Characterization of an enterobacterial common antigen (ECA) epitope recognized by anti-ECA-tetanus toxoid conjugate serum

The antigenic reactivity of both native and chemically modified enterobacterial common antigen (ECA) with anti-ECA-tetanus toxoid (TT) conjugate serum was investigated. The results obtained suggest that reduction of the carboxyl group of the mannosaminuronic acid component of ECA diminishes, but does not destroy its antigenic reactivity. Each of the sugar components were found to contribute to its reactivity with anti-ECA-TT conjugate serum, indicating that the trisaccharide repeating unit represents the ECA epitope. A nonasaccharide (trimer of the ECA repeating unit) inhibited antibody binding better than the hexasaccharide dimer, a finding which suggests that oligosaccharide conformation also makes a contribution to its inhibitory activity.     

Detection of enterobacterial common antigen on bacterial cell surfaces by colony-immunoblotting: effect of its linkage to lipopolysaccharide

Abstract A colony-immunoblotting procedure is described which allows a quick screening of high numbers of bacteria for their Enterobacterial Common Antigen phenotype. In this assay the intensity of reaction is dependent on the carrier to which ECA is linked and which anchors ECA in the outer membrane. Bacteria containing LPS-linked ECA react stronger in this and in other immunoassays than bacteria containing only the phospholipid-linked ECA.     

肠杆菌共同抗原的研究进展

肠杆菌共同抗原(Enterobacterial common antigen,ECA)是由多糖重复单元组成的多聚糖,几乎表达于所有肠杆菌细菌外膜,具有生物学功能。ECA由多基因协同作用而合成,这些基因在肠杆菌细菌基因组上成簇存在,形成ECA抗原基因簇。ECA是重要的毒力因子,在肠杆菌细菌入侵宿主、体内存活等过程中有一定作用。同时,ECA在维持细菌外膜渗透屏障、鞭毛表达、群集运动及抗胆酸胆盐等方面也有重要作用。此外,锚定在细菌脂多糖核心区的ECALPS还是细菌重要的表面抗原,能激发宿主产生高水平抗体,可以作为疫苗研究的靶点。结合笔者的研究,文中对ECA纯化、基因结构和合成、免疫特性、生物学功能及应用等方面进行了综述。     

The relationships between brain, serum, and whole blood ChE activity in the wood mouse (Apodemus sylvaticus) and the common shrew (Sorex araneus) after acute sublethal exposure to dimethoate

Inhibition of cholinesterase (ChE) activity produced by a single acute intraperitoneal administration of dimethoate was studied in the wood mouse, Apodemus sylvaticus, and the common shrew, Sorex araneus, under laboratory conditions. ChE values from serum and whole blood were compared with those obtained from brain in order to obtain a non-destructive tool for predicting the severity of brain acetylcholinesterase (AChE) inhibition. In addition, serum and brain inhibition following oral exposure to dimethoate was also measured in the wood mouse. Normal ChE activity was higher in the brain and whole blood of the shrews than in wood mice. There was no difference between species in serum ChE activity. Exposure to dimethoate caused a dose-dependent reduction in ChE activity and there was a significant recovery in activity with increasing time after administration. In both species, serum and whole blood were more sensitive than brain for revealing organophosphate-induced ChE inhibition and serum was more sensitive than whole blood. Statistically significant relationships were defined between whole blood and brain ChE activity and between serum and brain ChE activity. Compared with serum, whole blood ChE activity was the more accurate predictor of brain AChE levels. The relationships between brain and serum ChE activity did not appear to be affected by the route of administration of the pesticide.     

The relationships between brain,serum, and whole blood ChE activity in the wood mouse (Apodemus sylvaticus) and the common shrew (Sorex araneus) after acute sublethal exposure to dimethoate

Abstract

Inhibition of cholinesterase (ChE) activity produced by a single acute intraperitoneal administration of dimethoate was studied in the wood mouse, Apodemus sylvaticus, and the common shrew, Sorex araneus, under laboratory conditions. ChE values from serum and whole blood were compared with those obtained from brain in order to obtain a non-destructive tool for predicting the severity of brain acetylcholinesterase (AChE) inhibition. In addition, serum and brain inhibition following oral exposure to dimethoate was also measured in the wood mouse. Normal ChE activity was higher in the brain and whole blood of the shrews than in wood mice. There was no difference between species in serum ChE activity. Exposure to dimethoate caused a dose-dependent reduction in ChE activity and there was a significant recovery in activity with increasing time after administration. In both species, serum and whole blood were more sensitive than brain for revealing organophosphate-induced ChE inhibition and serum was more sensitive than whole blood. Statistically significant relationships were defined between whole blood and brain ChE activity and between serum and brain ChE activity. Compared with serum, whole blood ChE activity was the more accurate predictor of brain AChE levels. The relationships between brain and serum ChE activity did not appear to be affected by the route of administration of the pesticide.     

Humoral response to selected antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis in the course of yersiniosis in humans. I. Occurrence of antibodies to enterobacterial common antigen (ECA)

The antibodies against the Enterobacterial Common Antigen (ECA) were detected using the ELISA in 293 serum samples collected from 185 persons suspected for yersiniosis, as well as 115 serum samples from healthy individuals (blood donors). The presence of IgA antibody in diagnostically significant titres for ECA were detected by ELISA in 3.5%, IgG in 13.0%, and IgM in 5.2% of blood donors. Statistical analysis showed that the frequency of detecting antibodies for ECA among the patients with yersiniosis was significantly higher (p < 0.05) in relation to the blood donors. Most frequently the elevated antibody levels were detected among patients with reactive arthritis (IgA 29.2%, IgG 35.4%, IgM 16.7%) while the most infrequent among patients with abdominal pain in acute phase of yersiniosis (IgA 14.9%, IgG 25.3%, IgM 19.5%). The level of antibodies for ECA, together with age increased reaching its peak, on the average, among individuals aged 41 - 60 years. In majority of the individuals studied antibodies of the IgG class reached the level much higher in relation to those of the IgA and IgM classes. The obtained results showed that the detection of antibodies to ECA may be useful in serodiagnosis of Yersinia infections.     

Characterization of the lipid-carrier involved in the synthesis of enterobacterial common antigen (ECA) and identification of a novel phosphoglyceride in a mutant of Salmonella typhimurium defective in ECA synthesis

The polysaccharide chains of enterobacterial common antigen (ECA) consist of linear trisaccharide repeat units with the structure -->3)- alpha-d-Fuc4NAc-(1-->4)-beta-d-ManNAcA-(1--> 4)-alpha-d-GlcNAc-(1-->, where Fuc4NAc is 4-acetamido-4, 6-dideoxy-d-galactose, ManNAcA is N - acetyl-d- mannosaminuronic acid, and GlcNAc is N -acetyl-d-glucosamine. The major form of ECA (ECAPG) consists of polysaccharide chains that are believed to be covalently linked to diacylglycerol through phosphodiester linkage; the phospholipid moiety functions to anchor molecules in the outer membrane. The ECA trisaccharide repeat unit is assembled as a polyisoprenyl-linked intermediate which has been tentatively identified as Fuc4NAc-ManNAcA-GlcNAc- pyrophosphorylundecaprenol (lipid III). Subsequent chain-elongation presumably occurs by a block-polymerization mechanism. However, the identity of the polyisoprenoid carrier-lipid has not been established. Accordingly, the current studies were conducted in an effort to structurally characterize the polyisoprenyl lipid-carrier involved in ECA synthesis. Isolation and characterization of the lipid carrier was facilitated by the accumulation of a ManNAcA-GlcNAc- pyrophosphorylpolyisoprenyl lipid (lipid II) in mutants of Salmonella typhimurium defective in the synthesis of TDP-Fuc4NAc, the donor of Fuc4NAc residues for ECA synthesis. Analyses of lipid II preparations by fast atom bombardment tandem mass spectroscopy (FAB-MS/MS) resulted in the identification of the lipid-carrier as the 55-carbon polyisoprenyl alcohol, undecaprenol. These analyses also resulted in the identification of a novel glycolipid which copurified with lipid II. FAB-MS/MS analyses of this glycolipid revealed its structure to be 1,2-diacyl- sn -glycero-3-pryophosphoryl-GlcNAc-ManNAcA (DGP- disaccharide). An examination of purified ECAPGby phosphorus-31 nuclear magnetic resonance spectroscopy confirmed that the polysaccharide chains are linked to diacylglycerol through phosphodiester linkage. Thus, DGP-disaccharide does not appear to be an intermediate in ECAPGsynthesis. Nevertheless, although the available evidence clearly indicate that lipid II is a precursor of DGP-disaccharide, the function of this novel glycolipid is not yet known, and it may be an intermediate in the biosynthesis of a molecule other than ECAPG.      

人红细胞非凝集型单抗(自身全血凝集试验试剂)的研制及鉴定

应用杂交瘤技术 ,混合人O型红细胞免疫纯系BALB/C小鼠 ,包被的红细胞作为固相抗原 ,ELISA法筛选出识别红细胞表面共同抗原的抗体 ,然后利用Coombs原理 ,加入抗球蛋白试剂。用玻片法筛选出非凝集型抗体 ,得到 2株稳定分泌上述抗体的杂交瘤并进行初步鉴定 ,单抗上清血凝效价可达 1∶5 0～ 1∶10 0 ,腹水血凝效价为 1∶8× 10 4 ～ 1∶16× 10 4 。另外 ,本文特别讨论了凝集型单抗的Fab段作为非凝集型抗体应用的可能性。     

Ultrastructure of Kolmer's crystalloid in the pinealocytes of the common spiny mouse (Acomys cahirinus dimidiatus)

Light-and electron-microscopical observations revealed a rod-like inclusion body in the pinealocytes of the common spiny mouse (Acomys cahirinus dimidiatus). Ultrastructurally, the body is composed of a varying number of tubes whose wall consists of thin filaments aligned in helical arrangement. The inner and outer surface of the wall is covered with ribosome-like granules. Based on a morphological analysis it is concluded that the body described in the present investigation is identical with Kolmer's crystalloid of horizontal cells in the human and primate retina.This work represents a portion of a doctoral thesis (Dr. med.) submitted to the University of Bonn and was presented at the 87th meeting of the anatomische Gesellschaft in Mainz, Germany, March 1992     

Dual‐labeled chemiluminescence enzyme immunoassay for simultaneous measurement of total prostate specific antigen (TPSA) and free prostate specific antigen (FPSA)

The specificity for early diagnostic of prostate‐specific antigen (PSA) is low because the current technology mostly allows the detection of only one biomarker at one time. In this work, a dual‐labeled chemiluminescence enzyme immunoassay (CLEIA) for simultaneous measurement of total PSA (TPSA) and free PSA (FPSA) was proposed. Anti‐PSA McAb (Mab1) was immobilized on a microplate as the solid phase, horseradish peroxidase (HRP)‐labeled anti‐TPSA monoclonal antibody (McAb2) and alkaline phosphatase (ALP)‐labeled anti‐FPSA McAb3 were used as detection antibodies. Two chemiluminescence reactions of HRP with luminol and ALP with 4‐methoxy‐4‐(3‐phosphate‐phenyl)‐spiro‐(1,2‐dioxetane‐3,2′‐adamantane) (AMPPD) were used as the signal detecting system. Based on a sandwich model, the amount of FPSA and TPSA could be determined simultaneously. The effects of several physico‐chemical parameters were studied and optimized. Cross‐reactivities of six common tumor markers in serum were studied. The proposed method presented the sensitivity of 0.03 ng ml^?1 and 0.05 ng ml^?1 for FPSA and TPSA respectively, with low cross‐reactivities. Compared with the results from commercial chemiluminescent kits there was good correlation, indicating that this established method could be used to simultaneously to measure the concentrations of FPSA and TPSA in one serum sample and also could greatly facilitate the early diagnosis for PCa in clinical practice.     

A linkage map of common carp (Cyprinus carpio) based on AFLP and microsatellite markers

Common carp (Cyprinus carpio) is an important fish for aquaculture, but genomics of this species is still in its infancy. In this study, a linkage map of common carp based on Amplified Fragment Length Polymorphism (AFLP) and microsatellite (SSR) markers has been generated using gynogenetic haploids. Of 926 markers genotyped, 151 (149 AFLPs, two SSRs) were distorted and eliminated from the linkage analyses. A total of 699 AFLP and 20 microsatellite (SSR) markers were assigned to the map, which comprised 64 linkage groups and covered 5506.9 cM Kosambi, with an average interval distance of 7.66 cM Kosambi. The normality tests on interval map distances showed a non‐normal marker distribution. Visual inspection of the map distance distribution histogram showed a cluster of interval map distances on the left side of the chart, which suggested the occurrence of AFLP marker clusters. On the other hand, the lack of an obvious cluster on the right side showed that there were a few big gaps which need more markers to bridge. The correlation analysis showed a highly significant relatedness between the length of linkage group and the number of markers, indicating that the AFLP markers in this map were randomly distributed among different linkage groups. This study is helpful for research into the common carp genome and for further studies of genetics and marker‐assisted breeding in this species.     

Isolation of carcinoembryonic antigen N-terminal domains (N-A1) from soluble aggregates

Carcinoembryonic antigen (CEA) was identified as a prominent tumor-associated antigen in human colorectal cancer and it is still intensively investigated. However, its physiological role remains unclear. The CEA molecule is composed of seven highly hydrophobic, immunoglobulin-like domains, six of which contain a single disulphide bridge. The production of recombinant protein containing Ig-like domains in bacterial expression systems often results in partial degradation or insolubility due to aggregation hampering the analysis of their native structure and function. Here, we present a new method of expression and purification of CEA N-terminal domains (N-A1) fused to MBP in Escherichia coli. In order to optimize the expression and purification of CEA N-A1 domains we evaluated bacteria cultivation conditions, the length of N-A1 domains, fusion systems (GST- and MBP-tag), IPTG concentrations and protein purification conditions. We have found that MBP-N-A1 fusion protein digested with TEV protease forms soluble aggregates composed of N-A1 domains and incompletely digested MBP-N-A1 fusion protein. Using 1.25 M guanidinium chloride (GdmCl) as a component of the elution buffer we were able to achieve an almost complete dissociation of the aggregates. The dissociation was monitored by circular dichroism and fluorescence measurements. The CD spectra and Ellman's assay suggest that the conformation of N-A1 domains and their disulphide bonds are correct.     

The fertilization—induced Ca^2＋ oscillation in mouse oocytes is cytoplasmic maturation dependent

Mature eggs (at metaphase II stage) produce a series of Ca^2 oscillation at fertilization.To define whether the fertilization-induced Ca^2 oscillation is restrict to the metaphase II eggs and cell cycle dependent,mouse oocytes at prophase I (arrested at germinal vesicle stage),metaphase I,metaphase II,as well as the pronuclear embryos at interphase of the first mitotic division derived from fertilization of parthenogenetic activation were inseminated after removal of zona pellucida,The results show that the fertilization-induced Ca^2 oscillation is not specific to metaphase II eggs.This is supported by the fact that immature oocytes generated the Ca^2 oscillations at fertilization regardless of their nuclear progression from prophase I to metaphase I (in vitro matured) stage.More interestingly,it was first found that pronuclear embryos at interphase derived from parthenogenetic activation showed Ca^2 oscillations in response to fertilization while the zygotes at interphase did not after reinsemination or intracytoplasmic injection of sperm extracts which induce Ca^2 oscillations in MII eggs.This suggests that the ability of oocytes to generate Ca^2 oscillation in response to sperm penetration is not regulated in a cell cycle dependent manner but dependent on the cytoplasmic maturation.     

Proliferating cell nuclear antigen (PCNA/cyclin) immunocytochemistry as a labeling index in mouse lung tissues

Summary Proliferating cell nuclear antigen is expressed in cells from late G₁ through the S-phase of the cell cycle. Therefore, antibodies directed against this molecule should provide a probe for labeling immunocytochemically the nuclei of proliferating cells. Herein we demonstrate the feasibility and reliability of this technique by quantifying immunostained pulmonary nuclei. We applied polyclonal and monoclonal antisera to alveolar and bronchiolar pulmonary epithelial cells in various proliferative states in tissue-sections and in vitro. A/J mice had a slightly higher labeling index than C57BL/6J mice, and proliferation in both strains increased dramatically after butylated hydroxytoluene treatment produced compensatory hyperplasia of Type-II pneumocytes. Immunostaining in fetal and neonatal lung samples from mice was higher than in adults. Spontaneous lung adenomas had a higher labeling index than the surrounding normal lung tissue. In addition, new data contained herein demonstrate a strain difference in proliferation of bronchiolar epithelial cells, and quantify the extent to which BHT-induced lung damage increases these proliferative rates. This mammalian nuclear antigen did not cross-react with antiserum to a functionally related bacterial protein, the beta subunit of E. coli DNA polymerase-III holoenzyme.     

硫酸化茯苓多糖急性毒性实验研究

按10、8、6.4、5.12、4.1g/kg体重剂量的硫酸化茯苓多糖(SP)分别灌胃给药,通过观察小鼠的活动和急性毒性反应,记录小鼠的死亡数,用综合计算法计算半数致死量(LD50)等相关数据.结果表明硫酸化茯苓多糖灌胃给药的LD50为7.358/kg,LD50的95%平均可信限为(7.358±0.894)g/kg,属低毒性物质.该研究为开发应用硫酸化茯苓多糖新药提供科学的药理学依据.     

卵巢癌细胞系RMG-Ⅰ Lewis(y)抗原含量变化对其卡铂耐药性的影响

利用Lewis（y）抗原稳定高表达细胞株RMG-I-H,研究细胞表面Lewis（y）抗原含量变化与细胞对卡铂耐药性的关系．利用四甲基偶氮唑盐（MTT）法测定不同浓度卡铂作用后细胞系RMG-I—H及其对照组RMG-I、RMG-I—C的细胞生长抑制率（IR）;利用流式细胞仪（FCM）分析细胞凋亡,同时检测药物作用后细胞的凋亡比率;利用荧光染色法和透射电镜进一步观察细胞凋亡状态．结果表明,RMG-I-H的半数抑制浓度（IC50）为（58．07&#177;2．42）,明显高于对照组RMG—I（28．83&#177;3．57）和RMG-I-C（25．71&#177;8．24）的IC50（P〈0．01）,后两者间无统计学差异（P〉0．05）,流式细胞仪分析证实3组细胞均存在凋亡,经30mg／L、60mg／L卡铂处理后细胞再进行流式细胞仪检测,RMG—I-H的相对凋亡率分别为（20．43&#177;0．71m和（38．11&#177;0．33）％,为对照组的49％-63％,明显低于对照组（P〈0．05）,2个对照组间比较无统计学差异（P〉0．05）,荧光染色和透射电镜直接观察了细胞形态变化,每个浓度组RMG-I-H的凋亡程度均低于RMG-I和RMG-I-C．说明细胞表面Lewis（y）抗原含量增加的同时,卵巢癌细胞对卡铂的耐药性增强．     

The effect of oestradiol on the DNA synthesis in neonatal mouse uterus and cervix

Summary (³H)-Thymidine autoradiography was used to study the DNA synthesis in the stroma and epithelium in the uterus proper and the uterine cervix of neonatal mice treated with oestradiol. It was found that in the epithelium of the uterus proper the DNA synthesis is stimulated between 6 and 12 h after injection of oestradiol and decreases again at 18 h. In the stroma of the uterus proper the DNA synthesis is increased 18 h after oestradiol injection.In the epithelium and stroma of the uterine cervix the DNA synthesis decreases from 5 h and is strongly depressed 18 h after oestradiol treatment.This work has been supported by grants from the Norwegian Research Council for Science and the Humanities and from the Norwegian Cancer Society (Landsforeningen mot Kreft).     

The ovarian phenotype of the aromatase knockout (ArKO) mouse

Targeted disruption of exon 9 of the cyp19 gene gives rise to a non-functional aromatase enzyme incapable of converting androgens to oestrogens. The aromatase knockout (ArKO) mouse is, thus, characterised by a dysfunctional pituitary-gonadal axis, which manifests in non-detectable levels of oestrogen in serum. These mice also exhibit elevated levels of circulating gonadotrophins (luteinising hormone (LH) and follicle stimulating hormone (FSH)) and testosterone. The ArKO mouse is infertile due to folliculogenic disruption and a failure to ovulate. The age-dependent ovarian phenotype revealed a block in follicular development at the antral stage and a complete absence of corpora lutea. By 21–23 weeks of age haemorrhagic cystic follicles were present and by 1 year there were abnormal follicles, an absence of secondary and antral follicles and atretic primary follicles. Interstitial tissue remodelling was extensive and exemplified by an increase in collagen deposition and an influx of macrophages, coincident with the loss of follicles. In mice, maintained on a soy-free and, thus, phytoestrogen-free diet, the ovarian phenotype was accelerated and exacerbated. In conclusion, the ovarian phenotype of the ArKO mouse can be attributed to the altered hormonal environment brought about by the absence of aromatase and the failure of androgens to be converted to oestrogens in the presence of elevated gonadotropins.