Identification, isolation, and characterization of a species-specific 30-kDa antigen of Oesophagostomum dentatum

In the search for a serology tool for the diagnosis of nonpatent as well as patent infections with Oesophagostomum dentatum in pigs a water-soluble, unglycosilated antigen of about 30 kDa specific for the third-stage larvae of the parasite was purified by ion-exchange chromatography. In Western blots, the antigen was first detected by antibodies at day 7 postinfection. Cross-reactivity with O. quadrispinulatum, Ascaris suum, or Trichuris suis was not detected. It is suggested that this protein is a suitable tool for the species-specific serodiagnosis of O. dentatum infection in pigs. Received: 15 June 1998 / Accepted: 28 September 1998     

FPLC系统测定人红细胞HbA_1c的方法

采用快速蛋白液相层析系统（ＦＰＬＣ）及ＭｏｎｏＳ柱，建立人血红细胞中糖化血红蛋白组份ＨａＡ１ｃ的测定方法。用本法重复测定３份样品，平均批内变异系数为２．５％，平均批间变异系数为４．８％；与常用的微柱法比较，两者呈正相关（ｒ＝０．４１３０，Ｐ＜０．０５，ｎ＝３６）。本法操作简单，重复性好，自动化程度高，具有广泛应用前景     

Purification and characterization of a phospholipase A2 isoenzyme isolated from Lachesis muta snake venom

A new phospholipase A2 (PLA2) isoenzyme was isolated from Lachesis muta crude venom, and was named LM-PLA2-II. This enzyme was purified by gel filtration on a Sephacryl S-200 HR column followed by reverse-phase chromatography on a C2/C18 column. LM-PLA2-II consists of a single polypeptide chain with an apparent molecular mass of 18 kDa and an isoelectric point at pH 5.4. The amino terminal sequence of the enzyme revealed a high degree of homology with other PLA2s from several sources. LM-PLA2-II has a high indirect hemolytic activity and a potent inhibitory effect on platelet aggregation induced by ADP and collagen. It also produces a significant paw edema reaction in rats. The edematous response in rats was abolished by pretreatment with either indomethacin or dexamethasone, suggesting the involvement of cyclo-oxygenase. Pretreatment of LM-PLA2-II with p-bromophenacyl bromide abolished all of these actions, clearly indicating that the biological activities, including the edematogenic effect, are dependent entirely on its enzymatic activity.     

Regulation of enterocyte apoptosis by acyl-CoA synthetase 5 splicing

BACKGROUND AND AIMS: The constant renewal of enterocytes along the crypt-villus axis (CVA) of human small intestine is due to cell-inherent changes resulting in the apoptotic cell death of senescent enterocytes. The aim of the present study was to examine underlying molecular mechanisms of the cell death at the villus tip. METHODS: Characterization of human acyl-coenzyme A (CoA) synthetase 5 (ACSL5) was performed by cloning, recombinant protein expression, biochemical approaches, and several functional and in situ analyses. RESULTS: Our data show that different amounts of acyl-CoA synthetase 5-full length (ACSL5-fl) and a so far unknown splice variant lacking exon 20 (ACSL5-Delta 20) are found in human enterocytes. In contrast with the splice variant ACSL5-Delta 20, recombinant and purified ACSL5-fl protein is active at a highly alkaline pH. Over expression of ACSL5-fl protein is associated with a decrease of the anti-apoptotic FLIP protein in a ceramide-dependent manner and an increased cell-surface expression of the death receptor TRAIL-R1. Expression analyses revealed that the ACSL5-fl/ACSL5-Delta 20 ratio increases along the CVA, thereby sensitizing ACSL5-fl-dominated cells at the villus tip to the death ligand TRAIL, which is corroborated by functional studies with human small intestinal mucosal samples and an immortalized human small intestinal cell line. CONCLUSIONS: Our results suggest an ACSL5-dependent regulatory mechanism that contributes to the cellular renewal along the CVA in human small intestine. Deregulation of the ACSL5-fl/ACSL5-Delta 20 homeostasis in the maturation and shedding of cells along the CVA might also be of relevance for the development of intestinal neoplasia.     

Systematic development and validation of sanitization protocols for a chromatographic system designed for biotherapeutics purification

Production of protein therapeutics through the application of genetic engineering and biotechnology techniques requires comprehensive attention to good manufacturing practice and good laboratory practice (GMP/GLP) guidelines for product recovery and purification. Validated clean-in-place procedures are part of the master method and require analysis of microbial bioburden to assess the efficacy of cleaning protocols. This article describes the extensive microbial challenge of a chromatography system, the use of membrane filtration methods for high sensitivity microbial contamination measurement, and the effectiveness of sodium hydroxide and ethanol solutions in achieving multilog reduction of microbial contamination.     

Application of Dirofilaria immitis immunoreactive proteins in serodiagnosis

Dirofilariasis is a zoonotic global vector‐borne disease caused by Dirofilaria immitis. The present study focuses on the somatic and excretory/secretory (E/S) proteins released from adult D. immitis. We aimed to fractionate and identify adult D. immitis immunoreactive proteins. Somatic and E/S extracts were immunoblotted to identify the immunoreactive proteins. In the current study, we used matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐TOF/MS) to characterize the immunogenic proteins. Additionally, we used fast protein liquid chromatography (FPLC) to fractionate and evaluate the immunogenicity of the D. immitis secretome. The most immunoreactive proteins were between 10 and 48 kDa. Six proteins including polyprotein antigen, P22u, pepsin inhibitor Dit33, neutrophil chemotactic factor (DiNCF) precursor, glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and heat‐shock protein 70 (HSP70) were found in both somatic and E/S extracts. Eluting the FPLC column with NaCl resolved two peaks in which the immunoreactivities of the purified proteins were conserved. Characterization of these proteins could provide a novel perspective for understanding the pathogenesis and diagnosing of this disease.     

Efficient oxidation of promutagenic hydroxymethylpyrenes by cDNA-expressed human alcohol dehydrogenase ADH2 and its inhibition by various agents

Alkylated polycyclic aromatic hydrocarbons can be metabolically activated via benzylic hydroxylation and sulphation to electrophilically reactive esters. However, we previously found that the predominant biotransformation route for the hepatocarcinogen 1-hydroxymethylpyrene (1-HMP) in the rat in vivo is the oxidation of the side chain by alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases to the carboxylic acid. Inhibition of this pathway by ethanol (competing ADH substrate) or 4-methylpyrazole (ADH inhibitor) led to a dramatic increase in the 1-HMP-induced DNA adduct formation in rat tissues in the preceding study. In order to elucidate the role of individual ADHs in the metabolism of alkylated polycyclic aromatic hydrocarbons, we expressed the various members of the human ADH family in bacteria. Cytosolic preparations from bacteria expressing ADH2 clearly oxidized hydroxymethylpyrene isomers (1-, 2- and 4-HMP) with the highest rate. This form was purified to near homogeneity to perform detailed kinetic analyses. High catalytic efficiencies (V(max)/K(m)) were observed with HMPs. Thus, this value was 10,000-fold higher for 2-HMP than for the reference substrate, ethanol. The corresponding aldehydes were also efficiently reduced by ADH2. 4-Methylpyrazole inhibited the oxidation of the HMP isomers as well as the reverse reaction. Daidzein, cimetidine and the competing substrate ethanol were further compounds that inhibited the ADH2-mediated oxidative detoxification of 1-HMP.     

贲门癌术前短期化疗与远期生存的探讨

目的：探讨贲门癌术前短期化疗与远期生存的关系。方法：贲门腺癌患者６０例，随机分成用药组与对照组，每组３０人。用药组术前分次服用氟脲嘧啶多相脂质体，总剂量为５００ｍｌ。对两组手术切除标本进行大体和病理组织学检查，以及流式细胞荧光ＤＮＡ凋亡细胞率的检测。结果：与对照组比较，用药组肿瘤体积缩小。癌细胞发生显著的退行性变和坏死。ＤＮＡ凋亡细胞率明显高于对照组（Ｐ〈０．００１）。５年生存率，用药组为４０．０     

幽门螺杆菌中性白细胞激活蛋白与麦芽糖结合蛋白融合表达产物rMBP-NAP的分离纯化

目的：从E．coli TB1(pMAL-c2x-napA)细菌中分离纯化与麦芽糖结合蛋白融合表达的重组幽门螺杆菌中性白细胞激活蛋白rMBP-NAP,筛选幽门螺杆菌口服疫苗抗原组分。方法：0．3mmol／L IPTG在37℃,230r／min条件下诱导基因丁程菌E．coli TB1(pMAL-c2x-napA)3h。4℃ 4000g离心20min收获细胞。过柱缓冲液重悬细胞,一20℃冻仔过夜,在冰水浴中超卢破碎工程菌,4℃,9000g离心30min,十二烷基磺酸钠-聚炳烯酰胺凝胶电泳(SDS-PAGE)分析工程菌中rMBP-NAP可溶性;低相对分子质量蛋白Marker做标准对照,GeneTool测定诱导蛋白相对分子质量。FPLC分子排阻层析法分离rMBP-NAP;SDS-PAGE凝胶扫描法测纯化蛋白质的纯度,Bradford法检测蛋白质含量。结果：E．coli TB1(pMAL-c2x-napA)诱导表达的rMBP-NAP主要以可溶性形式存在于超声上清中,占上清中蛋白总量的39．35％,相对分子质量约为57000,与预期结果一致;分子大小排阻层析一步法纯化rMBP-NAP纯度可达91％,含量可达0.121g/L。结论：FPLC分子排阻层析可从大肠丁程菌超声菌液巾快速纯化rMBP-NAP。