Isolation of Naegleria fowleri from artificially heated water

Water samples were collected from artificially heated and ambient temperature water and processed for the presence of Naegleria fowleri. Samples taken from thermally altered habitats were positive 43% of the time, whereas ambient temperature habitats were positive 2% of the time. The data suggests that thermally altered aquatic systems in the south-eastern United States may provide habitats conducive to the proliferation of N. fowleri.     

Diagnosis of the primary amoebic meningoencephalitis due to Naegleria fowleri

Trophozoites of the free-living amoeba, Naegleria fowleri, were isolated from the cerebrospinal fluid of meningoencephalitis patient. The infecting agent was identified as N. fowleri based on morphologic, serologic and molecular techniques carried out on the isolated organisms.     

A genetic approach to species criteria in the amoeba genus Naegleria using allozyme electrophoresis

The present study employs allozyme electrophoresis to characterize and inter-relate 61 isolates of Naegleria. Diploidy was confirmed, with heterozygotes observed at 29 of the 33 loci established and in all but two isolates. With a single exception, isolates clustered at two levels of similarity, either below 21% or above 52%. It is argued that such a major discontinuity provides a sound biological basis for a species concept in Naegleria. On this basis the present species-level taxonomy does not reflect the genetic diversity of the genus. The study recognized 18 genetic groups of species rank. The subspecies N. australiensis italica deserves specific rank; additional thermophilic species not closely related to N. fowleri and N. lovaniensis are recognized; and N. gruberi as currently conceived is a complex of 10 species, at least five of which are represented in the formal culture collections. Most species are genetically too different for relationships to be elucidated by allozyme electrophoresis, supporting the view that some of the times of divergence within the genus are extremely ancient.     

Detection, quantification and identification of fungal extracellular laccases using polyclonal antibody and mass spectrometry

This study presents a combined method to analyze extracellular fungal laccases using a new anti-laccase antibody together with the identification of tryptic laccase peptides by mass spectrometry (nanoLC–ESI–MS/MS). The polyclonal anti-laccase antibody LccCbr2 was raised against peptides designed from the copper binding region II of fungal laccases using in silico data obtained from GenBank database. As a consequence, detection requires denaturation of the enzymes due to the stable conformation of the copper binding region II. The specificity of the antibody was shown with denatured laccase Lcc1 of Coprinopsis cinerea and laccase of Hypholoma fasciculare. LccCbr2 detected amounts as low as 5 ng of highly purified laccase, indicating a possible use of the antibody for quantification of laccase proteins. Denatured extracellular laccases from culture supernatants of the basidiomycetes C. cinerea, H. fasciculare, Lentinula edodes, Mycena sp., Piriformospora indica, Pleurotus cornucopiae, Pleurotus ostreatus, Pycnoporus cinnabarinus, Trametes versicolor and furthermore the ascomycete Verpa conica were detected with apparent molecular masses between 60 and 70 kDa by LccCbr2. The identity of extracellular laccases from C. cinerea, H. fasciculare, P. ostreatus, P. cinnabarinus and T. versicolor were verified by tryptic peptides using nanoLC–ESI–MS/MS.     

Identical properties of transmembrane synaptic vesicle protein Mr 100,000 in Torpedo and Mr 86,000 in bovine brain

Synaptic vesicles derived from the Torpedo electric organ and bovine cerebral cortex contain concanavalin A binding transmembrane glycoproteins of M_r 100,000 and 86,000, respectively. Their isolelectric points range from 5.5 to 6.0. On deglycosilation both glycoproteins yield identical products of M_r 62,000. The fully glycosilated and the deglycosilated proteins from both Torpedo and bovine brain are recognized by the monoclonal anti-SV2 antibody (Buckley and Kelly, J. Cell Biol. 100, 1284–1294, 1985) as well as by a monospecific IgG fraction raised against Torpedo vesicles and immunopurified against the bovine brain M_r 86,000 glycoprotein. This is shown by Western blotting as well as by immunoaffinity isolation with one antibody and immunodetection with the other antibody. Furthermore on immunohistochemical analysis of the Torpedo electric organ both antibodies recognize exactly the same nerve terminal ramifications. It is concluded that the glycoproteins of M_r 100,000 in Torpedo and of M_r 86,000 in bovine brain are corresponding proteins with different degrees of glycosilation.     

The immune response of a Cyprinid fish to infections of the acanthocephalan Pomphorhynchus laevis

1972. The immune response of a Cyprinid fish to infections of the acanthocephalan, Pomphorhynchus laevis. International Journal for Parasitology 2: 459–469. Chub (Leuciscus cephalus) were found to produce precipitating antibody in response to natural and experimental infections of an acanthocephalean Pomphorhynchus laevis, although no apparent manifestation of resistance was evident The parasites matured only in the chub although they occurred in five other species of naturally infected fish. Furthermore, this was the only fish species which responded, at least by humoral antibody formation to the parasite. Preliminary experiments indicated that the helminth antigen was an excretory or secretory product and was probably produced only by mature adult worms. The antibody was detected in both the serum and intestinal mucus of the chub. The presence of antibody in the mucus of fish might suggest the presence of a secretory antibody system although the precipitins appeared to have similar chemical characteristics to IgM-type antibody.     

Molecular analysis of the gene encoding a protein component of the Eikenella corrodens adhesin complex that is close to the carbohydrate recognition domain

A monoclonal antibody against a lectin-like substance (LS) of Eikenella corrodens (Ec) was used for screening the Ec DNA library. Three positive clones that carried an identical 12-kb segment were obtained. A 25-kDa protein, which specifically binds to the antibody, was overproduced in all of the Escherichia coli clones. Deletion analysis showed that the gene encoding the 25-kDa protein was located within a 1.2-kb segment. The nucleotide (nt) sequence of this segment contained an open reading frame encoding a protein of 24 600 Da. We purified the 25-kDa protein from the cloned E. coli strain. The sequence of the first 10 amino acids (aa) from the N-terminus of the purified 25-kDa protein agreed with that deduced from the nt sequence. Since the monoclonal antibody used in this study inhibits the physiological activity of EcLS, we concluded that the 25-kDa protein is a component of the adhesin complex, which is located near the carbohydrate recognition domain of lectin in EcLS.     

抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因小鼠乳腺生物反应器的制备与验证

目的:构建抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因动物乳腺特异性表达载体并制备和验证抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因小鼠乳腺生物反应器模型。方法:利用PCR法扩增出抗人p185~(erbB2)人鼠嵌合抗体ChAb26的重链基因H和轻链基因L,然后分别将嵌合抗体重链基因H和嵌合抗体轻链基因L连接到乳腺特异性表达质粒pBC1,从而构建抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因动物乳腺特异性表达载体pBC1-H和pBC1-L。分别将抗p185~(erbB2)人鼠嵌合抗体ChAb26乳腺特异表达载体pBC1-H和pBC1-L线性化,然后使用原核显微共注射法获得8只转基因FVB小鼠,通过鼠尾直接PCR鉴定其转基因阳性。通过RT-PCR、荧光定量PCR鉴定转基因小鼠乳腺组织中抗p185~(erbB2)人鼠嵌合抗体ChAb26的mRNA表达。使用小鼠乳汁采集器收集其乳汁并通过Western blot和夹心ELISA等实验鉴定抗p185~(erbB2)人鼠嵌合抗体ChAb26是否获得表达。结果:经测序验证,抗p185~(erbB2)人鼠嵌合抗体ChAb26的嵌合重链基因H和嵌合轻链基因L分别与乳腺特异表达质粒pBC1正确正向连接。鼠尾直接PCR结果显示所获8只转基因FVB小鼠均为转基因双阳性小鼠,且抗p185~(erbB2)人鼠嵌合抗体ChAb26的重链基因H和轻链基因L在它们的后代中稳定遗传,它们的后代中转基因小鼠双阳性率约为30%; RT-PCR和荧光定量PCR的结果显示,转基因双阳性小鼠及其双阳性后代的乳腺组织中存在抗p185~(erbB2)人鼠嵌合抗体ChAb26的mRNA表达; Western blot和ELISA等实验结果显示,转基因双阳性小鼠乳汁中存在抗p185~(erbB2)人鼠嵌合抗体ChAb26的蛋白质表达,而且抗p185~(erbB2)人鼠嵌合抗体ChAb26与羊抗人κ链抗体和羊抗人Ig G Fc-HRP抗体均能特异性结合。结论:成功构建抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因动物乳腺特异性表达载体pBC1-H和pBC1-L和制备了抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因小鼠乳腺生物反应器模型,为今后抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因牛乳腺生物反应器的研究奠定了理论和技术基础。     

Parasite antigens and host antibodies in Ostertagia circumcincta infection of the sheep

and 1972. Parasite antigens and host antibodies in Ostertagia circumcincta infection of the sheep. International Journal for Parasitology, 2: 449–457. An allergenic component was separated from Ostertagia circumcincta antigens and specific reaginic antibody was separated from the corresponding 7S antibodies in sheep sera. Further evidence was obtained that the immunoglobulin class defined as IgG1A contains the reaginic or homocytotropic antibodies in sheep. Both the IgG1A antibody and P.C.A. levels continued to increase after the expulsion of the parasites, whereas the levels of anti-Ostertagia IgG1 did not.     

促进大肠杆菌周质空间小分子抗体表达的菌种构建方法*

目的:构建一株表达TNF-α Fab'抗体的大肠杆菌工程菌,并设计一种高效实用的策略以促进大肠杆菌周质空间的可溶性Fab'抗体表达。方法:首先,通过更换不同表达载体,改变轻链和重链顺序,更换信号肽,共表达分子伴侣(Skp)、二硫键合成酶(Dsbc)、肽基辅氨酰顺反异构酶(PPIB)、二硫键异构酶(hPDI)、核酸酶(Nuclease),以评估对Fab'抗体表达量的改善。其次,纯化表达的Fab'抗体。通过周质提取、Q阴离子交换柱净化、苯基柱捕获、Protein L柱亲和三步纯化方案得到高纯度的Fab'抗体。最终将纯化后的Fab'抗体进行亲和力测定。结果:提高正确组装的Fab'抗体表达量的策略有——将目的蛋白构建至pET-30a载体;重链在前、轻链在后;轻、重链采用相异的信号肽;共表达hPDI。周质提取液中的Fab'抗体浓度达到588.0mg/L提取液,纯化后产量可达28.2mg/L发酵液,总回收率为32.0%,纯度为90.9%。Fab'抗体亲和力为(5.8±3.0)×10^-9mol/L,体外细胞学活性IC₅₀为(5.2±2.4)×10^-11mol/L。结论:通过大肠杆菌工程菌分子构建方式的优化,得到了一株高效表达可溶性Fab'抗体的工程菌株,为可溶性小分子抗体的规模化生产奠定了研究基础。     

广叶绣球菌β-D-葡聚糖对巨噬细胞RAW264.7免疫调节作用受体TLR4和TLR2的影响

确定广叶绣球菌β-D-葡聚糖对巨噬细胞RAW264.7的免疫调节作用受体,探索广叶绣球菌β-D-葡聚糖的免疫调节机制。采用MTT法测定不同浓度广叶绣球菌β-D-葡聚糖对巨噬细胞RAW264.7增殖活力的影响,筛选出促进巨噬细胞增殖能力最强的浓度。用筛选出的β-D-葡聚糖浓度作用巨噬细胞RAW264.7;TLR4抗体和TLR2抗体分别作用巨噬细胞RAW264.7 1h,再用含有β-D-葡聚糖的细胞培养液培养。收集细胞培养上清和细胞,检测细胞培养上清中NO、IL-6、TNF-α、IFN-β的生成量;提取细胞内总RNA,采用RT-PCR测定巨噬细胞TLR4 mRNA表达量;提取巨噬细胞总蛋白,采用蛋白免疫印迹western blot测定TLR4的蛋白表达。广叶绣球菌β-D-葡聚糖能够促进巨噬细胞RAW264.7增殖,增加NO、IL-6、TNF-α、IFN-β的生成量,提高TLR4 mRNA表达和蛋白表达,差异极显著（P<0.01）。TLR4抗体作用细胞后,NO、IL-6、TNF-α、IFN-β的生成量明显下降,差异极显著（P<0.01）。TLR2抗体作用细胞后,NO、IL-6、TNF-α、IFN-β的生成量下降,但差异不显著。广叶绣球菌β-D-葡聚糖可以通过细胞表面受体TLR4激活信号转导通路,增强下游细胞因子的释放,从而调节巨噬细胞RAW264.7的免疫功能。TLR2可能不是广叶绣球菌β-D-葡聚糖的免疫受体。     

The sheep antibody response to repeated infection with Lucilia cuprina.

, , and 1992. The sheep antibody response to repeated infection with Lucilia cuprina. International Journal for Parasitology 22: 1169–1174. The specific serum antibody responses of sheep exposed to 10 consecutive infections of L. cuprina have been analysed by enzyme-linked immuno-sorbent assay and immunoblotting using monoclonal antibodies specific for sheep immunoglobulin isotypes. Recognition of a number of larval excretory-secretory products by IgM antibodies appeared to be non-specific. IgG₁ was the major antibody class stimulated by the infection protocol and marked increases in antibody to specific excretory-secretory antigens were observed. Three molecules of 35, 30 and 25 kDa were particularly recognized although the extent of recognition of these molecules varied considerably between individual sheep serum. A pooled serum composed of sera collected after five to seven infections significantly inhibited larval growth in in vitro cultures when compared to a sera pool consisting of sera collected both prior to infection and after infections 1 and 2. The degree of inhibition was greater when serum with high specific antibody titre was used.     

圈养华南虎钩端螺旋体感染情况调查

钩端螺旋体病(Leptospirosis)为致病性钩端螺旋体引起的人畜共患传染病,在世界范围内分布(Levett,2001),可感染鼠科、猴科和犬科等多种动物(Desvars et al.,2012).人或动物通过接触带菌动物的粪便、尿液污染的水和土壤而感染(娄银莹等,2019),可造成黄疸、急性肾功能衰竭、出血性素质...     

Characterization and purification of the biologically active v-sis protein generated in silkworm larvae with the Bombyx mori nuclear polyhedrosis virus vector system

The v-sis oncogene of simian sarcoma virus encodes a protein which is homologous to the human platelet-derived growth factor B-chain. The biologically active v-sis protein was expressed in silkworm larvae using the Bombyx mori nuclear polyhedrosis virus vector system. The v-sis protein purified from infected silkworm larvae is a 30 kDa disulfide-linked homodimer. Mitogenic activity of the v-sis protein was comparable to that of PDGF and inhibited by the pretreatment with anti-PDGF antibody. These results show that the recombinant v-sis protein is biologically and antigenically related to PDGF.     

Studies of Listeria monocytogenes-antibody binding using electro-orientation

An electro-optical (EO) approach has been used for studies of Listeria monocytogenes–antibody binding. The EO analyzer, which has been developed at the State Research Center for Applied Microbiology, Obolensk, was used as a basic instrument for EO measurements. AC electro-kinetic effects depend on dielectric properties of bioparticles, their composition, morphology, the medium, and the frequency of applied electrical field. Electro-orientational spectra were used for discrimination of bacteria before and after selective binding with antibodies. The measurements were performed using a discrete set of frequencies of the orienting electric field (10, 100, 250, and 500 kHz). During biospecific interactions an antibody is bound to the microorganism causing a change in the dielectric properties of the microorganism–antibody complex and the EO signal reaches its maximum at 100–200 kHz. It has been shown that the biospecific interactions of L. monocytogenes cells with anti-Listeria antibody in the presence of Escherichia coli K-12, and Azospirillum brasilense Sp7 change the EO signals significantly. Thus, the determination of the presence of particular bacteria within a mixed sample may be achieved by selection and matching of antibodies specific to individual bacterium types and by comparing spectra of bacterium in the presence and in the absence of specific antibody.     

Mechanism of coupling periodate-oxidized nucleosides to proteins

This paper describes a mechanism of coupling periodate-oxidized nucleosides to proteins. Each of the dialdehyde groups of a periodate-oxidized nucleoside is shown to couple to lysine residues on different protein molecules through Schiff bases, thereby cross-linking different protein molecules, forming a polymer. This is in contrast to the previous model in which nucleosides were suggested to couple to proteins through a morpholine structure. The cross-linked structure of the nucleoside-antigen, significantly different when compared to the native protein, may affect the specificity and the efficiency of antibody production.

Nucleoside-antigen Periodate oxidation Schiff base Protein cross-linking Polymerization Antibody production     

Isolation, initial characterization and serological studies of glycolipid antigens from Babesia bovis-infected erythrocytes

Serological analysis of Babesia bovis-derived glycolipids by ELISA and the indirect fluorescent antibody technique demonstrated the existence of their antigenic and immunogenic activities not only in B. bovis but also in B. bigemina infections. This indicates that serological cross-reactivity of B. bovis and B. bigemina relates to glycolipids. The negative ELISA reaction obtained with Anaplasma marginale antisera suggested the specificity of the reaction to the genus Babesia. Fractionation of these glycolipids by Florisil Sep-Pak column chromatography with subsequent HPTLC immunostaining and Orcinol staining suggested the presence of carbohydrate antigenic determinants in B. bovis glycolipids.     

Sieve element specific labeling with an anti-idiotypic monoclonal antibody mimic of the phytotoxin dothistromin

A monoclonal antibody, 12C9, an anti-idiotypic mimic of dothistromin, a toxin produced by Dothistroma pini, was found to label the cell wall of sieve elements in a number of different plant tissues and species. The antibody labeled apple leaf tissue, tobacco leaf mid vein, leaf and meristem, and Coprosma robusta leaf mid vein. Labeling was restricted to cell walls of sieve elements and did not label the companion cells or the lumen of the cells. The antibody labeled over a wide range of dilutions. This antibody could be used to differentiate sieve elements from other types of phloem. It could also be used to co-localize sieve elements and microorganisms such as phytoplasmas stained with DAPI.     

Production of monoclonal antibodies against target schistosomal antigen secreted in the urine of Schistosoma mansoni-infected patients

This study was undertaken to develop an immunodiagnostic test of active human schistosomiasis mansoni using a monoclonal antibody which targets urinary schistosomal antigen. Polyclonal antisera raised in rabbits against the processed urine of Schistosoma mansoni-infected patients showed very high and significant reactivity with ES product of ova compared with other different S. mansoni antigens. The monoclonal antibody (4.23) was reactive with repetitive epitopes of S. mansoni soluble egg antigen and ES product of ova with molecular mass range of 65–23 kDa and 80–23 kDa, respectively. It recognised different stages of the parasite life-cycle, with no cross reaction with Fasciola or hydatid antigen. MAbs were characterised by isotyping, immunoelectrophoresis, SDS–PAGE and the enzyme-linked immunoelectrotransfer blot technique, ELISA, and their recognition of carbohydrate or protein antigenic epitopes by periodate oxidation and trichloroacetic acid treatment of the antigen. It was used for detection of circulating schistosomal antigen in an antigen capture antibody sandwich ELISA on sera and urines of 58 S. mansoni-infected patients, 17 S. haematobium-infected patients, 15 parasite-free negative healthy controls and sera from 13 schistosomiasis-free patients harbouring Fasciola or hydatid infections. The percentage sensitivity of the assay in the serum of S. mansoni-infected patients was 98.4% and in urine 94.8%. A positive correlation was found between the number of faecal S. mansoni eggs and the circulating antigen, both in serum and in urine. Antigen circulating in urine correlated with that in the sera of S. mansoni patients. These data provide a sensitive and non-invasive method almost comparable with the use of sera for immunodiagnosis of schistosomiasis and an indirect way to reflect the intensity of infection. Australian Society for Parasitology.