Nucleotide sequence of the staphylococcal enterotoxin C3 gene sequence comparison of all three type C staphylococcal enterotoxins

Summary The structural gene entC3, which encodes staphylococcal enterotoxin C3 was cloned from the genome of Staphylococcus aureus FRI-913 and sequenced. The primary amino acid sequence of the toxin was deduced from the nucleotide sequence data. entC3 contains 801 by and encodes a precursor protein of 266 amino acids. Glutamic acid was found to be the N-terminus of mature enterotoxin C3. Thus, the first 27 residues of the toxin precursor comprise the signal peptide, and the mature toxin contains 239 amino acids with a molecular weight of 27 563 daltons. Enterotoxin C3 differs from enterotoxin C2 by four amino acids and from enterotoxin C1 by nine residues. The 167 C-terminal residues of the three toxins are identical, except for one conservative amino acid substitution in enterotoxin C3. The degree of immunological relatedness among the three Type C enterotoxins is proportional to their molecular relatedness. This study also provides evidence that the N-termini of Type C enterotoxins determine subtype-specific antigenic epitopes, while more conserved C-terminal regions determine biological properties and cross-reactive antigenic epitopes shared with other pyrogenic toxins.     

茄科劳尔氏菌单克隆抗体的制备及其特性鉴定

茄科劳尔氏菌(Ralstonia solanacearum,RS)是番茄、茄子、辣椒、马铃薯等茄科蔬菜青枯病害的致病菌。为实现对RS的快速高效检测,以茄科劳尔氏菌株1.76免疫BALB/c小鼠,经细胞融合后利用间接酶联免疫吸附分析(Enzyme-linked Immunosorbent Assay,ELISA)筛选出3株能稳定分泌抗茄科劳尔氏菌株1.76的单克隆杂交瘤细胞株1C1、1B3和9D7。然后利用小鼠体内诱生腹水,1C1、1B3和9D7效价分别为1:1 024 000、1:64 000、1:256 000。采用饱和硫酸铵沉淀及Protein-G亲和层析法纯化腹水,经SDS-PAGE鉴定显示纯化后的单克隆抗体(Monoclonal Antibodies,mAb)纯度较高。纯化后单克隆抗体(2 mg/mL)效价分别为1:17 529、1:35 819、1:50 000,抗体亚型均为IgG1。对3株抗体进行特异性检测结果显示,1C1和9D7均不能与RS-5结合,1B3不能结合1.74和RS-5。此外,检测结果还表明3株单克隆抗体与桑肠杆菌JX-6、苏云金芽胞杆菌SYJ及实验室现有11株燕麦嗜酸菌卡特莱兰亚种、燕麦嗜酸菌西瓜亚种、玉米细菌性条斑菌、嗜酸菌魔芋亚种,梨火疫病菌QB0809、 XL-4,玉米细菌性枯萎病菌QB0241、QB0242,水稻细菌性谷枯病菌QB0017、QB0753、QB0755均无交叉情况。此次茄科劳尔氏菌抗体的制备,为后期青枯病菌的快速检测提供参考。     

Monoclonal antibodies to the two most basic papaya proteinases

The proteinases fromCarica papaya include papain, isoenzymes of chymopapain and two proteinases A and B distinguished by their unusually high pI. The identity of one of the most basic proteinases has been questioned. The present report describes the preparation and characterisation of two monoclonal antibodies that react specifically with papaya proteinases A and B respectively and a third that identifies a common structural feature found in papain and proteinase A.     

Monoclonal antibodies againstWuchereria bancrofti microfilarial excretory-secretory antigens

A battery of monoclonal antibodies were produced againstWuchereria bancrofti microfilarial excretory-secretory antigens and their specificity was studied using different filarial antigens. Among the 1116 wells plated out, 42 % of the wells developed hybrids and 5 % of the hybrids showed antiWuchereria bancrofti microfilarial excretory-secretory antigens. Specificity studies on the antibodies produced from 63 cloned and expanded hybrids showed 10 clones which were specifically positive only toWuchereria bancrofti microfilarial excretory-secretory antigens.     

Monoclonal antibodies against human chondrocytes

Summary Cell-specific antigens are mainly found in cells or membrane surfaces rather than in the surrounding matrix. However, until now it was not possible to produce antibodies specific for cellular structures of chondrocytes. In 1989, Lance (Immunol. Lett. 21:63–73; 1989) first established specific monoclonal antibodies for human articular chondrocytes tested only by immunofluorescence. Studies describing the specificity of these five antibodies (HUMC 1–5) and their relevance for immunohistological analysis of cartilage tissue were not available until now. Therefore, the aim of the following study was to investigate the distribution of HUMC 1, 2, 3, 4, and 5 in mesenchymal cellsin vivo andin vitro immunohistochemically. Further investigations concentrate on the localization of chondrocyte specific antigens using immunoelectron microscopy. Immunohistological studies showed positive immunostainings with all five antibodies in human chondrocytesin vivo andin vitro. A cross-reaction with human fibroblasts and osteoblasts for the antibodies HUMC 2 and HUMC 5 was observed. furthermore, a parallel loss of immunoreactivity for HUMC 1, HUMC 3, and HUMC 4 was observed in cultured chondrocytes indicating that the specific antigens vanish during differentiation observedin vitro. Subsequent immunoblot analysis employing collagens as antigens did not show any reactivity. Using immunoelectron microscopy, gold particle labeling was observed in intracytoplasmatic vesicles of isolated chondrocytes. Our results indicate that HUMC 1, HUMC 3, and HUMC 4 are specific for cartilage cells and might be suitable for immunohistological analysis of different cartilage tissues and pathologically altered chondrocytes.     

Intranasal vaccination with a double mutant of staphylococcal enterotoxin C provides protection against Staphylococcus aureus infection

Staphylococcus aureus expresses a repertoire of factors including staphylococcal exotoxins (SEs), exoenzymes, and numerous cell-associated components that contribute to the pathogenesis of disease. We constructed and expressed a nontoxic double mutant SEC (dmSEC), devoid of superantigenic activity, and investigated the ability of intranasal vaccination with dmSEC plus cholera toxin (CT) adjuvant to protect mice against S. aureus infection. Mice were vaccinated with dmSEC and inoculated with a viable S. aureus clinical isolate strain. The survival rate in the immunized mice was higher, and bacterial counts in the organs were significantly lower than those in the control group. Intranasal vaccination with dmSEC induced the production of SEC-specific antibodies such as IgG1, IgG2b and IgA. dmSEC-vaccinated mice elicited significantly higher titers of interleukin-4 (IL-4) and IL-10, and lower levels of interferon-gamma (IFN-gamma) after challenge with S. aureus compared with the control group. Furthermore, the sera from dmSEC-immunized mice significantly inhibited IFN-gamma and tumor necrosis factor-alpha production in vitro. These results indicate that intranasal vaccination with dmSEC devoid of superantigenic properties induces systemic immune responses and provides protection against S. aureus infection.     

抗金黄色葡萄球菌感染的新药研究进展

金黄色葡萄球菌因抗菌药耐药性而引起广泛关注,其耐药株感染所引起的皮肤和软组织感染、肺炎、菌血症以及骨髓炎等增加了患者的发病率和死亡率。金黄色葡萄球菌通常通过携带多种耐药基因、毒力基因或形成生物被膜等方式快速适应生存环境而产生耐药性,使得常规抗菌药物难以达到理想的治疗效果,因此,金黄色葡萄球菌感染的治疗一直是一个难题。从抗菌肽、植物次生代谢物及其他独特作用机制的新药等三方面进行阐述,以期为抗金黄色葡萄球菌感染的新药研发提供新的思路。     

Development of Panel of Monoclonal Antibodies Specific to Urochordate Cell Surface Antigens

Monoclonal antibodies are an important tool in the study of botryllid ascidians’ immunology and developmental biology. Here we describe the development of a panel of 38 monoclonal antibodies that are specific to Botryllus schlosseri (Ascidiacea; subfamily Botryllinae) cell surface antigens. Many of these hybridomas recognize (by enzyme-linked immunosorbent assay and immunohistochemistry) epitopes of Botrylloides subpopulations (SP) II and III from the Mediterranean coast of Israel and show, on blood cell smear assays, reactions with subsets of Botryllus circulating blood cells. Fluorescence-activated cell sorting analyses using antibodies positive for botryllid tissues revealed up to 3.6% positive cells. ELISA screenings were performed with 64 new monoclonal antibodies on 5 different individual botryllid ascidian colonies (B. schlosseri, Botrylloides). The positive antibodies in this panel identified a large number of different antigenic determinants, some of which distinguish Botryllus versus Botrylloides colonies, and other, different colonies within these two species, or different cell types within tissues, embryos, and buds of individual colonies. Only 21 monoclonal antibodies tested positive with all colonies. Cross-reactivity with at least one Botrylloides colony was recorded in 49 hybridomas that identified Botryllus cells. This wide panel of monoclonal antibodies is the first such detailed set of monoclonals available for studies on botryllid ascidians.     

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。     

Monoclonal antibodies raised against post-translational domains of the electroplax sodium channel

Summary Eleven monoclonal antibodies were identified that recognized eel electroplax sodium channels. All the monoclonal antibodies specifically immunostained the mature TTX-sensitive sodium channel (M _r 265,000) on immunoblots. None of the monoclonal antibodies would precipitate the in vitro translated channel core polypeptide in solution. One monoclonal antibody, 3G4, was found to bind to an epitope involving terminal polysialic acids. Extensive digestion of the channel by the exosialidase, neuraminidase, or partial polysialic acid removal bythe endosialidase, endo-N-acetylneuraminidase, destroy the 3G4 epitope, 3G4 is, therefore, a highly selective probe for the post-translationally attached polysialic acids. Except for this monoclonal antibody, the epitopes recognized by the remaining antibodies were highly resistant to extensive N-linked deglycosylation. Thus, the monoclonal antibodies may be directed against unique post-translationally produced domains of the electroplax sodium channel, presumably sugar groups that are abundant on this protein (Miller, J.A., Agnew, W.S., Levinson, S.R. 1983.Biochemistry 22:462–470). These monoclonal antibodies should prove useful as tools to study discrete post-translational processing events in sodium channel biosynthesis.     

Monoclonal antibodies against surface components of the mechano-and chemosensitive cnidocil complex of Hydra vulgaris

Summary Two monoclonal antibodies (Gc3.2 and Bd 2.2) against surface components of the cnidocil complex of Hydra vulgaris have been produced. In indirect immunofluorescence and in immunogold-labelling, the Gc 3.2-antibody stains the complete surface of all nematocytes, whereas other cellular surfaces are not labelled. The Bd 2.2-antibody, in contrast, produces only a small band of fluorescence on isolated cnidocils. This pattern of fluorescence and the corresponding immunogold-labelling indicate that the Bd 2.2-antibody exclusively binds to those intermembrane connectors that link the cnidocil and stereovillar cone in situ. In isolated and decnidociliated nematocytes, the tips of the stereovilli are also labelled by the Bd 2.2-antibody. Physiological experiments suggest that the Bd 2.2-antibody disturbs the reconstitution of intermembrane connectors during cnidocil regeneration. These data confirm the hypothesis that the intermembrane connectors are formed by two identical subunits located at the cnidociliar and stereovillar surfaces.     

快速检测金黄色葡萄球菌肠毒素A基因方法的建立与应用

目的建立一种快速准确定量检测金黄色葡萄球菌肠毒素A的方法。方法以femB、SEA基因分别作为金黄色葡萄球菌菌株、肠毒素A的靶序列,设计合成引物和TaqM an探针;收集腹泻患者大便标本分离的金黄色葡萄球菌68株,并定量检测其肠毒素A。结果TaqM an探针荧光聚合酶链反应检测金黄色葡萄球和肠毒素A的灵敏度均为1.0×102拷贝。68株金黄色葡萄球菌中检出产肠毒素A菌株11例(16.2%,11/68),CT值为13.5~20.6。结论TaqM an探针荧光聚合酶链反应能够准确快速检测金黄色葡萄球菌肠毒素A。     

Monoclonal antibodies specific for cell culture mycoplasmas

Summary Mycoplasma infection of cell cultures is still a major problem in some laboratories. Although several methods can be used for their detection, identification is normally by serological procedures. As no commercial source for the necessary antibodies is available we have prepared monoclonal antibodies to the five mycoplasma species that account for the majority of cell culture infections. These antibodies have been characterized by the growth inhibition test (GIT), immunofluorescence, and enzyme linked immunosorbent assay (ELISA) and have shown perfect correlation in all tests when compared to conventional antisera raised in rabbits or donkeys. In addition, a monoclonal antibody toMycoplasma pneumoniae was produced.M. pneumoniae is an infrequent cell culture contaminant but is a human pathogen, and the monoclonal antibody described here could be useful in the clinical diagnosis ofM. pneumoniae infection in man. These studies were supported by Grants Al-15748 from the National Institute of Allergy and Infectious Diseases, and GM20138-07 from the National Institutes of Health, Bethesda, MD.     

Monoclonal antibodies in the cytodiagnosis of serous effusions

In order to assess the value of immunocytochemical staining as a method of discriminating between benign reactive mesothelial cells and malignant epithelial cells in serous effusions, we have studied the reactions of a panel of commercially available antibodies on cells harvested from 83 pleural and peritoneal fluids and compared the results with the clinical and cytological diagnoses. The antibodies used were raised against cytokeratin (PKK1), epithelial membrane antigen (EMA), carcino-embryonic antigen (CEA), pregnancy specific B1-glycoprotein (SP1) and leucocyte common antigen (LCA). Anti-CEA was positive in 16 of 39 effusions (41%) containing carcinoma cells. Pregnancy specific B1-glycoprotein (SP1) was positive in 33% of the same samples. Mesothelial cells did not stain with these antibodies. Thus anti-CEA and SP1 can be used to discriminate between benign mesothelial and malignant epithelial cells in effusions. Anti-PKK1 stained both benign reactive mesothelial cells and malignant epithelial cells and cannot be used to discriminate between these two cell types. Strong positive staining of malignant cells was noted with anti-EMA. However, as occasional weak staining of mesothelial cells was also noted, strong staining with this antibody may be regarded as suspicious but not conclusive of malignancy.     

Monoclonal antibodies directed against plasmid-encoded released proteins of enteropathogenic Yersinia

Abstract Yersinia enterocolitica of serotypes O:3, O:8, O:9 and O:5,27 and Yersinia pseudotuberculosis of serotypes I and III release plasmid-encoded proteins into calcium-deficient medium. Mouse monoclonal antibodies were elicited against plasmid-encoded released proteins of Y. enterocolitica of serotype O:9. As shown by immunoblot analysis the monoclonal antibody Mab9–200 recognized the 46-kDa protein of Y. enterocolitica of serotypes O:3, O:9 and O:5,27, the 58-kDa protein of Y. enterocolitica of serotype O:8 and the 67-kDa protein of Y. pseudotuberculosis of serotypes I and III. Mab9–15 reacted with the 36-kDa protein of Y. enterocolitica of serotypes O:9, O:3 and O:8, and the 34-kd protein of Y. enterocolitica of serotype O:5,27 and Y. pseudotuberculosis of serotypes I and III. The 25-kDa proteins of Y. enterocolitica of serotypes O:3, O:9, O:8 and O:5,27, but not those of Y. pseudotuberculosis were recognized by the monoclonal antibody Mab-128. This species-specific recognition of epitopes could not be achieved by mouse polyclonal antibodies.     

Multiplex PCR for simultaneous detection of enterococcal genes vanA and vanB and staphylococcal genes mecA, ileS-2 and femB

The experimental transfer of the vanA gene cluster from Enterococcus faecalis to Staphylococcus aureus has raised fears about the occurrence of such genetic transfer in clinical isolates of methicillin-resistant staphylococci. Recently, infections by a S. aureus strain carrying the enterococcal vancomycin resistance vanA gene cluster were reported. The possible emergence and dissemination of these strains is a serious health threat and makes optimization of prevention strategies and fast detection methods absolutely necessary. In the present study, we developed a PCR protocol for simultaneous detection of enterococcal vanA and vanB genes , the staphylococcal methicillin and mupirocin resistance markers mecA and ileS-2, and identification of S. aureus. As no vancomycin-resistant S. aureus isolates were available for our study, we used mixtures of enterococcal and staphylococcal colonies that harbored the different resistance markers to show that these genes could be detected simultaneously. This protocol could be used to facilitate the detection and identification of predictable S. aureus or methicillin-resistant strains carrying vanA or vanB.     

Monoclonal antibodies to beet soil-borne virus

Four monoclonal antibodies (MCA) were obtained to the ‘Ahlum’ serotype of beet soil-borne virus (BSBV). On ELISA plates which had been precoated with polyclonal antibodies (PCA) all four MCA detected this serotype with a higher sensitivity than alkaline phosphatase-labelled PCA. Three of the MCA were specific for the ‘Ahlum’ serotype, but a fourth one also detected the distantly related ‘Wierthe’ serotype. Cross-reactions with wheat soil-borne or oat golden stripe furoviruses were not observed. One of the MCA reacted with an epitope which is exposed along the entire length of the BSBV particles, whereas two others were specific for epitopes which are exposed on one particle extremity only. Although these latter two epitopes occur apparently on the same extremity of the particles, they seem to be different, because one is found only on the particles of the ‘Ahlum’ serotype, whereas the other one is present also on the particles of the ‘Wierthe’ serotype. The fourth MCA is specific for a cryptotope which is not exposed on the intact virus particles, but presumably on some degradation product or precursor of the viral coat protein present in crude sap preparations. All four epitopes are SDS-labile; they are not detected on denatured viral coat protein on Western blots.     

Improved high-performance liquid chromatographic separation of peptidoglycan isolated from various Staphylococcus aureus strains for mass spectrometric characterization

Reversed-phase high-performance liquid chromatography (RP-HPLC) of muropeptides, obtained by muramidase digestion of peptidoglycan in combination with amino acid analysis and plasma desorption time-of-flight mass spectrometry is today by far the best tool to analyze the fine structure of the peptidoglycans. Here we report further improvements of the RP-HPLC separation of muropeptides for analyzing the peptidoglycans of various methicillin-resistant strains of Staphylococcus aureus, with emphasis on a more detailed characterization of the interpeptide bridge of the peptidoglycans of this species.     

Nucleotide sequence of the staphylococcal enterotoxin C1 gene and relatedness to other pyrogenic toxins

Summary The nucleotide sequence for the structural gene entC1 encoding staphylococcal enterotoxin C1 was determined. The gene contained 801 bp and coded for a protein of 266 amino acids. Of these, 27 comprised the signal peptide. Cleavage of the signal peptide resulted in a mature protein with 239 amino acids and a calculated molecular weight of 27496. The nucleotide sequence of entC1 shared considerable homology (74% and 59%, respectively) with genes encoding enterotoxin B and streptococcal pyrogenic exotoxin A. A similar degree of amino acid homology was observed after alignment of the respective proteins. Thus, certain regions of these three toxin molecules possess structural similarities that may be responsible for shared biological properties.     

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