSIONS: The in vitro model we used, demonstrates that IL-4, but mostly IL-10, efficiently suppresses TNF-alpha secretion in LPMNC from CD patients, with a dose-response curve similar to results obtained in vivo. Resistance at high rhIL-10 concentrations was associated with a change in the phenotype of TNF-alpha-secreting cells.     

糖蛋白PAGE分离后的糖基显色法

鸡卵清蛋白通过SDS-PAGE分离后,用过碘酸-希夫(PAS)显色法可以使卵清蛋白显红色,而用糖苷酶去除卵清蛋白的糖基可以去除相应的红色。考马斯亮兰染色结果显示,去糖基后的卵清蛋白分子量略有减小。可见,PAS可方便地使糖蛋白呈现红色。     

Synthesis of recombinant human interleukin-2 via controlled feed of lactose-complex media in fed-batch cultures of Escherichia coli BL21(DE3)[pT7-G3IL2]

Using lactose as an inducer, recombinant human interleukin-2 (rhIL-2) was synthesized with an N-terminus fusion partner, G3 (three tandem-arranged glucagon peptides) in fed-batch cultures at high cell concentration (60–90 g l^–1) of Escherichia coli BL21(DE3) [pT7-G3IL2]. With batch additions of lactose (4 × 13.5 g), the fusion rhIL-2 was synthesized up to 9.3 g l^–1. However, if all the lactose (54 g) was added at once to the culture, synthesized fusion rhIL-2 decreased to 5.4 g l^–1 with a decreased cell growth rate. A statistical optimization of the production medium containing glucose, yeast extract, and lactose led to fusion rhIL-2 being produced at > 9 g l^–1.     

人白细胞介素11的定点聚乙二醇修饰

利用人白细胞介素11（hIL-11）无半胱氨酸（Cys）残基这一特点,通过定点突变将一个Cys残基引入hIL-11的N末端。然后,利用与Cys 巯基特异性反应的mPEG-马来酰亚胺将mPEG偶联到预先选定的位点,经层析纯化得到hIL-11的定点PEG修饰物。利用依赖型细胞株7TD1测定其生物学活性,结果表明,其体外生物学活性保持原有hIL-11活性的30%左右。定点聚乙二醇修饰方法为定向改造hIL-11,提高其药效的应用研究打下基础。     

Modulation of hematologic and immunologic effects of high dose chemotherapy by interleukin-2 in a murine tumor model.

To determine if intensive chemotherapy consisting of cyclophosphamide (C), etoposide (E), and cisplatin (P) (CEP) may be usefully combined with recombinant human interleukin-2 (rhIL-2), we examined a murine tumor model designed to approximate a common clinical situation: macroscopic, drug-resistant cancer. Using C57BL/6 mice with extensive tumor burden 10 days after intravenous B16 melanoma cell injection, we observed (1) C, E, and P synergize to enhance survival but do not cure mice at the highest tolerable dose (C = 200 mg/kg, E = 60 mg/kg, and P = 3 mg/kg); (2) rhIL-2 at 3 x 10(5) U (subcutaneously) daily for 4 days administered 10-18 days after B16 injection significantly improves survival; (3) CEP plus rhIL-2 is more effective than CEP alone only when rhIL-2 is administered before CEP; (4) CEP suppresses IL-2-induced lymphokine-activated killer cell activity in the spleen; and (5) rhIL-2 protects mice incompletely from the immunologic and hematologic suppression of CEP. Our results suggest that intensive chemotherapy combined with rhIL-2 may be beneficial. The success of any such combination may be schedule dependent.     

Heating of an ovalbumin solution at neutral pH and high temperature

The thermal denaturation, aggregation, and degradation of hen egg white ovalbumin dissolved in distilled and deionized water (60 mg/ml, pH 7.5) was investigated by differential scanning calorimetry (DSC), polyacrylamide gel electrophoresis (PAGE), and viscosity measurement. Two independent endothermic peaks were observed up to 180 degrees C by the DSC analysis. The first peak appeared at around 80 degrees C, corresponding to the denaturation temperature of ovalbumin. The second peak occurred around 140 degrees C due to the degradation of protein molecules as judged from the analysis by SDS-PAGE. The viscosity of the ovalbumin solution increased dramatically above 88 degrees C and maintained almost the same value up until heating to 140 degrees C. The increase in viscosity after heating to 88 degrees C was due to the denaturation and subsequent aggregation of ovalbumin molecules as observed by SDS-PAGE. The decrease in viscosity of the samples heated above 150 degrees C appears to have been the result of degradation of the ovalbumin molecules.