麒麟菜硫酸酯多糖体外抗病毒作用的研究

目的 采用体外细胞培养法，研究琼枝和异枝麒麟菜硫酸酯多糖对HSV-1和CVB3的抗病毒活性．并初步探讨其作用机制。方法采用MTT比色法观察琼枝和异枝麒麟菜硫酸酯多糖对Vero细胞的细胞毒性，用细胞病变（CPE）抑制法观察其对由HSV-1和CVB。引起CPE的抑制作用；采用分组实验初步探讨了多糖样品的抗病毒作用机制。结果和结论琼枝和异枝麒麟菜硫酸酯多糖能明显抑制由上述两种病毒引起的CPE，对Vero细胞的毒性很低，异枝粗多糖和异枝多糖A2组分（ESPA和ESPA2）对HSV-1的抗病毒效果均优于阿昔洛韦（ACV），本实验采用的全部多糖样品对CVB。的抗病毒作用也都优于病毒唑，而异枝麒麟菜硫酸酯多糖抗CVB3效果更为显著。从分组实验结果可初步推知，麒麟菜硫酸酯多糖样品不仅具有直接杀灭病毒作用，而且还可能通过进入细胞内部或吸附在细胞表面，发挥其抑制或杀伤病毒的作用。     

酸处理的表皮生长因子对小鼠胎肝细胞BNL CL.2生物活性的影响

目的 探讨酸抑制剂对表皮生长因子（ＥＧＦ）活性的保护机理。方法用含有胃蛋白酶的盐酸培养重组人ＥＧＦ，用高压液相分析酸和胃蛋白酶中ＥＧＦ的稳定性，用３Ｈ－ＴｄＲ法测定酸降解的ＥＧＦ对胎肝细胞的体外生物活性的影响，用ＦＡＣＳ检测小鼠胎肝细胞经酸降解的ＥＧＦ诱导４８ｈ后ＥＧＦ的变化。结果所有含胃蛋白酶的标本中，当ｐＨ＜４．０时，ＥＧＦ被降解成为分子量较小的形式。经盐酸降解的该因子受体（ＥＧＦ）对小鼠胎肝细胞的增殖效应明显低于未用酸降解的ＥＧＦ处理组，未用酸降解的ＥＧＦ处理的小鼠胎肝细胞的ＥＧＦＲ表达也明显高于酸降解的ＥＧＦ处理组。结论 在ｐＨ＜４．０时，经酸处理的ＥＧＦ稳定性较差，能被迅速降解成分子量较小的形式，其活性也大大减弱，对小鼠胎肝细胞的促增殖作用明显低于未经酸降解的ＥＧＦ组。用抑酸药调节胃液中适当的ｐＨ值，对维持ＥＧＦ的稳定性，促进对胃粘膜的保护有一定作用。     

麒麟菜提取物对HepG2、H22肝癌细胞的抗癌作用及机制研究

目的从麒麟菜中提取天然海藻色素糖蛋白(NSPG),并观察其抗肿瘤作用。方法应用溶剂浸提法从麒麟菜中提取NSPG,应用IR、UV、液相-质谱法、凝胶色谱法测定NSPG样品的分子量及结构特征等。采用MTT比色法测定NSPG作用后HepG2人肝癌细胞、小鼠H22肝癌细胞的体外活性。结果本研究建立了常温下从麒麟菜中提取NSPG的方法,并测定其理化性质。人HepG2肝癌细胞体外实验结果显示,50、100 mg/L NSPG组培养48 h的细胞增殖活性分别为0.62±0.02、0.54±0.01,低于空白对照组(0.87±0.01),差异有统计学意义(P<0.05);小鼠H22肝癌细胞体外实验结果显示,NSPG对肿瘤细胞的增殖活性随NSPG浓度的升高而逐渐降低,抑制率逐渐升高,低、中剂量组的增殖活性、抑制率与高剂量组比较差异有统计学意义(P<0.05)。结论 NSPG对HepG2人肝癌细胞、小鼠H22肝癌细胞表现出不同程度的肿瘤抑制作用。     

短尾蝮蛇毒中磷脂结合抗凝蛋白的分离纯化及鉴定

目的从短尾蝮蛇毒中分离纯化一种抗凝蛋白,并对其生化性质进行研究。方法利用阳、阴离子交换、凝胶过滤的方法分离纯化这种抗凝蛋白,用活化部分凝血活酶时间(APTT)测定其抗凝活性,用SDS-PAGE测定其蛋白相对分子量,用等电聚焦法测定蛋白的等电点.用薄层析方法确定抗凝蛋白与磷脂酰胆碱结合。结果从短尾蝮蛇中分离纯化出的抗凝蛋白是二聚体,相对分子量为24.0×103(非还原)和14.6×103(还原)。等电点为pH 5.2。该蛋白具有精氨酸酯酶活性,能明显地延长活化的部分凝血活酶时间(APTT),其抗凝活性与磷脂结合有关。结论此方法成功地从短尾蝮蛇毒中纯化出一种抗凝蛋白。因其能够与磷脂结合,又具有明显的抗凝活性,因此把该蛋白称为磷脂结合抗凝蛋白(phospholip id-b ind ing anticoagu lation prote in,PBAP)。     

松罗酸的提取和抗癌活性研究

由长松萝中分离得以萝酸粗品、精品并制成钠盐。３种试品的Ｂｒｉｎｅｓｈｒｉｍｐ生物活性测定结果ＬＣ５０均小于５０，松萝酸钠活性最高。松萝酸粗品（５０－１２０ｍｇ／ｋｇ）对小鼠Ｓ８０肉瘤的抑制率大于６５％，表明松萝酸具有较强的抗癌活性。     

蝮蛇蛇毒类凝血酶的研究

为研制蝮蛇抗栓酶的换代产品，开发单一组分的类凝血酶制剂。采用离子交换和亲和层析等现代纯化方法，从东北白眉蝮蛇蛇毒中分离出了单一组分的类凝血酶。半成品经高效液相色谱凝胶柱测定，纯度大于90％，最高可达97.94％，分子量38200D，SDS－PAGE电泳测定为一条带，分子量为42100D，活性为1137BU／mg，比活性为1705BU／mg·pro，不含出血毒和神经毒，冻干成品制剂全部符合注射用品标准。     

猪脾脏中的应激免疫抑制蛋白类似物(英文)

目的:研究猪脾脏中的一种具有免疫抑制活性的蛋白质,纯化该蛋白并研究其与临床疾病的关系。方法:1)稀酸提取;2)超滤透析分离分子量大于M_r30 000的分子;3)FPLC免疫亲和层析纯化;4)T淋巴细胞转化及ELISA检测其功能活性;5)SDS-PAGE检测其分子量及纯度。结果:该蛋白能抑制由Con A诱导的淋巴细胞增殖。其分子量在190 000 左右。该蛋白对某些来源于免疫细胞系的细胞,具有明显的选择性抑制作用。结论:成功部分纯化这种蛋白,并证明其与应激免疫抑制蛋白有相似的理化、免疫原性和功能活性。这一研究为进一步大量纯化该蛋白提供了实用和稳定的生物学来源,并为研究它与肿瘤、自身免疫性等临床疾病关系开辟了一条新的研究途径。     

感觉神经33.1kDa特异蛋白二级结构及其神经营养活性探讨

目的纯化大鼠脊神经感觉纤维33．1kDa特异蛋白(SSP-33．1)，并分析其二级结构及其神经营养活性。方法采用双向电泳技术比较了大鼠脊髓背根神经纤维和腹根神经纤维中蛋白质表达差异，用DEAE-Sephacel阴离子交换层析和高效液相凝胶过滤的方法纯化了其中一种蛋白质，并通过圆二色谱初步分析了该蛋白质的二级结构；又通过经典的神经营养活性的模型-鸡胚背根节(DRG)体外培养模型，测定该蛋白的生物学活性。结果纯化的蛋白质分子量为33．1，等电点为5．52，其二级结构中α螺旋占20．8％，β片层占54．8％，转角占7．3％，无规卷曲占17．1％。体外实验表明该蛋白质能促进鸡胚背根神经节突起的生长。结论纯化了大鼠感觉神经33．1kDa特异蛋白，该蛋白质二级结构以β片层为主，体外实验表明该蛋白质对体外培养的鸡胚背根神经节有神经营养活性。     

人胎盘抗凝蛋白的分离纯化及鉴定

目的　从人胎盘中分离纯化一种胎盘抗凝蛋白 (theanticoagulationprotein ,PAP) ,并对其生化性质进行研究。方法　用饱和硫酸铵分步盐析、阴离子交换、凝胶过滤和亲和层析方法分离纯化人胎盘抗凝蛋白 ,用活化部分凝血活酶时间 (APTT)测定其抗凝活性 ,用SDS 聚丙烯酰胺凝胶电泳(SDS PAGE)测定其蛋白分子量 ,用等电聚焦法测定蛋白的等电点。结果　从人胎盘中分离纯化出相对分子质量约为3 4 9× 1 0 4,等电点约为pH4 9的单聚体抗凝蛋白。该蛋白能延长APTT时间 ,且延长的时间与剂量成线性关系 ,得率占胎盘总重量的 0 0 1‰。结论　此方法成功地从人胎盘中纯化出一种抗凝蛋白。该蛋白的性质与膜联蛋白Ⅴ比较相似 ,可能是属于膜联蛋白家族     

人粒细胞集落刺激因子基因重组及在E.coli中的表达和鉴定

将RT－PCR扩增的人粒细胞集落刺激因子（hG－CSF）结构基因与表达载体pJGW1重组，转化含有pGP1－2质粒的E.coliDH5α，进行温度诱导表达。经SDS－PAGE分析，rhG－CSF表达量占菌体总蛋白量的30％以上。将其复性，经CM－52FF离子交换层析纯化，纯化后的rhG－CSF（纯度＞98％）经SDS－PAGE测定分子量约为19kD；经胰酶裂解、反相HPLC分析肽谱具有8条特异肽段；等电聚焦测定PI值约为5．8；免疫印迹实验证实其与标准hG－CSF具有相同的抗原反应特异性；NH2-末端氨基酸分析与文献报道一致，采用小鼠白血病细胞株NFS－60测定活性为I×108IU／mg。     

Isolation and characterization of β-galactoside specific lectin from Korean mistletoe (Viscum album var.coloratum) with lactose-BSA-Sepharose 4B and changes of lectin conformation

Lectins and its A- and B-chains from Korean mistletoe (Viscum album var.coloratum) were isolated by affinity chromatography on the Sepharose 4B modified by lactose-BSA conjugate synthesized by reductive amination of ligand (lactose) to ε-amino groups of lysine residues of spacer (BSA) after reduction by NaCNBH₃. The lactose-BSA conjugate was coupled to Sepharose 4B activated by cyanogen bromide. The molecular weight determined by SDS-PAGE were a 31 kD of A-chain and a 35kD of B-chain. Amino acid analysis andN-terminal sequencing were performed. The effects of pH, temperature and guanidine chloride on the conformation of the lectin were investigated by measuring its intrinsic fluorescence and compared with its hemagglutinating activities. Blue shift was detected on the acidic pH and there was a close relationship between activities and conformation of the lectin. Under denaturing conditions, the tryptophan emission profile of lectin showed typical denaturational red shift which also correspond to the conformations and activity of lectin.     

Isolation and characterization of lectins from stem and leaves of Korean mistletoe (Viscum album var.coloratum) by affinity chromatography

We attempted to isolate and characterize the lectins from stem and leaves of Korean mistletoe (Viscum album var.coloratum) by affinity chromatography. Lectin I was isolated only from stem. Lectin II was not isolated from Korean mistletoe, whereas lectin III was isolated from the stem and leaves. The hemagglutinating activity of lectin I was 16HU and inhibited by D-galactose, lactose, and N-acetyl-D-galactosamine. The lectin I has molecular weight of 60,000D being composed of two basic subunits with molecular weights of 32,000 D and 28,000 D which are linked by a disufide bond. The lectin III from stem has molecular weight of 66,000D being two basic subunits which have molecular weights of 34,000D and 29,000D and are linked by a disufide bond. The activity of lectin I was stable at the pH range of 4.00∼8.50, and at a wide range of temperature (0∼42°C). The lectin I showed more potent mitogenic activity to murine lymphocytes than concanavalin A.     

Physico-chemical properties of the lectin from winged bean (Psophocarpus tetragonolobus (L.) DC) tubers

A galactose specific lectin from the tubers of winged bean, Psophocarpus tetragonolobus (L.) DC, has been isolated and purified to homogeneity by chromatography on DEAE-cellulose, Biogel P-60, and CM-Sephadex C-50 columns. It is a glycoprotein containing 6% total sugar, comprising mannose, fucose, and xylose in a ratio of 7:2:1, and 0.85% amino sugars. The M_r of the lectin by SDS-PAGE and analytical ultracentrifugation is 29000. In contrast, gel filtration gave an M_r of ca. 48 000. The lectin has a pI value of ～ 9.5 and a sedimentation coefficient value of 3.0. The purified lectin agglutinates both trypsinized and untrypsinized erythrocytes of human (types A, B, and AB), rabbit, and rat erythrocytes but not human (type O) and sheep erythrocytes. The hemagglutinating activity of the lectin is inhibited by D(+) galactose and other galactose containing oligosaccharides, and its derivatives. The sugar specificity of the lectin appears to be directed to glycosides containing non-reducing α-d -galactopyranosyl residues; v-d -galactosides are poor inhibitors. The amino acid composition of the lectin has revealed that it is rich in aspartic acid and poor in sulphur-containing amino acids. Lysine alone appears to be the NH₂-terminal residue of the lectin. The purified lectin is fairly stable over a wide range of pH and temperature. The lectin is found to be mitogenic by transforming normal human lymphocytes at a concentration of 0.1 mg/mL.     

Spectrin degradation in intact red blood cells by phenylhydrazine

The effects of phenylhydrazine on intact red cells and on red cell ghost membrane proteins were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In intact red cells 1 mM phenylhydrazine induced a marked decrease in intensity of the alpha- and beta-bands of spectrin without the formation of high molecular weight materials. Phenylhydrazine was also responsible for cross-linking of hemoglobin, which is apparent by the appearance of two new broad bands on the gel. Membrane glycoproteins were unaffected. Electrophoretic patterns of cytoskeletal proteins from phenylhydrazine-treated red cells obtained on two-dimensional SDS-polyacrylamide gels and stained with Coomassie blue or fluorescently labeled with monobromobimane indicated the presence of a new band between bands 4.2 and 5 at 60-65 kilodaltons (K). An immunoelectrophoretic blotting procedure utilizing polyclonal IgG antibodies for alpha- and beta-spectrin of the red cell cytoskeletal proteins revealed that the band observed at 60-65 K in the two-dimensional SDS-PAGE studies reacted with the antibodies. The presence or absence of glucose in the incubation medium and modification of oxyhemoglobin to met- or carboxyhemoglobin in the red cells did not protect the phenylhydrazine-mediated degradation of the major cytoskeletal proteins. Metal chelators and antioxidants had no effect on membrane protein changes. Ghost red cell proteins did not undergo changes at 1 mM phenylhydrazine in the presence or absence of hemoglobin, although at 5 mM phenylhydrazine the appearance of a faint high molecular weight band was observed. These results indicate that spectrin degradation without significant polymerization can be induced by phenylhydrazine.     

Identification of human lung mast cell kininogenase as tryptase and relevance of tryptase kininogenase activity

We have described previously the IgE-mediated release of kininogenase activity from purified human lung mast cells. Using supernatant fractions from mast cells stimulated with anti-IgE in the presence of deuterium oxide, we have purified this kininogenase to homogeneity by gel filtration and heparin-agarose chromatography and have demonstrated that it is identical to tryptase, the major neutral protease of human lung mast cells. Thus, tryptase and kininogenase activities co-chromatographed through both purification steps with equivalent yields. The final purified kininogenase was free of detectable chymotryptic and carboxypeptidase activities and was identified as tryptase on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), amino acid composition and inhibition profile. Three such preparations of tryptase were all capable of releasing kinin from each of two different preparations of purified, single-chain, human low molecular weight kininogen. Interestingly, kinin generation was optimal at pH 5.5 and was enhanced by heparin, which has been reported to stabilize tryptase. SDS-PAGE analysis of kininogen hydrolysis by tryptase revealed the formation of a diffusely stained region in the molecular weight range of 60,000-65,000, rather than a discrete heavy chain band. Under optimal conditions, the three tryptase preparations released 10-12 micrograms kinin/hr/mg but released only 2 micrograms kinin/hr/mg at pH 7.2. HPLC analysis revealed that the kinin released was bradykinin. We conclude that the kininogenase activity from human lung mast cells is attributable to tryptase. The unique pH optimum of this reaction of a serine protease, however, raises doubts as to the physiologic significance of this activity.     

Bioactive proteins from marine sponges: Screening of sponge extracts for hemagglutinating, hemolytic, ichthyotoxic and lethal properties and isolation and characterization of hemagglutinins

D. Mebs, I. Weiler and H. F. Heinke. Bioactive proteins from marine sponges: screening of sponge extracts for hemagglutinating, hemolytic, ichthyotoxic and lethal properties and isolation and characterization of hemagglutinins. Toxicon23, 955–962, 1985. — Aqueous extracts of 48 sponge species from the Red Sea, the Australian Barrier Reef and the Florida Keys were screened for hemagglutinating, hemolytic, ichthyotoxic and lethal activities. Forty two per cent of the sponge species exhibited agglutinating properties to human erythrocytes of ABO groups. From four species (Haliclona sp., Cinachyra tenuifolia, Callyspongia viridis, Terpios zetekl) the hemagglutinating factors were isolated by gel filtration and affinity chromatography. A molecular weight of 24,000 was determined for the pure hemagglutinin from Haliclona sp. by SDS electrophoresis and of 22,000 for the semipure hemagglutinin from Cinachyrat tenuifolia by gel filtration. These hemagglutinins were inhibited by d-lactose, but not by d-melibiose or other oligosaccharides, indicating that they may react with terminal d-galactose β1 → 4 residues. The other semipure hemagglutinins were not inhibited by various sugars tested. Hemolytic activity to human erythrocytes was present in about 15% of the sponge extracts, showing a close relationship to ichthyotoxic activity. More than half of the sponge extracts caused toxic symptoms in mice when injected i.p. Using various concentrations death occurred within 12–48 hr. The lethal factors seem to be related to components of low molecular weight in the sponge extracts.     

N-terminal amino acid sequences and some characteristics of fibrinolytic/hemorrhagic metalloproteinases purified from Bothrops jararaca venom.

We determined the N-terminal amino acid sequences of the fibrinolytic/hemorrhagic metalloproteinases (jararafibrases I, III and IV) purified from Bothrops jararaca venom. The N-terminal amino acid sequences of jararafibrase I and its degradation products were identical to those of jararhagin, another hemorrhagic metalloproteinase purified from the same snake venom. Together with enzymatic and immunological properties, we concluded that those two enzymes are identical. The N-terminal amino acid sequence of jararafibrase III was quite similar to C-type lectin isolated from Crotalus atrox, and the protein had a hemagglutinating activity on intact rat red blood cells.     

Chemical and biological characterization of the galactose binding lectins from Trichosanthes kirilowii root tubers

Three galactose binding isolectins have been isolated from Trichosanthes kirilowii root tubers. Two of the isolectins, TK-I and TK-II, are similar in many aspects including molecular weight, amino acid composition, NH₂-terminal amino acid residue, blood group and carbohydrate specificities, immunodiffusion and immunoelectrophoretic behavior, hemagglutinating and insulinomimetic activities, and in possessing subunits with different molecular weights. Compared to TK-I and TK-II, lectin TK-III has a larger molecular weight, subunits with the same molecular weight, a single and distinctive NH₂-terminal amino acid residue, a different isoelectric point and lower hemagglutinating activity. The three lectins share common antigenic determinants in their structures. β-Linked terminal oligosaccharides containing D-galactose inhibit hemagglutination induced by the lectins with a higher potency than α-linked oligosaccharides. The lectins are non-mitogenic and did not inhibit the concanavalin-A induced mitogenic response of lymphocytes.