CT抗原KM-HN-1 HLA-A * 0201限制性表位预测及其与HLA-A * 0201结合强度分析

目的通过对CT抗原（cancer-testis antigen）KM-HN-1进行HLA-A＊0201限制性表位预测，并对候选表位肽与HLA-A＊0201分子结合亲和力及复合物稳定性进行分析，为探索基于KM-HN-1的免疫治疗奠定基础。方法利用基于蛋白酶体剪切位点特异性的算法PAProc及基于肽MHC-I结合的算法BIMAS和SYFPEITHI对KM-HN-1进行HLA-A＊0201限制性表位预测．合成KM-HN-1相关候选表位肽KM-HN-I321-329（KLLPFRETV），KM-HN-I303-211，（FLPTAPPNV），KM-HN-I629-637。（TLLQIIETV），KM-HN-I87-95（ILNKSIIEV），KM-HN-I538-596。（QMMEALDQL）及阳性对照肽HBVcAg18-27（FLPSDFFPSV）；对这些合成肽与HIA-A＊0201分子结合亲和力及其复合物稳定性根据文献报道的方法进行分析。结果KM-HN-I321-329（KLLPERETV）结合亲和力最低，KM-HN—I203-211（FLPTAPPNV）结合亲和力最高，其余3条肽结合亲和力介于2者之间；稳定性实验（DC50）结果显示：KM-HN-I538—546（QMMEALDQL）DC50小于2h，KM—HN-I321-329（KLLPERETV）的DC50介于2～4h之间，KM-HN-I87-95。（ILNKSIIEV）的DC50介于6～8h之间，KM-HN-I233-211（HLPTAPPNV）及KM-HN-I629—633（TLLQIIETV）的DC50均大于8h。结论基于蛋白酶体剪切位点特异性的算法及基于肽MHC-I结合的算法对KM-HN-1进行HLA-A＊0201限制性表位预测，结合候选表位肽与HLA-A＊0201分子结合的亲和力与复合物稳定性实验分析，为该抗原HLA-A＊0201限制性表位的鉴定奠定了基础。     

Characterization of Porcine Zona Pellucida Antigens by Immunoaffinity Chromatography and by High-Pressure Liquid Chromatography

ABSTRACT: In the present work, 500 and 50,000 porcine zonae pellucidae were solubilized using Lithium-3,5-diiodosalicylate. The zona antigens were purified by immunoaffinity chromatography (IAC) on immobilized antizona immunoglobulin G (IgG). The antizona-IgG was raised by immunization of female rabbits with 500 heat-solubilized porcine zonae. Four antigens could be detected following IAC: ZP I/1 (M_r = 42,000), ZP II/1 (M_r = 67,000), ZP II/2 (M_r = 32,000), ZP III/1 (M_r = 17,000). In a parallel experiment, 50,000 zonae were solubilized in a similar manner and the mixture was analyzed by high-pressure liquid chromatography (HPLC) using a protein column. Altogether, 9 protein peaks that contained the antigens ZP I/1, ZP II/1, ZP II/2, and ZP III/1 could be detected following HPLC. The carbohydrate composition is characteristic for O-glycosidic-glycoproteins. ZP II/1 and ZP II/2 are probably in close association within the zona. Based on the reaction of the antigens with antibodies induced by intact and heat-solubilized zonae, it is postulated that only ZP I/1 and ZP II/l are expressed on the surface in intact zonae.     

Multidimensional Chromatographic and Hyphenated Techniques for Hydrophilic Copolymers, 1

Summary: The chromatographic analysis of hydrophilic copolymers is complicated due to the fact that in most cases aqueous eluents must be used. In aqueous eluents different polar and ionic effects may disturb the selective interactions between the macromolecules and the stationary phase making it impossible to separate such copolymers with regard to chemical composition. Therefore, 2D chromatography combining a separation according to composition with a separation according to molar mass has been applied mostly to polymers that are soluble in organic solvents. The present contribution describes experimental approaches to analyze such hydrophilic copolymers by 2D‐chromatography. For a model polymer system resulting from the copolymerization of methacrylic acid and a poly(ethylene glycol) macromonomer, it is shown that different analytical techniques including SEC, LC‐CC, MALDI‐TOF MS and 2D chromatography can be used to analyze the different parameters of molecular heterogeneity of such copolymers.

2D separation of poly(MPEG‐MM 2), 1^st dimension: LC‐CC, 2^nd dimension: SEC.     

运用变性高效液相色谱对肺炎克雷伯菌产ESBL进行基因分型

目的 通过运用变性高效液相色谱（DHPLC）技术对前期研究已确认产超广谱β-内酰胺酶（ESBL）的肺炎克雷伯菌临床分离株TEM型质粒进行基因分型,试图建立一种方便快捷的用于ESBL分子诊断及其流行病学监测的新方法.方法 利用PCR技术从肺炎克雷伯菌临床分离株中扩增出TEM型质粒的编码序列,扩增产物运用DHPLC技术进行分析,分析提示,异常的样本通过测序确定其基因突变的类型,最后通过比对确定其基因型.结果 共分析了101例肺炎克雷伯菌临床分离株,全部样本均扩增出TEM型质粒的编码序列,经过DHPLC分析,52例（51.4%）样本表现为单一的洗脱峰,其形态与TEM-1标准菌株的峰型相一致,测序确定它们的碱基序列亦相一致,不存在变异,为TEM-1型;49例（48.6%）样本表现为异常的洗脱峰,它们均为双峰,形态一致,但异源双链峰的高度有差异,测序结果表明它们均存在四种相同的基因突变,在NCBI网站比对后确定为TEM-116;测序结果还提示,部分样本中TEM-1和TEM-116混合存在,其比例的不同表现为DHPLC时异源双链峰高度的差异;文献检索表明,本次确定的TEM-116为一新的基因亚型,为国内首次报道.结论 DHPLC具有简便快捷、高通量和自动化的特点,重复性好,不仅可对已知突变作出即时诊断,还可发现新的基因亚型,不失为一种较好的ESBL分子诊断方法及其流行病学监测手段.     

人初乳分泌片的纯化和兔抗人分泌片免疫血清的制备及应用

目的：获得足够量的分泌片（SC）及其相应的抗血清，用于研究SC的功能与SC在人和某些物种黏膜组织内的分布。方法：在已有的凝胶过滤和离子交换层析的基础上，进一步通过亲和层析和凝胶过滤分离纯化人的SC并进行相应的鉴定。用免疫组织化学技术检测了SC在部分小鼠组织中的表达。结果：获得了免疫纯的SC，并制备了相应的兔抗人SC的免疫血清；免疫组化显示，SC在小鼠小肠和子宫内膜处有表达。结论：利用改进的方法可以获得免疫纯的SC，进一步证实人和小鼠SC之间的交叉反应性。     

Renewal of the membrane complex of Schistosoma mansoni is closely associated with lipid metabolism

Metabolic pathways leading to lipid biosynthesis in four different developmental stages of Schistosoma mansoni were explored and quantified by incubation in the presence of labeled precursors in a chemically defined medium. At the schistosomulum stage and in male, female, or paired worms, glycerol and oleate incorporation into neutral lipids, mainly in the form of triacylglycerols, was greater than into phospholipids, whereas in 11-and 15-day-old worms, synthesis mainly led to phospholipids. Incorporation into phospholipids was recovered largely in phosphatidylcholine, and distribution into other phospholipids depended on the developmental stage. Incorporation of choline and ethanolamine into their respective phospholipids represented up to 15% of the parasitic phospholipid content. The formation of phosphatidylcholine by phosphatidylethanolamine methylation occurred mainly in the immature parasitic stages. Inositol incorporation was also measurable, whereas [14C]serine incorporation was low or undetectable. Addition of 1-palmitoyl-2-[14C]oleyl phosphatidylcholine revealed a very high uptake of this phospholipid by the immature stages but further metabolism was not detectable. In contrast, adult S. mansoni were completely unable to take up or absorb this exogenous phospholipid. The most striking aspect of this study was the relatively high metabolic activity in 11-day-old worms and the lower but sustained activity on day 15 and at the schistosomulum stage. By comparison, biosynthetic activity in adult S. mansoni, on which research studies have been focused until now, was very low. We also discuss the participation of lipid metabolism in the constant renewal of the membrane complex which is essential to parasitism by S. mansoni.     

巨噬细胞源性神经营养因子的初步分离

本文制备了巨噬细胞条件培养基（ＭφＣＭ），并应用快速自动比色微量分析法检测ＭφＣＭ对体外培养的生后７ｄＳＤ大鼠小脑皮质神经元的作用。结果表明，分子量大于１０ｋＤ的ＭφＣＭ对神经元（细胞密度１×１０６／ｍｌ）的作用，与对照组比较有明显的神经营养活性（Ｆ＜０．０５）；用盖玻片培养法表明，该组份有促进神经元突起生长的作用。ＭφＣＭ经ＳｅｐｈａｃｒｙｌＳ－１００－ＨＲ凝胶层析和生物活性鉴定，获得了具有神经营养活性的第二峰洗脱液。此洗脱液经ＳＤＳ－聚丙烯酰胺凝胶电泳分析，证明ＭφＣＭ中神经营养活性成份的蛋白质其分子量为３１ｋＤ－６８ｋＤ之间。     

Isolation of equine infectious anemia virus glycoproteins. Lectin affinity chromatography procedures for high avidity glycoproteins

Lectin affinity chromatography procedures were evaluated for the isolation of enveloped virus glycoproteins. The major glycoprotein of equine infectious anemia virus (E1AV) bound to concanavalin A (Con A)-Sepharose through interactions which could not be reversed by α-methylglucoside, but elution could be accomplished with buffers containing guanidine hydrochloride or sodium dodecyl sulfate. These denaturants, however, also released about one-half of the Con A protein from the Sepharose matrix. This degradation does not appear to have been recognized previously, as denaturants are frequently employed for the isolation of virus glycoproteins from Con A-Sepharose. In contrast, the virus glycoprotein bound equally well to Sepharose-bound Lens culinaris (lentil) lectin affinity columns and was effectively eluted with buffer containing 0.2 M α-methylglucoside. The lentil lectin-Sepharose procedure described is rapid, inexpensive and results in the efficient separation and recovery of EIAV glycoproteins. Thus, lentil lectin-Sepharose can provide a useful alternative to Con A-Sepharose for isolating other high avidity glycoproteins from viral envelopes or cell membranes.     

The affinity of anti-HBc antibodies in acute and chronic hepatitis B infection.

Antibodies to hepatitis B core antigen (anti-HBc) are found in the sera of all individuals infected with hepatitis B virus. A role for these antibodies has been suggested in determining the outcome of infection. In this study, the affinity of anti-HBc antibodies in asymptomatic virus carriers was compared with that of antibodies present in the sera of patients with chronic liver disease. Persistently infected individuals with no evidence of clinical disease were found to have anti-HBc antibodies of greater affinity, compared with the chronic liver disease group. Sera from patients with chronic hepatitis contained high levels of low-affinity antibody whereas antibody levels in asymptomatic carriers were significantly lower. These findings are discussed in relation to the predicted role of anti-HBc antibodies in mediating hepatitis B virus-related hepatocellular injury.     

Biochemical characterization of I-Ak proteins from mutant antigen-presenting B-cell--B-lymphoma hybridomas

Chemically induced mutants of an I-Ak,d expressing antigen-presenting B-cell--B-lymphoma hybridoma have recently been generated by immunoselection in vitro and were found to possess alterations in some of their serologically and functionally defined I-Ak region dependent functions. In order to identify at the structural level the origin of the differences in serological and functional properties of these mutants, I-Ak molecules from several of these mutant hybridomas were compared biochemically to wild-type I-Ak polypeptides by two-dimensional gel electrophoresis and high-pressure liquid chromatographic tryptic peptide analyses. Two-dimensional gel electrophoresis indicated that no major structural alterations, resulting in changes in mol. wt or charge, had occurred in the Ak alpha or Ak beta polypeptides from the mutant cells. Likewise, Ak alpha peptide maps of the mutants were indistinguishable from the normal Ak alpha peptide maps. However, two of the three mutants studied did exhibit one additional peptide in their Ak beta peptide maps. These results suggest that the major deficiencies in T-cell-activating functions of these mutants are a result of a limited alteration in the Ak beta polypeptide primary structure.