Indirect detection of anti-acetylcholinesterase compounds in microcolumn liquid chromatography using packed bed reactor with immobilized human red blood cell acetylcholinesterase and choline oxidase

The inhibiting compounds were separated by micro-column liquid chromatography in the mobile phase containing the natural substrate acetylcholine. A home-made packed bed microbioreactor system containing immobilized enzyme acetylcholinesterase (ACHE) in human red blood cell membrane and choline oxidase (CHO) from alcaligenes was used for the post-column conversion of acetylcholine to hydrogen peroxide which was detected by an electrochemical detector. The inhibition effect of the solutes caused a decrease in the acetylcholinesterase activity, a decrease in the formation of hydrogen peroxide and also a decrease in the response corresponding to the concentration of the solutes. The rate of the enzyme regeneration was also recorded. The micro-system was compared with a conventional LC system comprising commercially prepared enzyme reactor. The stability of the enzymes is at least 3 weeks at ambient temperature. The limit of detection depends on biological activity of inhibition and for galanthamine was 1 pmol.     

Development and Intrauterine Fate of Normal and Abnormal Human Conceptuses

Using the data from the Kyoto Collection of Human Embryos, three main topics related to normal and abnormal development of human embryos are discussed. 1) Wide variability was noted in developmental stage of human embryos at any given gestational age. This was true not only for the estimated ovulation age but also for ‘coital’ age in single coital cases. Such diversity in human prenatal development may be, at least in part, ‘normal’ biological variability and it should be taken into account when assessing the teratogenic risk of environmental agents to human embryos. 2) At the early postimplantation period prior to major organogenesis, the percentage of morphologically abnormal embryos is high (> 30%), which supports the clinical finding that a substantially large proportion of human conceptuses are eliminated at an early stage of pregnancy, often without the knowledge of the mother. The fate of undifferentiated abnormal embryos is not certain and should be studied. 3) Life-table estimates for normal and abnormal human conceptuses showed that more than 10% of all embryos recognizable at 5 weeks gestation are malformed or ‘potentially’ malformed. Because of selective intrauterine death of malformed embryos and fetuses, the proportion of the malformed drops to 2.4% by age 8 weeks and 1% at term. The cumulative intrauterine mortality rate of malformed conceptuses was estimated to be 93%, while the corresponding rate for normal conceptuses was 18%.     

Antibody-dependent neutrophil-mediated parasite killing in non-lethal rodent malaria

The effects of administrating recombinant human granulocyte colony-stimulating factor (rhG-CSF) and passively transferring immune serum on infection with an attenuated variant of Plasmodium berghei XAT (Pb XAT), in severe combined immunodeficiency (SCID) mice were examined. In immune competent (C.B-17) mice, the attenuated parasite infection was inevitably self-resolving and degenerating forms inside erythrocytes appeared, coinciding with the drop in parasitaemia, whereas SCID mice were unable to control parasite growth and all the mice died. Continuous administration with rhG-CSF caused neutrophilic granulocytosis in both SCID and C.B-17 mice. The effect of rhG-CSF on the infection in C.B-17 mice was to suppress the course of the parasitaemia at an early phase whereas it had no effect in SCID mice. When immune serum was transferred on the day of infection, the prepatent period was prolonged two days in both SCID and C.B-17 mice. When administration with rhG-CSF was combined with transfer of immune serum, SCID mice showed four days delay in patency and degenerating parasites were seen during the course of parasitaemia, although the infection was ultimately fatal. C.B-17 mice similarly treated showed a seven day delay in the onset of the patent parasitaemia which was of a lesser magnitude and shorter in duration compared with control mice. On the other hand, when C.B-17 mice were splenectomized three weeks before infection and then treated with rhG-CSF and immune serum, no degenerating parasites were seen during the infection and all mice died with high parasitaemias. These results show that antibody-dependent neutrophil-mediated parasite killing may occur in the spleen of mice infected with P. berghei XAT.     

Multiplex PCR analysis of in vivo-arising deletion mutations in the hprt gene of human T-lymphocytes

A multiplex polymerase chain reaction (PCR) procedure was adapted for the rapid and efficient evaluation of deletions of the hypoxanthine guanine phosphoribosyltransferase (hprt) gene in human T-lymphocytes. The hprt clonal assay was used to isolate in vivo-arising hprt-deficient T-cells from six healthy males. Mutant frequencies ranged from 9-27 × 10^?6. Simple crude cellular extracts from 223 mutants were analyzed for hprt gene deletion. Sixteen (7.2%) were found to be due to total gene deletion and 22 (9.9%) were due to partial gene deletion. The relatively high frequency of total gene deletions was caused by replicate isolates of a single mutational event as shown by single-strand conformation polymorphism (SSCP) analysis of rearranged T-cell receptor (TCR)-γ genes. Eighteen of the 22 partial hprt gene deletion mutants were determined to be of independent origin based on a unique hprt mutation or SSCP-TCR-γ pattern. One-half (9/18) of the partial deletion mutants involved all or part of exon 4 alone, suggesting that this region of the hprt gene is prone to deletion. The small deletions effecting exon 1 (1 mutant), exon 2 (2 mutants), and exon 4 (6 mutants) would not have been detected by conventional Southern blot analysis and may represent a new, previously unrecognized class of mutations. The ready isolation of such intragenic deletions will allow the characterization of breakpoint junctions and may provide insights into the important processes of DNA breakage and rejoining. © 1994 Wiley-Liss, Inc.     

聚焦层析分离人肝精氨酸酶同工酶组分

用聚焦层析结合DEAE-Sephadex A-50离子交换层析,从正常人肝浸液中分离得两个精氨酸酶同工酶组分.经聚丙烯酰胺凝胶电泳(PAGE)鉴定从聚焦层析得到的第一峰同工酶组分为一条蛋白带,第二峰具有精氨酸酶活性的蛋白组分的主要蛋白带也极明显。各蛋白质成分向阴极电泳的前后顺序与从聚焦层析洗脱的先后次序一致。两型同工酶的比活力分别约为2050U/mg及790U/mg。用SDS-PAGE检测两型同工酶均由两种亚基组成,且分子量近似。     

IgE and atopy in perinatally HIV-infected children

Elevated serum immunoglobulin E (IgE) and increased prevalence of atopy is reported in patients infected with human immunodeficiency virus (HIV). The elevated serum IgE may be attributed to polyclonal stimulation of B cells or IgE production against allergens, viruses, fungi and bacteria. This study investigates the prevalence of atopy in perinatally HIV-infected children, and the relationships between serum IgE (and other serum immunoglobulins) with atopy, CD4+ cell count and HIV-disease stage. Serum immunoglobulin levels, epicutaneous skin test for common aeroallergens, clinical Centers for Disease Control and Prevention (CDC) classification, CD4+ cell counts and allergy history were extracted from the charts of perinatally HIV-infected children on highly active antiretroviral therapy. The prevalence of atopy (52%) and the pattern of aeroallergen sensitivity were comparable with the US pediatric population. Serum IgE levels did not correlate with clinical disease stage. However, in non-atopic patients, serum IgE levels increased with disease progression (p = 0.02). There was an inverse relationship between the prevalence of elevated serum IgE levels and atopy with progression of disease (p = 0.019). Serum IgE did not correlate with atopy, CD4+ cell count, or duration of HIV infection or levels of serum immunoglobulins. This is the first study to show no increased prevalence of atopy in perinatally HIV-infected children compared with the general population. In advanced stages of HIV, elevated serum IgE may be specific for antigens other than those known as allergens.     

Blockade of superoxide generation prevents high-affinity immunoglobulin E receptor-mediated release of allergic mediators by rat mast cell line and human basophils

BACKGROUND: Previous studies have shown that rat peritoneal mast cells and mast cell model rat basophilic leukaemia (RBL-2H3) cells generate intracellular reactive oxygen species (ROS) in response to antigen challenge. However, the physiological significance of the burst of ROS is poorly understood. OBJECTIVE: The present study was undertaken to investigate the role of superoxide anion in mediator release in rat and human cell systems. METHODS: RBL-2H3 cells were directly stimulated with anti-rat FcepsilonRI alpha-subunit monoclonal antibody (mAb). For the analysis of human cell system, leucocytes were isolated by dextran sedimentation from healthy volunteers or from patients, and challenged either with anti-human FcepsilonRI mAb or with the relevant antigens. Superoxide generation was determined by chemiluminescence-based methods. The releases of histamine and leukotrienes (LT)s were determined by enzyme-linked immunosorben assay (ELISA). RESULTS: Cross-linking of FcepsilonRI on RBL-2H3 cells or on human leucocytes from healthy donors by the anti-FcepsilonRI mAb resulted in a rapid generation of superoxide anion, as determined by chemiluminescence using superoxide-specific probes. Similarly, leucocytes from patients generated superoxide anion in response to the challenge with the relevant allergen but not with the irrelevant allergen. Furthermore, diphenyleneiodonium (DPI), a well-known inhibitor of flavoenzymes suppressed the superoxide generation and the release of histamine and LTC4 induced by the anti-FcepsilonRI mAb or by allergen in parallel. CONCLUSION: These results indicate that both RBL-2H3 cells and human basophils generate superoxide anion upon FcepsilonRI cross-linking either by antibody or by allergen challenge and that blockade of the generation prevents the release of allergic mediators. The findings strongly support the role of superoxide generation in the activation of mast cells and basophils under both physiological and pathological conditions. The findings suggest that drugs regulating the superoxide generation have potential therapeutic use for allergic disorders.     

人β-防御素3基因在COS-7细胞中的表达

目的：构建人β—防御素3(hBD3)的真核表达载体，建立稳定表达hBD3的细胞株。方法：用EcoR Ⅰ酶切合有hBD3全长基因的pGEM-hBD3重组质粒，获得其编码区全长序列，将其连接入EcoR Ⅰ预处理过的pcDNA3中。转化大肠杆菌，酶切鉴定筛选出插入方向正确的转化子。采用脂质体转染法将重组pcDNA3-hBD3真核表达载体导入COS-7细胞，用G418进行抗性筛选，用RT-PCR和Western印迹检测目的基因hBD3的mRNA和蛋白表达水平。结果与结论：构建的pcDNA3-hBD3真核表达载体转染COS-7细胞后可稳定表达hBD3的mRNA和蛋白，且蛋白主要以分泌形式存在于培养上清中。     

大学生蠕形螨感染的调查分析

目的调查广州医学院2001级大学生人体蠕形螨的感染情况,探讨蠕形螨感染与皮肤疾病、宿舍分布的关系。方法采用透明胶纸粘贴法对468名在校大学生蠕形螨感染进行调查,同时观察其面部皮肤状况,询问其所住宿舍状况并记录于调查表内。结果广州医学院2001级大学生468人,检出人体蠕形螨者85人,感染率18.16%。男学生感染率为18.37%(52/283人),女学生为17.84%(33/185人),两者无显著性差异(P>0.05)。从学生来源地看,城镇学生感染率为15.94%(51/320人),农村学生为22.97%(34/148人),两者无显著性差异(P>0.05)。感染者的感染度以每条透明胶纸1～3条虫为多,占87.06%(74/85人)。检获的螨大多为毛囊蠕形螨,占70.59%(60/85人),单一感染皮脂蠕形螨仅15.29%(13/85人),两者混合感染为14.12%(12/85人)。面部有痤疮、毛囊炎或毛囊扩张者蠕形螨感染率均高于面部皮肤正常、有痒感者(P<0.05);有痒感者蠕形螨感染率与面部皮肤正常者无显著性差异(P>0.05)。感染蠕形螨者具有显著的宿舍聚集性(P<0.01)。结论广州医学院2001级大学生人体蠕形螨感染较为普遍。感染大多为毛囊蠕形螨,少数为皮脂蠕形螨及两者混合感染,两种蠕形螨的感染度都属轻度。蠕形螨感染可能是痤疮、毛囊炎或毛囊扩张等皮肤疾病的致病因素之一。蠕形螨感染可以通过同     

思他宁对人胰腺癌细胞株ASPC-1的生长调控

目的:观察思他宁对人胰腺癌细胞ASPC-1的生长作用.方法:细胞培养,分别加入不同浓度的思他宁.应用MTT法来观察细胞增殖程度;放免法测定细胞内cAMP含量;荧光法测定[Ca2 ]i.结果:不同浓度的思他宁(10-11～10-6mol/L)均能有效地抑制ASPC-1的生长,能抑制ASPC-1细胞内cAMP生成和细胞内钙离子水平,cAMP生成量和细胞内钙离子水平与思他宁浓度呈负相关.结论:思他宁能抑制ASPC-1细胞的生长,经过特异性受体调节.