Identity of hemolysins produced by Bacillus thuringiensis and Bacillus cereus

A hemolysin (Bt-hemolysin) produced by Bacillus thuringiensis var. kurstaki HD-1 producing crystalline toxin(s) was purified by successive treatments of ammonium sulfate (45-65%) and column chromatography using DEAE-cellulose, Sephadex G-75 and KB-002 (a hydroxyapatite column for fast protein liquid chromatography). A hemolysin (Bc-hemolysin) produced by B. cereus HG-6A was also purified by the same procedure. The purified Bt-hemolysin and Bc-hemolysin, both of which are thiol-activated hemolysins, were biologically, physicochemically and immunologically identical. These findings provide further evidence of the similarity of B. thuringiensis, which is being used as a biological insecticide, to B. cereus, a toxigenic organism of food poisoning.     

Monoclonal Antibody #5-2-26 Recognizes the Phosphatase-Sensitive Epitope of Rabies Virus Nucleoprotein

We prepared monoclonal antibodies (MAbs) against the rabies virus N protein, among which one antibody (MAb 5-2-26) was shown to lack reactivity with the phosphatase-treated N protein. The MAb was able to recognize the sodium dodecyl sulfate (SDS)-denatured N protein. The MAb did not recognize the N-protein analogues produced in Escherichia coli (E. coli), indicating that the N-gene products were not normally processed in E. coli after translation. On the other hand, the MAb reacted normally with N-gene products produced in COS-7 cells, but not with those produced in the presence of K-252a (a protein kinase inhibitor of a broad spectrum). The MAb displayed weak cross-reactivity with the Triton-insoluble network structures composed of several components, while another phosphoprotein (M1) of the virus was not recognized at all. These results suggest that MAb 5-2-26 preferentially recognizes a phosphatase-sensitive linear epitope of N protein, which may enable further investigations to be conducted on the mechanism of N-protein phosphorylation and its role(s) in virus replication.     

Comparison of hemolysins of Vibrio cholerae non-O1 and Vibrio hollisae with thermostable direct hemolysin of Vibrio parahaemolyticus

Hemolysin (Vh-rTDH) produced by Vibrio hollisae and hemolysin (NAG-rTDH) produced by Vibrio cholerae non-O1 were characterized and compared with hemolysin (Vp-TDH) produced by Vibrio parahaemolyticus. These three hemolysins are each composed of two subunits and have similar, but not identical, molecular weights. The amino acid compositions of Vp-TDH and NAG-rTDH are similar, but are different from that of Vh-rTDH. The three hemolysins showed similar lethal toxicities to mice. The effects of temperature on hemolysis and the time dependencies of hemolysis by the three hemolysins were similar. The three were concluded to be immunologically related, but not identical, and to have common and also unique antigenic determinants.     

Production and characteristics of hemolysins of Escherichia coli

Snyder, Irvin S. (University of Iowa, Iowa City), and Nancy A. Koch. Production and characteristics of hemolysins of Escherichia coli. J. Bacteriol. 91:763-767. 1996.-Filterable and nonfilterable hemolysins were produced by Escherichia coli in beef-heart infusion, casein hydrolysate, and chemically defined media. Differences were found among the three media in the time of appearance and in the relationship between production of these two hemolysins. The nonfilterable hemolysin produced in the three media, as well as the filterable hemolysin produced in the beef-heart infusion medium, were destroyed in 1 hr at 56 C. The filterable hemolysin produced in the casein hydrolysate and chemically defined media was unaffected by exposure to 56 C for 1 hr. Formalin inactivated the hemolysins produced in all three media. The optimal pH for activity of the nonfilterable hemolysin varied with time of production, whereas the optimal pH for the filterable hemolysin was constant. Certain carbohydrates were required for the production of filterable hemolysin by E. coli grown in casein hydrolysate and chemically defined media.     

Evaluation of the ligand specificity of hemolymph hemoagglutinins and hemolysins of gastropod and bivalve molluscs

The study deals with evaluation of ligand specificities of hemoagglutinins and hemolysins of hemolymph of three species of gastropods(Planorbius corneus, Lymnaea stagnalis, andAchatina fulica) and one species of bivalve molluscs(Anodonta cygnea). The hemoagglutinin titer was estimated from hemoagglutination reaction, the hemolysin titer, spectrophotometrically, from release of hemoglobin from sheep erythrocytes. The ligand specificity of hemoagglutinins and hemolysins was evaluated by an inhibitory modification of the hemoagglutination reaction. It has been shown that hemoagglutinins and hemolysins of pulmonary gastropod molluscs have different spectra of ligand specificity to the substances of the carbohydrate nature. Hemoagglutinins and hemolysins of the bivalve molluscAnodonta cygnea have the same spectrum of ligand specificity. Among the ligands of these lectins, terminal N-acetylated sugars and monosaccharides predominate. Components of bacterial cell walls, lipopolysaccharide and phosphorylcholine, have been revealed among specific ligands of hemolymph hemoagglutinins and hemolysins.     

Characterization of Recombinant Terrelysin,a Hemolysin of <Emphasis Type="Italic">Aspergillus terreus</Emphasis>

Fungal hemolysins are potential virulence factors. Some fungal hemolysins belong to the aegerolysin protein family that includes cytolysins capable of lysing erythrocytes and other cells. Here, we describe a hemolysin from Aspergillus terreus called terrelysin. We used the genome sequence database to identify the terrelysin sequence based on homology with other known aegerolysins. Aspergillus terreus mRNA was isolated, transcribed to cDNA and the open reading frame for terrelysin amplified by PCR using specific primers. Using the pASK-IBA6 cloning vector, we produced recombinant terrelysin (rTerrelysin) as a fusion product in Escherichia coli. The recombinant protein was purified and using MALDI-TOF MS determined to have a mass of 16,428 Da. Circular dichroism analysis suggests the secondary structure of the protein to be predominantly β-sheet. Results from thermal denaturation of rTerrelysin show that the protein maintained the β-sheet confirmation up to 65°C. Polyclonal antibody to rTerrelysin recognized a protein of approximately 16.5 kDa in mycelial extracts from A. terreus.     

The clinical significance of HAMA in patients treated with mouse monoclonal antibodies

Twenty-four patients were analyzed for the development of HAMA (human antimouse antibodies) after being treated with repeated doses (200–500 mg) of the mouse monoclonal antibody (MAb) 17-1A. All patients developed anti-17-1A IgG antibodies, and most of them also developed IgM antibodies. In only two patients could immune complexes be demonstrated. Allergic reactions were rare (1.9%). In an extended study, a further 19 patient were analyzed for an idiotypic response. Forty-one out of 43 patients developed antiidiotypic antibodies (ab₂), and 20 of these also antianti-idiotypic antibodies (ab₃). Ab₃ ⁺ patients responded significantly better (p=0.01) and survived longer (p<0.001) compared to ab₃ ⁻ patients. In this study, we showed that MAb 17-1A could be repeatedly given on a safe basis. The development of high titers of HAMA did not cause significant clinical problems when further repeated infusions of MAb 17-1A were given. The development of an idiotypic response also indicate that the induction of HAMA might be beneficial and not harmful to the patient.     

Catabolite repression of the synthesis of inducible polygalacturonase and pectinesterase byAspergillus niger sp.

Several recent reports have described large numbers of monoclonal antibodies that cross-react with toxins A and B ofClostridium difficile; this suggests that the toxins share major epitopes. Our results show that monoclonal antibodies (MAb) against other antigens bind nonspecifically to both toxins. Therefore, we believe that the cross-reacting MAb bind by this manner and not by a true immune reaction.     

Biochemical and immunological characterization of pea nuclear intermediate filament proteins

In immunoblot assays, at least three putative nuclear intermediate filament (NIF) proteins were detected in nuclear envelope-matrix (NEM) and lamin (L1) fractions of nuclei from plumules of dark-grown pea (Pisum sativum L.) seedlings. These NIF proteins had apparent molecular masses of ca. 65, 60, and 54 kDa (also referred to as p65, p60, and p54), and appeared as multiple isoelectric forms, with pIs ranging from ca. 4.8 to 6.0. Polyclonal and monoclonal antibodies were raised to the 65-kDa NIF protein bands excised from gels after electrophoresis. These anti-pea antibodies were specifically cross-reactive with the pea nuclear p65, p60, and p54 proteins and also with chicken lamins. Sequence alignment of peptide fragments obtained from the 65- and 60-kDa pea NIF proteins showed similarity with animal intermediate filament proteins such as lamins and keratins and with certain plant proteins predicted to have long coiled-coil domains. These pea NIF proteins were further purified and enriched from the NEM fraction using methods similar to those used for isolating animal lamins. When negatively stained and viewed by transmission electron microscopy, the filaments in the pea lamin (L1) fraction appeared to be 6–12 nm in diameter. As assayed by immunofluorescence cytochemistry using a confocal laser-scanning microscope, fixed pea plumule cells displayed uniform as opposed to peripheral nuclear staining by several of the antibody preparations, both polyclonal and monoclonal. This report describes the biochemical and immunological properties of these pea NIF proteins.Abbreviations IF Intermediate filament - L Lamin fraction - LM Lamina-matrix fraction - MAb JLA20 Anti-chicken actin monoclonal antibody - MAb LN43 Anti-human lamin B2 monoclonal antibody - MAb PL19 Anti-pea lamin #19 monoclonal antibody - MAb TIB 131 Anti-intermediate filament monoclonal antibody - N Nuclei fraction - NEM Nuclear envelope-matrix fraction - NIF Nuclear intermediate filament - PAb PL3 Anti-pea lamin #3 polyclonal antibody     

A common allergenic epitope of Bermuda grass pollen shared by other grass pollens

The present study disclosed the cross-reactivity between Bermuda grass pollen (BGP) and other grass pollens using monoclonal antibodies (MAbs) and polyclonal antiserum. MAb 9–13, directed against a group of minor allergens of BGP (Cyn d Bd68K, 48K, 38K) was found to cross-react with extracts of ten other grass pollens. Immunoblotting assays illustrated that MAb 9–13 cross-reacted with multiple components of most of these pollens, and the major cross-reactive components had molecular weights of 29–36 kD. The cross-reactivity between BGP andLol pI, the group I allergen of rye grass pollen, was further evaluated;Lol pI was recognized by MAb 9–13, but not by our MAbs/polyclonal antiserum againstCyn dI, the major allergen of BGP. These results suggest that the epitope recognized by MAb 9–13 is a common (C) epitope shared byLol pI andCyn d Bd68K, 48K, 38K, andCyn dI does not share significant antigenicity withLol pI. In a modified radio-allergosorbent test, IgE antibodies in the serum of BGP-allergic patients reacted mildly with C-epitope-bearing components of both BGP and rye grass pollens, and this binding could be blocked specifically by MAb 9–13. This suggests that in addition to an antigenic cross-reaction, the C epitope can also lead to an allergenic cross-reaction.     

CD8+ T Lymphocytes Are the Major Cell Population Involved in the Early Gamma Interferon Response and Resistance to Acute Primary Toxoplasma gondii Infection in Mice

Gamma interferon (IFN-γ) is known to be a major mediator influencing host defense against Toxoplasma (T.) gondii. To evaluate lymphocyte populations involved in this cytokine-mediated early resistance to T. gondii, the effects of in vivo administration of monoclonal antibodies (MAbs) against T-cell subsets and anti-asialo GM1 antibody on the course of infection and IFN-γ response were investigated in mice infected acutely with this parasitic protozoan. A single injection of anti-CD8 MAb on day ?1 or day 4 severely exacerbated the infection, in accordance with a marked suppression of endogenous IFN-γ production. Moreover, the administration of anti-IFN-γ MAb on day 0 but not later than day 4 resulted in a total abrogation of resistance to T. gondii, suggesting that endogenous IFN-γ produced during the first several days of infection is critical for the generation of antitoxoplasmal resistance in mice. In contrast, no significant increase in mortality was observed when injected with either anti-CD4 MAb or anti-asialo GM1 antibody on day ? 1, while these antibodies reduced significantly the ability of mice to produce IFN-γ. Indeed, simultaneous depletion of CD4⁺ and CD8⁺ cells had no greater suppressive effect on host defense and endogenous IFN-γ production than depletion of CD8⁺ cells alone. Together, these results suggest that CD8⁺ T cells play a central role for resolution of acute toxoplasmosis by participating in endogenous IFN-γ production. The possible role of early produced IFN-γ in the development of protective immune response to T. gondii is also discussed.     

Use of monoclonal antibody to potato leafroll virus for detecting groundnut rosette assistor virus by ELISA*

Three of 10 monoclonal antibodies (MAbs) produced to potato leafroll luteovirus (PLRV) were found to react in triple antibody sandwich ELISA (TAS-ELISA) with groundnut rosette assistor luteovirus (GRAV), though none reacted with four other luteoviruses (barley yellow dwarf, bean leaf roll, beet western yellows or carrot red leaf)- The most effective PLRV MAb, SCR 6, was used in TAS-ELISA to detect isolates of GRAV from groundnut plants with chlorotic, green and mosaic forms of rosette from Nigeria and Malawi. The test also detected GRAV in extracts of single Aphis craccivora.     

Production and Characterization of Monoclonal Antibodies against the Hemolysin BL Enterotoxin Complex Produced by Bacillus cereus

A total of five hybridoma cell lines that produced monoclonal antibodies against the components of the hemolysin BL (HBL) enterotoxin complex and sphingomyelinase produced by Bacillus cereus were established and characterized. Monoclonal antibody 2A3 was specific for the B component, antibodies 1A12 and 8B12 were specific for the L₂ component, and antibody 1C2 was specific for the L₁ protein of the HBL enterotoxin complex. No cross-reactivity with other proteins produced by different strains of B. cereus was observed for monoclonal antibodies 2A3, 1A12, and 8B12, whereas antibody 1C2 cross-reacted with an uncharacterized protein of approximately 93 kDa and with a 39-kDa protein, which possibly represents one component of the nonhemolytic enterotoxin complex. Antibody 2A12 finally showed a distinct reactivity with B. cereus sphingomyelinase. The monoclonal antibodies developed in this study were also successfully applied in indirect enzyme immunoassays for the characterization of the enterotoxic activity of B. cereus strains. About 50% of the strains tested were capable of producing the HBL enterotoxin complex, and it could be demonstrated that all strains producing HBL were also highly cytotoxic.     

Some properties of Vibrio vulnificus hemolysin

Some properties of hemolysin produced by Vibrio vulnificus were investigated. The hemolysin was heat labile, and the hemolytic activity was inhibited by adding cholesterol or divalent cations. Cholesterol inhibited the temperature-independent hemolysin-binding step, suggesting that cholesterol made up the binding site of the cell membrane, whereas the divalent cations inhibited the temperature-dependent membrane-degradation step. However, the V. vulnificus hemolysin was stable to oxygen and sulfhydryl reagents and was not inactivated by antiserum against streptolysin O, suggesting that the V. vulnificus hemolysin differs from oxygen-labile hemolysins which bind to cholesterol. The V. vulnificus hemolysin seems to be one of the exceptional cholesterol-binding hemolysins.     

Killer toxin secretion through the cell wall of the yeastPichia anomala

A secreted killer toxin was detected through the cell wall ofPichia anomala cells by ultrastructural immunodetection with a specific monoclonal antibody (MAb KT4). MAb KT4 was successively detected by colloidal gold labeled streptavidin and biotinylated anti-mouse F(ab')₂ antibodies. The antigenic determinants of the toxin were localized throughout the cytoplasm and the cell wall of killer yeast cells. The Lowicryl K4M-immunogold method gave very satisfactory results and showed that the killer toxin was somewhat concentrated in the yeast cell wall layers before being exported into the medium. In agreement with previous reports, the binding of MAb KT4 suggested that theP. anomala killer toxin secretion did not result from a homogeneous diffusion across the yeast cell wall.Abbreviations G15 gold particles of 15 nm - IEM immunoelectron microscopy - IFA immunofluorescence assay - MAb monoclonal antibody - PBS phosphate buffered saline - SAM/F(ab)₂ sheep antibodies anti-mouse F(ab)₂ - SBB Sabouraud buffered broth     

Establishment of Monoclonal Antibodies Specific for Bacillus subtilis DB9011

Bacillus subtilis DB9011 is a strain with useful functions for agriculture. To establish a method for the discrimination of this strain from others, monoclonal antibodies (MAbs) were prepared. Although two established MAbs (MAb9B6 and MAb14D2) cross-react with some other Bacillus strains in ELISA, only B. subtilis DB9011 vegetative cells are recognized by both MAbs. MAb14D2 recognizes flagellin, a 34-kDa unit protein of flagella. The two MAbs established will provide powerful tools with which detailed analysis of this bacterial strain can be obtained under environmental conditions.     

Monoclonal Antibodies againstAntigens of Hydra vulgaris and Hydra oligactis

The goal of the study was to obtain a panel of monoclonal antibodies (MAb) against antigens of freshwater polyps of the genus Hydra. Hybrid mice F₁(Balb/c × SJL/J) were immunized with cell membrane fraction of H. vulgaris and three months later their splenocytes were fused with cultured mouse myeloma cells 653A. Testing of culture fluids in ELISA with immobilized H. vulgaris cells, 82 hybridomas producing MAb were revealed. Study of MAb specificity in ELISA with H. vulgaris and H. oligactis cells indicated that 22% of them recognized only H. vulgaris antigens. About 50% of MAb recognized equally antigens of the both species. The rest of MAb reacted with H. vulgaris and H. oligactis antigens to different degree. Eight hybridomas producing MAb of all three above groups were adapted for growth as ascitic tumors. The distribution of antigens binding these MAb was studied in indirect immunofluorescence on fixed polyps, living or fixed cells, and on paraffin- embedded sections. Among the best studied MAb, of the greatest interest were the following reagents. One of them (1A10) revealed an antigen on surface membranes of ectodermal epithelial cells of H. vulgaris. The second one (1G10) was specific of the antigen located in mesoglea and basal cytoplasmic areas of ectodermal and entodermal epithelial cells of the both hydra species. The MAb 4G3 interacted with cytoplasmic antigen of ectodermal epithelia-muscular cells of the both hydra species. MAb 4H1 revealed nematocytes in H. vulgaris and H. oligactis. The data obtained indicate that in two species of hydra the epitopes binding the same MAb might be located in cells of different types.     

Differences between macrophage migration inhibitions by lymphokines and muramyl dipeptide (MDP) or lipopolysaccharide (LPS): migration enhancement by lymphokines

To investigate the repertoire of molecules which are associated with cytolytic T-lymphocyte (CTL)-mediated killing, function-blocking monoclonal antibodies (MAb) have been selected and characterized. Spleen cells from rats immunized with secondary mouse CTL were fused with mouse myeloma cells. Antibodies secreted by 2400 hybrid cultures were selected solely by their ability to block CTL-mediated killing in a mouse anti-rat xenogeneic system. Fifteen cultures with antibodies which blocked CTL-mediated killing were chosen for cloning and further characterized by immunoprecipitation and immunofluorescence flow cytometry. One group of five monoclonal antibodies recognized the Lyt-2,3 molecule of 35,000 M_r. The second group of six MAb recognized the LFA-1 antigen containing two subunits of 180,000 and 95,000 M_r. One MAb giving only partial inhibition of killing was an IgM anti-Thy-1. It strongly agglutinated CTL. The target antigens defined by three other MAb were not definitively identified. Competition in cell binding between anti-Lyt-2,3 and anti-LFA-1 MAb suggested that their blocking effect in cytolysis is due to binding to distinct and spatially separate molecules on effector cells. The results of direct screening for functional blockade support the important role of Lyt-2,3 and LFA-1 molecules in T-cell-mediated cytolysis.     

Development of monoclonal antibody against isoquinoline alkaloid coptisine and its application for the screening of medicinal plants

In the development of immunoassay technique, the design of hapten containing a functional group suitable for protein conjugate is the key step for the preparation of antibodies against small molecules. Coptisine (MW 320), a bioactive constituent of Berberis and Coptis species, is small as an immunogen. In addition, coptisine has no reactive group in molecule for conjugating with a protein. To overcome this problem, 9-O-carboxymethyl-berberrubine was designed and conjugated with carrier protein. In order to confirm its immunogenicity, the ratio of hapten in the conjugate was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). After immunization, hybridomas secreting antibodies against coptisine were produced by fusing splenocytes with mouse myeloma cell line, P3-X63-Ag8-653. Among hybridomas, the clone 2A1 secreting anti-coptisine monoclonal antibody (MAb) 2A1-9E-1 was obtained through the limited dilution method. The MAb-based enzyme-linked immunosorbent assay (ELISA) against coptisine was developed and characterized. The linear range of the assay in this ELISA method was extended from 1.56 to 25 μg ml⁻¹ possessing the detection limit of 1.56 μg ml⁻¹. The established ELISA using MAb 2A1-9E-1 was applied for the survey of isoquinoline alkaloids in various medicinal plants.     

Production and Characterization of Monoclonal Antibodies against Vegetative Cells of Bacillus cereus

Two monoclonal antibodies (MAbs) against Bacillus cereus were produced. The MAbs (8D3 and 9B7) were selected by enzyme-linked immunosorbent assay for their reactivity with B. cereus vegetative cells. They reacted with B. cereus vegetative cells while failing to recognize B. cereus spores. Immunoblotting revealed that MAb 8D3 recognized a 22-kDa antigen, while MAb 9B7 recognized two antigens with molecular masses of approximately 58 and 62 kDa. The use of MAbs 8D3 and 9B7 in combination to develop an immunological method for the detection of B. cereus vegetative cells in foods was investigated.