Feeding,egg production,and egg hatching success of the copepods Acartia tonsa and Temora longicornis on diets of the toxic diatom Pseudo-nitzschia multiseries and the non-toxic diatom Pseudo-nitzschia pungens

In 1987, there was an episode of shellfish poisoning in Canada with human fatalities caused by the diatom Pseudo-nitzschia multiseries, which produced the toxin domoic acid. In order to examine whether domoic acid in this diatom serves as a grazing deterrent for copepods, we compared feeding rates, egg production rates, egg hatching success and mortality of the calanoid copepods Acartia tonsa and Temora longicornis feeding on unialgal diets of the toxic diatom P. multiseries and the similarly-sized non-toxic diatom Pseudo-nitzschia pungens. Copepods were collected in summers of 1994, 1995 and 1996 from Shediac Bay, New Brunswick, Canada, near Prince Edward Island, the site of the 1987 episode of domoic acid shellfish poisoning. Rates of ingestion of the toxic versus the non-toxic diatom by A. tonsa and T. longicornis were similar, with only one significantly different pair of values obtained in 1994, for which A. tonsa had a higher mean rate of ingestion of the toxic than the non-toxic diatom. Thus, domoic acid did not appear to retard grazing. Analyses of copepods with high performance liquid chromatography (HPLC) revealed that copepods accumulated domoic acid when feeding on P. multiseries. Egg production rates of copepods when feeding on P. multiseries and P. pungens were very low, ranging from 0 to 2.79 eggs female^–1 d^–1. There did not appear to be differential egg production or egg hatching success on diets of the toxic and non-toxic diatoms. Mortality of females on the toxic diet was low, ranging from 0 to 20%, with a mean of 13%, and there was no apparent difference between mortality of copepods feeding on toxic versus non-toxic diatoms. Egg hatching success on both diets, although based on few eggs, ranged between 22% and 76%, with a mean percentage hatching of 45%. Diets of the non-toxic diatom plus natural seawater assemblages supplemented with dissolved domoic acid, revealed similar rates and percentages when compared to previous experiments. In summary, none of the variables measured indicated adverse effects on copepods feeding on the toxic compared to the non-toxic diatom.     

Formation of domoic acid and fatty acids inPseudonitzschia pungens f.multiseries with scale of culture

Pseudonitzschia pungens f.multiseries was cultured in 20-L polycarbonate carboys, 350-L fibreglass columns and 500-L plastic bags to determine the effects of medium enrichment and scale of culture on cell yield, production of cellular domoic acid and formation of fatty acids, particularly the potential tracer acid 16:4n-1. Cell concentrations were highest in seawater enriched with stock levels of nitrate and phosphate, but with double the stock level of silicate, at all scales of culture. Cellular toxin in 20, 350 and 500-L cultures averaged 0.32, 0.04 and 2.56 pg cell^-1 and was independent of medium used. The order of magnitude difference in levels of cellular toxin was considered to reflect the varying levels of irradiance within the culture vessels. Support was given to this by the significant difference in content of total cellular fatty acids, due principally to the algal storage acid 16: 1n-7, which is known to be influenced by irradiance. Levels of cellular domoic acid correlated significantly with total fatty acids in 350 and 500-L cultures. Bag cultures producing significantly higher levels of cellular domoic acid provided lower relative proportions of 16:4n-1, which limited its use as a tracer for food-web studies.     

Domoic acid production by Pseudo-nitzschia australis and Pseudo-nitzschia calliantha isolated from North Chile

The morphology and toxicity of the four ubiquitous species belonging to the genus Pseudo-nitzschia found in mixed blooms of phytoplankton from northern Chilean waters were studied. The phytoplankton samples and cultures obtained were identified by scanning electron microscopy, revealing the presence of Pseudo-nitzschia australis, P. calliantha, P. pseudodelicatissima and P. subfraudulenta. This is the first report of P. calliantha in northern Chile. Toxin analyses using the LC–MS method confirmed the presence of domoic acid in P. australis and P. calliantha. Domoic acid was not detected in cultures of P. subfraudulenta. This study therefore confirms P. australis and P. calliantha as an unequivocal source of domoic acid in Chilean waters. P. australis is probably the most important producer of amnesic shellfish toxin in view of its domoic acid content. However, more research is needed to evaluate the potential for toxin production in P. pseudodelicatissima.     

Comparison of two domoic acid-producing diatoms: a review

In the past five years, awareness of domoic acid has increased from localized problems in Canada to outbreaks along both North American coasts. The phycotoxin domoic acid causes Amnesic Shellfish Poisoning (ASP) in humans and can be fatal. The known species of phytoplankton responsible for production of domoic acid include some pennate diatom species of the genus Nitzschia, sensu latu, which form stepped chains typical of the Pseudonitzschia. These diatoms are widely distributed, but their life histories and population dynamics are poorly understood. This review addresses histories of occurrences, morphology, geographical distributions, seasonal patterns, growth requirements, domoic acid production, and trophic interactions, with emphasis on a comparison of Pseudonitzschia pungens f. multiseries (Hasle) Hasle and Pseudonitzschia australis Frenguelli. Through continued research it will become possible to provide guidelines for regulatory agencies that protect both the consumer and the seafood industry.     

Monoclonal antibodies directed against protoplasts of soybean cells

Splenocytes, derived from mice that had been immunized with protoplasts prepared from suspension cultures of root cells of Glycine max (L.) Merr. (SB-1 cell line), were fused with a murine myeloma cell line. The resulting hybridoma cultures were screened for the production of antibodies directed against the soybean protoplasts and were then cloned. One monoclonal antibody, designated MVS-1, was found to bind to the outer surface of the plasma membrane on the basis of several criteria: (a) agglutination of the protoplasts; (b) binding of fluorescence-labeled immunoglobulin on protoplasts yielding a ring staining pattern with prominent intensity at the edges; and (c) saturable binding by protoplasts of ¹²⁵I-labeled Antibody MVS-1. The antigenic target of Antibody MVS-1, identified by immunoblotting techniques, contained a polypeptide of relative molecular mass (M_r) approx. 400000 under both reducing and non-reducing conditions. When the antigenic target of Antibody MVS-1 was chromatographed in potassium phosphate buffer, the position of elution corresponded to that of a high-molecular-weight species (M_r 400000). These results provide the protein characterization required for the analysis of the mobility of Antibody MVS-1 bound to the plasma membrane of SB-1 cells.Abbreviations D diffusion coefficient - M_r relative molecular mass - PBS phosphate-buffered saline (8.00 g NaCl, 1.15 g Na₂HPO₄, 0.20 g NaH₂PO₄ per 1 L, pH 7.2) - TPBS phosphate-buffered saline containing 0.5% Tween-20 - TX-100, TX-114 Triton X-100, X-114 - SDS sodium dodecyl sulfate     

猪肺炎支原体Mhp366-N蛋白多克隆抗体的制备及应用

[背景]猪肺炎支原体是猪的一种重要的病原.该菌的研究工具较少,特别是缺少开展其致病机制研究需要的抗体.[目的]制备猪肺炎支原体Mhp366-N蛋白抗体并确定其应用范围和使用时的最佳稀释倍数.[方法]Escherichia coli BL21 (DE3)-pET28a(+)-mhp366-N重组菌诱导表达Mhp366-N...     

The N-glycolyl form of mouse sialyl Lewis X is recognized by selectins but not by HECA-452 and FH6 antibodies that were raised against human cells

E-, P- and L-selectins critically function in lymphocyte recirculation and recruiting leukocytes to inflammatory sites. MECA-79 antibody inhibits L-selectin-mediated lymphocyte adhesion in several species and does not require sialic acid in its epitope. Many other antibodies, however, recognize human selectin ligands expressing N-acetylneuraminic acid but not mouse selectin ligands expressing N-glycolylneuraminic acid, suggesting that difference in sialic acid in sialyl Lewis X leads to differential reactivity. We found that HECA-452 and FH6 monoclonal antibodies bind Chinese hamster ovary (CHO) cells expressing N-acetylneuraminyl Lewis X oligosaccharide but not its N-glycolyl form. Moreover, synthetic N-acetylneuraminyl Lewis X oligosaccharide but not its N-glycolyl oligosaccharide inhibited HECA-452 and FH6 binding. By contrast, E-, P- and L-selectin bound to CHO cells regardless of whether they express N-acetyl or N-glycolyl form of sialyl Lewis X, showing that selectins have a broader recognition capacity than HECA-452 and FH-6 anti-sialyl Lewis x antibodies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparative study of epitopes recognized by two monoclonal antibodies that protects mice against Trichomonas vaginalis challenge

Trichomonas vaginalis is a flagellated protozoan which infects the urogenital tract of humans. Previous studies have demonstrated that monoclonal antibodies (MAbs) against a 62 kDa proteinase (4D8 and 1A8) decreased cytoadherence of the parasite to epithelial cells in vitro and passive inoculation of mice with two MAbs 24 h before the intraperitoneal challenge resulted in different grade of protections to T. vaginalis infection. In the present paper we describe the characterization of the epitopes recognized by MAbs 4D8 and 1A8. The epitopes were characterized by heat treatment, trichloroacetic acid precipitation, beta-mercaptoethanol treatment, enzymes proteolysis and periodate oxidation. The results showed that the two MAbs 4D8 and 1A8 each react with a different protein epitope of repetitive nature found on the same excretory-secretory molecules of T. vaginalis and it could explain the variation in the protection grade obtained in the challenge experiments.     

金黄色葡萄球菌黏附素ClfA等抗体抑制该菌黏附牛乳腺上皮细胞效果的比较

【目的】为从免疫的角度比较和分析黏附素分子细胞外纤维蛋白原结合蛋白(Extracellular fibrinogen-binding protein,EfB)和纤维连接蛋白结合蛋A(Fibronection binding protein,FnBP)对聚集因子A(Clumping factor A,ClfA)抑制牛源金黄色葡萄球菌(Staphylococcus aureus,SA)黏附牛乳腺原代上皮细胞作用的增强效果。【方法】本试验分离培养牛乳腺上皮细胞并鉴定;将真核重组质粒EfB和FnBPA分别与ClfA组合免疫新西兰大白兔,并利用免疫后抗体体外抑制2株SA分离株侵染牛乳腺上皮细胞,采用平板计数法定量检测与比较不同免疫组合诱导抗体对黏附的抑制作用;对SA和乳腺上皮细胞分别进行荧光染色,观察不同免疫组合诱导的抗体对黏附抑制效果的差异。【结果】成功分离培养了牛乳腺原代上皮细胞;证明了构建的ClfA、FnBPA和EfB真核重组表达质粒均可在细胞中表达,且在免疫实验兔后可诱导特异性抗体的产生;细菌平板计数和荧光染色观察的结果表明,黏附素分子单独和组合免疫的抗体对该菌不同菌株(GY278和GY309)的黏附抑制能力不同,ClfA抗体的黏附抑制能力最强,FnBPA分子A区的黏附抑制能力优于D区。FnBPA、EfB分别与ClfA联合免疫抗体对黏附的抑制程度高于黏附素分子单独免疫组,FnBPA分子A区对ClfA黏附抑制的增强作用优于D区,FnBPA-A区与Efb相比对ClfA的黏附抑制增强差异不显著。【结论】FnBPA-A、FnBPA-D和EfB分别与ClfA联合免疫可不同程度地影响ClfA的黏附抑制效果,该结果为以黏附素分子为靶点的牛乳腺炎疫苗的研究提供了实验数据。     

Production of monoclonal antibodies against surface antigens of spores from arbuscular mycorrhizal fungi by an improved immunization and screening procedure

Monoclonal antibodies (mAbs) were produced against surface antigens of chlamydospores of arbuscular mycorrhizal (AM) fungi by immunizing mice with crushed or complete spores. The intrasplenic approach proved to be superior to the intraperitoneal method of immunization with regard to the amount of antigen required for the immune response. The hybridoma technology was combined with an improved screening procedure, applying an immunogold-silver staining technique to semi-thin sections of spores. In this way, mAbs to surface antigens on the outer wall could be selected. Two mAbs were raised against Glomus etunicatum and G. scintillans spores. Cross-reactivities of the antibodies to other structures of the fungus, to other species of Glomus and to other soil-borne fungi were tested with indirect immunofluorescent labelling. The mAbs did not react with non-AM fungi. One mAb (A5B1) selectively recognized G. etunicatum, another (D12F11) exhibited limited interspecies cross-reactivities. One further mAb (H8F7), which reacted with spores of all AM fungi but not with other fungi, was shown to be specific for Bacillus mycoides. The implications are discussed.     

A membrane-located polypeptide ofUlva sp. which may be involved in HCO 3 - uptake is recognized by antibodies raised against the human red-blood-cell anion-exchange protein

Polypeptides present in a membrane fraction of the marine macroalga Ulva sp. were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tested for cross-reactivity with antibodies raised against the human red-blood-cell anion exchanger (AE1). A polypeptide of ca. 95 kDa was identified with a monoclonal, as well as two polyclonal (one against the C-terminus and one against the whole protein) antibodies, indicating that it shares homologous domains with AE1. These findings complement an earlier study which indicated that a plasmalemma-bound, disulfonic stilbenesensitive, protein was functionally involved in HCO ₃ ^- transport into the photosynthesizing cells of Ulva (Z. Drechsler et al. 1993, Planta 191, 34–40). It is thus suggested here that a similar protein has evolved, and has been conserved, in marine photosynthetic organisms and mammalian red blood cells for the purpose of HCO ₃ ^- transport.Abbreviations AE1 anion exchanger 1 (of red blood cells) - CI inorganic carbon This paper is in partial fulfillment of a Ph.D. study by R. Sharkia. Supported by the Israel Academy of Sciences, grant 441/93 (to S.B.)     

trans-Stilbene degradation by Arthrobacter sp. SL3 cells

trans-Stilbene degradation was examined by the reaction using resting cells of microorganisms isolated through the enrichment culture using trans-stilbene. The strain SL3, showing the highest trans-stilbene-degrading activity, was identified as Arthrobacter sp. One of the reaction products was identified to be cis,cis-muconic acid. Arthrobacter sp. SL3 cells also transformed benzaldehyde, benzoic acid and catechol into cis,cis-muconic acid, suggesting that one benzene ring of trans-stilbene was converted into cis,cis-muconic acid via benzaldehyde formed by its C_α=C_β bond cleavage.     

Monoclonal antibodies identify common and differentiation-specific antigens on the plasma membrane of guard cells of Vicia faba L.

Monoclonal antibodies were raised against the plasma membrane of Vicia faba L. guard cells by immunizing either with total membranes from purified guard-cell protoplasts or with sealed, predominantly right-side-out plasma-membrane vesicles prepared from abaxial epidermes of V. faba by aqueous two-phase partitioning. Hybridoma screening was performed by enzyme-linked immunosorbent assay using polystyrene-adsorbed plasma-membrane vesicles as solid phase and by indirect immunofluorescence analysis using unfixed, immobilized protoplasts in a microvolume Terasaki assay. A range of monoclonal antibodies was characterized and is reported here. One monoclonal antibody, G26-6-B2, is guard-cell-specific and does not react with mesophyll-cell protoplasts of the same species. It binds to a periodate-resistant but trypsin-labile epitope, probably a differentiation-specific plasma-membrane protein.Abbreviations ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate - GCP guard cell protoplast(s) - Ig immunoglobulin - MAB monoclonal antibody - MCP mesophyll-cell protoplast(s) - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Tissue-specific antigens secreted by suspension-cultured callus cells of Prunus avium L.

A number of antigenic components are secreted into the medium by P. avium callus cells derived from different tissues and grown in suspension culture. These antigens have been detected using antiserum raised in rabbits to a protein fraction secreted by P. avium leaf callus. One antigen is specific to leaf tissue and is secreted by callus cells derived from stem, pistil and anthers as well as leaves. A second antigen is, in intact organs, restricted to styles of a particular self-incompatibility (S) genotype, but is also secreted by callus cells derived from the leaf. Another antigen, apparently not organ-specific, is secreted by all calli tested, including Rosa (cv. Paul's Scarlet).     

Isolation, expression and characterization of two single-chain variable fragment antibodies against an endo-polygalacturonase secreted by Sclerotinia sclerotiorum

Canola is a very important economic crop in the world and canola stem rot caused by Sclerotinia sclerotiorum (Lib.) de Bary, a necrotrophic, highly destructive and non-host-specific fungus, can reduce yield significantly. This fungus secretes numerous cell wall degrading enzymes including an endo-polygalacturonase, SSPG1d, which has been detected at early stages of infection. In this report we describe the isolation of two recombinant antibodies of the single-chain variable fragment (ScFv) format from RNA of mice immunized with recombinant SSPG1d (rSSPG1d) or a peptide derived from SSPG1d (peptide 3796) that was predicted to be antigenic. The ScFvs were isolated using the established phage display technology. These recombinant antibodies were expressed, purified and refolded to functional antibodies with a yield of 120–500 μg per liter of cell culture. Recombinant antibodies were characterized using various techniques including enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Of the two ScFvs, it appears that only ScFv-rSSPG1d is able to detect whole SSPG1d produced by the fungus. Thus our results indicate that this ScFv may have utility in the detection of the SSPG1d enzyme in an antibody-based diagnostic test for S. sclerotiorum infection.     

Production of α-keto acids: 2. Immobilized whole cells of Providencia sp. PCM 1298 containing -amino acid oxidase

A number of bacteria belonging to the genera Proteus, Providencia, Pseudomonas and Erwinia have been tested for their capacity to oxidize -amino acids to their corresponding α-keto acids. Members of the Proteus and the Providencia genera were active towards various -amino acids. Immobilized cell preparations of Providencia sp. PCM 1298 were shown to form up to 80 mg α-keto-γ-methiol butyric acid from -methionine per g of gel preparation (containing 4% w/w cells) per day. The productivity was highly dependent on the size of the beads. Oxygen appeared to be the rate-limiting substrate and oxygen transfer rates of 3–4 μmol cm⁻² h⁻¹ were calculated. The entrapment of activated charcoal to remove H₂O₂ formed during the oxidation extended the half-life of the immobilized biocatalyst considerably. A decrease in -amino acid oxidase [ -amino acid: oxygen oxidoreductase (deaminating); EC 1.4.3.2] activity during operation could be compensated for by reinoculation of the alginate-entrapped cells in fresh growth medium, allowing use of these preparations of immobilized bacterial cells for more than one month.     

Anaerobic oxidation of toluene (analogues) to benzoate (analogues) by whole cells and by cell extracts of a denitrifying Thauera sp.

Toluene and related aromatic compounds can be mineralized to CO₂ under anoxic conditions. Oxidation requires new dehydrogenase-type enzymes and water as oxygen source, as opposed to the aerobic enzymatic attack by oxygenases, which depends on molecular oxygen. We studied the anaerobic process in the denitrifying bacterium Thauera sp. strain K172. Toluene and a number of its fluoro-, chloro- and methyl-analogues were transformed to benzoate and the respective analogues by whole cells and by cell extracts. The transformation of xylene isomers to methylbenzoate isomers suggests that xylene degradation is similarly initiated by oxidation of one of the methyl groups. Toluene oxidation was strongly, but reversibly inhibited by benzyl alcohol. The in vitro oxidation of the methyl group was coupled to the reduction of nitrate, required glycerol for activity, and was inhibited by oxygen. Cells also contained benzyl alcohol dehydrogenase (NAD⁺), benzaldehyde dehydrogenase (NADP⁺), benzoate-CoA ligase (AMP-forming), and benzoyl-CoA reductase (dearomatizing). The toluene-oxidizing activity was induced when cells were grown anaerobically with toluene and also with benzyl alcohol or benzaldehyde, suggesting that benzyl alcohol or benzaldehyde acts as inducer. The other enzymes were similarly active in cells grown with toluene, benzyl alcohol, benzaldehyde, or benzoate. This is the first in vitro study of anaerobic oxidation of an aromatic hydrocarbon and of the whole-cell regulation of the toluene-oxidizing enzyme.Dedicated to Prof. Achim Trebst     

Neospora caninum: antibodies directed against tachyzoite surface protein NcSRS2 inhibit parasite attachment and invasion of placental trophoblasts in vitro

Polyclonal and monoclonal antibodies to native Neospora caninum tachyzoite surface protein NcSRS2 were generated and tested in vitro for their ability to neutralize tachyzoite attachment to and invasion of host cells. Host cells included Vero cells and a newly cloned, immortalized ovine trophoblast cell line obtained from primary cultures of ovine placenta. The ovine trophoblasts had morphology consistent with fetal trophoblasts and expressed mRNA for interferon-tau, a marker for trophoblasts. Native NcSRS2 was used to immunize mice to obtain monospecific anti-NcSRS2 polyclonal serum and anti-NcSRS2 monoclonal antibodies. Compared to irrelevant antibodies, monospecific anti-NcSRS2 serum and two anti-NcSRS2 monoclonal antibodies, 100.2.4.4 and 119.4.9.10, significantly blocked invasion of tachyzoites into both trophoblasts and Vero cells. Parasite attachment, assessed by IFA, was significantly reduced by anti-NcSRS2 mAb 100.2.4.4 and monospecific serum. The findings provide rationale to investigate a role for antibodies to NcSRS2 in prevention of N. caninum transplacental transmission in vivo.     

Connexin32 protects against vascular inflammation by modulating inflammatory cytokine expression by endothelial cells

Gap junctions (GJs) play an important role in vascular function, stability, and homeostasis in endothelial cells (ECs), and GJs are comprised of members of the connexin (Cx) family. GJs of vascular ECs are assembled from Cx37, Cx40, and Cx43, and we showed that ECs also express Cx32. In this study, we investigated a potential role for Cx32 during vascular inflammation. Expression of Cx32 mRNA and protein by human umbilical venous ECs (HUVECs) decreased following treatment with tumor necrosis factor (TNF)-α, but lipopolysaccharide (LPS) and interleukin (IL)-1β did not affect Cx32 expression. Intracellular transfer of an inhibitory anti-Cx32 monoclonal antibody significantly enhanced TNF-α-induced monocyte chemotactic protein (MCP)-1 and IL-6 expression, but overexpression of Cx32 abrogated TNF-α-induced MCP-1 and IL-6 expression. LPS treatment of Cx32 knock-out mice significantly increased the serum concentrations of TNF-α, interferon-γ, IL-6 and MCP-1, compared to wild-type littermate mice. These data suggest that Cx32 protects ECs from inflammation by regulating cytokine expression and plays an important role in the maintenance of vascular function.