Hemagglutinin specificity of Cl. botulinum types A, B, and F in reaction with erythrocytes of various animals]

The following differences were revealed in the haemagglutination reaction with the erythrocytes of man, sheep, rabbit, chicks and mice between the haemagglutinins of Cl. botulinum, types A, B and F, having a close affinity with one another: haemagglutinin of type A actively reacted with the erythrocytes of man, sheep, rabbit, rats and chicks; haemagglutinin of type B reacted only with the erythrocytes of man and rabbits; haemagglutinin of type F failed to react with any of the types of the erythrocytes tested; only with the use of erythrocytes treated with neuraminidase was it possible to establish the presence of haemagglutinin fraction in Cl. botulinum, type F. Treatment of human erythrocytes with neuraminidase and proteolytic enzymes caused a marked increase in the sensitivity of the haemagglutination reaction in haemagglutinins of Cl. botulinum, types A, B and F.     

Evidence for the production of a novel proteinaceous hemolytic exotoxin by dinoflagellate Alexandrium taylori

We found that a whole cell suspension of Alexandrium taylori, which is toxic to Artemia, causes species-specific hemolysis against mammalian erythrocytes. Among the erythrocytes tested, rabbit and guinea-pig erythrocytes were highly sensitive, but human, sheep, and cattle erythrocytes were insensitive. The cell-free culture supernatant also showed potent hemolytic activity toward rabbit erythrocytes as seen in whole cell suspension. The hemolytic activity in the culture medium gradually increased with increase in cell number during exponential growth phase, and relatively high activity was maintained even after reaching the death phase. These results suggest that the hemolytic substance is actively released into the medium from A. taylori cells rather than simple leakage from ruptured or dead cells, and a part of them are steadily accumulated in the medium during the algal growth. Chemical characterization with ultrafiltration and trypsin-treatment suggested that the hemolytic substance released into the medium is protein-like compound with molecular weight more than 10,000 Da. The ammonium sulfate precipitated fraction obtained from the cell-free supernatant of A. taylori showed cytotoxic effect on HeLa cells as well as the hemolytic activity in a similar concentration range on a protein content basis. Our results suggest that A. taylori produces a novel proteinaceous hemolytic exotoxin.     

Investigation of the cyt gene in Bacillus thuringiensis and the biological activities of Bt isolates from the soil of China

The presence of cyt genes was investigated in 80 type strains of Bacillus thuringiensis and 143 isolates obtained from soil samples of China by PCR amplification using two pairs of primers for the cyt1 and cyt2 genes. Three type strains of serotypes H11ac, H14 and H36, eight isolates belonging to H3, H14, H18 and H21, and one isolate of unknown serotype harbored cyt genes. We also tested the cytolytic activity for mammal cells, the hemolytic activity for sheep erythrocytes and insecticidal activity against mosquitoes of five isolates that contained cyt genes but did not belong to B. thuringiensis serovar israelensis. The protein profiles of the five isolates were different from those of the type strains of B. thuringiensis serovar israelensis, and among the five isolates, only Y-5 showed mosquitocidal activity against larvae of Culex quinquefasciatus. All five of the isolates exhibited hemolytic activity, but only three could cause the cell death of A549 cells. The cytopathological changes induced by NX-4 in some A549 cells were characterized with cell-ballooning.     

Haemagglutinin activity in Acrididae (grasshopper) haemolymph

The haemolymph of Acrididae causes haemagglutination of human and animal erythrocytes. Thirteen of seventeen species tested had detectable activity and gave agglutination titres in the range 2–64, Melanoplus bivittatus, and M. sanguinipes showed greatest activity. Haemagglutinin activity is continuously present in male and female insects from 4th instar and throughout adulthood. Females contain slightly more activity than do males. M. sanguinipes haemolymph agglutinates rabbit, calf, human (all ABO types) guinea pig, mouse, chicken, cat, pig and sheep erythrocytes. Rabbit red cells are agglutinated most strongly and sheep and chicken cells least. M. sanguinipes haemolymph also agglutinates the protozoan Nosema locustae, a natural grasshopper pathogen. Preabsorption of haemolymph with different erythrocyte types selectively removes haemagglutinin activity suggesting the presence of multiple or heteroagglutinins. M. sanguinipes haemagglutinin is inhibited by glycoproteins, simple carbohydrates and carbohydrate derivatives. The inhibitory pattern is complex and among the sugars tested only mannose and derivatives of mannose are exclusively non-inhibitory. Haemolymph haemagglutinin activity is destroyed by heat and EDTA. It is totally precipitated by dialysis against water and may be partially recovered in phosphate or Tris buffer. Activity is stable in frozen haemolymph.     

Counterimmunoelectrophoresis and opportunistic fungal infections

Sera from 35 apparently normal humans, 37 compromised human patients, 30 hedgehogs and 30 sheep, were examined for precipitating antibodies to four opportunistic fungi — Absidia corymbifera, Aspergillus fumigatus, Candida albicans and Rhizopus arrhizus — using Counterimmunoelectrophoresis (CIE).Precipitins to A. fumigatus were almost exclusively confined to specimens obtained from the compromised human group (51% of those examined) while Candida precipitating antibodies were detected in the sera of both normal (26%) and compromised (49%) humans and in 10% of the hedgehog specimens. Serum precipitins against the two phycomycetes included in the investigations were rare.Because of the complexity of most fungal antigen extracts, it appears essential that sera be tested against a number of different antigen concentrations if CIE is to be used with confidence in fungal serology.     

Monoclonal antibodies that react with human band 3 residues 542–555 recognize different conformations of this protein in uninfected andPlasmodium falciparum infected erythrocytes

A monoclonal antibody generated against synthetic peptides patterned on amino acids 542–555 of human band 3, designated 1F4, specifically immunostainedPlasmodium falciparum-infected erythrocytes and inhibited the cytoadherence ofP. falciparum-infected erythrocytes to C32 amelanotic melanoma cells. 1F4 did not recognize intact band 3 protein on immunoblots, however it was reactive towards proteolytic fragments of band 3.The binding region of another murine monoclonal antibody previously reported to recognize the membrane spanning domain of human band 3, designated B6, was found to also recognize residues 542–555, however its properties differed from 1F4. Mab B6 recognized both infected and uninfected red cells, and reacted only with intact band 3 on immunoblots. Mab B6 was without effect on cytoadherence.These results demonstrate that monoclonal antibodies reactive against a common peptide sequence may bind to different conformations of the peptide sequence and suggest that the adherent competency ofP. falciparum-infected erythrocytes may result from a change in the surface topography of human band 3 protein.Abbreviations ELISA Enzyme-Linked Immunosorbent Assay - KLH Keyhole Limpet Hemocyanin - PBS Phosphate Buffered Saline - Mab Monoclonal Antibody - PMSF Phenylmethyl sulfonyl fluoride - i.p. intraperitoneum - TBS Tris Buffered Saline - H₂DIDS dihydro 4,4-diisothocyanostilbene-2,2-disulfonic acid - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid     

Time-dependent mode of immunization augments suppressed antibody responses in spleen cell cultures from mice experimentally infected with Trypanosoma cruzi

Mice infected with Trypanosoma cruzi develop immunosuppressed responses to heterologous antigens. Experiments were performed using infected mice in the acute stage of infection to assess immunoregulatory activities during induction of direct plaque-forming cells (DPFC) to sheep erythrocytes (SRBC). After normal or infected mice were primed with SRBC, their spleen cells were restimulated 4 days later with SRBC in Mishell-Dutton cultures and found to mount hyperaugmented IgM anti-SRBC responses. It was also demonstrated that T-cells derived from normal mice primed in vivo 4 days previously with SRBC, and subsequently added to cultures of spleen cells from T. cruzi-infected mice, enhanced anti-SRBC DPFC responses in a dose-dependent fashion. These results show that functional help provided by T-cells activated during an in vivo priming and exposed to an in vitro challenge dose of antigen (SRBC) in a time-dependent mode can overcome the effect of immunosuppression in the spleen cell cultures from T. cruzi-infected mice.     

Kinetics of Experimental Infection of Sheep with Ehrlichia ruminantium Cultivated in Tick and Mammalian Cell Lines

Inoculation of sheep with Ehrlichia (previously Cowdria) ruminantium which has been cultivated in mammalian endothelial cell cultures is almost always followed by a severe clinical reaction, whereas inoculation of the agent cultivated in tick cell lines usually does not provoke a clinical response, but may result in seroconversion and/or protection against subsequent challenge with virulent stabilates. A quantitative, real-time PCR assay was developed to determine the kinetics of infection (rickettsaemia) in sheep inoculated with tick cell- and mammalian cell-derived E. ruminantium (Gardel isolate). The method and initial results are described, and the significance of the findings is discussed in relation to the clinical responses of the sheep. This revised version was published online in July 2006 with corrections to the Cover Date.     

Degradation of keratinous materials by the plant pathogenic fungus <Emphasis Type="Italic">Myrothecium verrucaria</Emphasis>

In this paper it is described for the first time the capability of Myrothecium verrucaria to grow in submerged and solid state cultures using poultry feathers as the only substrate. The fungus produced a protease with an unusual keratinolytic activity among plant pathogenic fungi. Its crude protease hydrolyzed keratinous substrates at pH 9.0 and 40 °C in the following order: poultry feather keratin > sheep wool keratin > human nail keratin > human hair keratin. Protease activity was highly sensitive to phenylmethyl sulphonyl fluoride (PMSF) indicating that the enzyme belonged to the serine protease family.     

The monoclonal antibody,anti-asialoglycophorin from human erythrocytes,specific forβ-d-Gal-1-3-α-d-GalNAc-chains (Thomsen-Friedenreich receptors)

The monoclonal antibody 22.19 of IgM class obtained after immunization of BALB/c mice with asialoglycophorin of human erythrocyte membranes is described. The specificity of this antibody for -d-Gal-1-3--d-GalNAc- disaccharide chains (Thomsen-Friedenreich receptors) was established by studying its reactivity against various erythrocytes, glycoproteins and oligosaccharides and by comparison with two lectins, peanut agglutinin andVicia graminea lectin, which recognize these disaccharide chains.Abbreviations PNA peanut agglutinin - VgL Vicia graminea lectin - TF Thomsen-Friedenreich - HSA human serum albumin - MoAb monoclonal antibody     

Purification and characterization of a novel haemagglutinin from Chlorella pyrenoidosa

We previously identified a strong haemagglutination activity in the freshwater unicellular green alga, Chlorella pyrenoidosa. Here, we sought to purify and characterize the haemagglutinin associated with this activity. Ammonium sulfate precipitation, gel filtration on sephacryl S-200 and DEAE-Sepharose ion-exchange chromatography were used to purify the haemagglutinin, which was designated CPH (Chlorella pyrenoidosa haemagglutinin). The molecular weight of CPH was estimated as 58 kDa by SDS-PAGE and 60 kDa by gel filtration of the native protein, indicating that this haemagglutinin exists as a monomer. The haemagglutinin activity of CPH was inhibited by glycoproteins, especially yeast mannan, but not by monosaccharides or disaccharides, indicating that CPH is carbohydrate-specific. In addition to the composition of CPH shown to be rich in glycine and acidic amino acids, heamagglutinating activity of CPH was insensitive to variations in pH or the presence of divalent cations, and atomic force microscopy revealed that the protein is rod-shaped. These results indicate that the characteristics of CPH are consistent with its identification as a haemagglutinin, and suggest that CPH may be a viable candidate for applications in a variety of biomedical fields.     

High level of soluble expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> and characterisation of the CyaA pore-forming fragment from a <Emphasis Type="Italic">Bordetella pertussis</Emphasis> Thai clinical isolate

Bordetella pertussis adenylate cyclase toxin-haemolysin (CyaA) can permeabilise erythrocytes by forming lytic pores. Here, a gene segment encoding CyaA pore-forming (CyaA-PF) domain cloned from genomic DNA of B. pertussis Thai isolate was over-expressed in Escherichia coli as a 126-kDa soluble protein which cross-reacted with anti-RTX monoclonal antibody. By co-expressing with acyltransferase CyaC, the CyaA-PF protein was found palmitoylated at Lys⁹⁸³. Unlike E. coli lysate with the non-acylated form, the lysate containing acylated CyaA-PF exhibited high haemolytic activity against sheep erythrocytes. This study presents that the recombinant CyaA-PF protein comprising pore-forming domain can be expressed separately as soluble native-folded precursor that conserves at least part of its functionality.     

Interactions between Cylindrocarpon destructans and ectomycorrhizas of Picea abies with Laccaria laccata and Paxillus involutes

Summary In paired cultures with two mycorrhizal fungi, the root pathogen Cylindrocarpon destructans (Zinsm.) Scholten had an inhibitional effect on mycelial growth of Laccaria laccata (Scop, ex Fr.) Bk. & Br. but was inhibited itself by Paxillus involutes (Batsch.) Fr. A similar pathogen-symbiont interaction scheme was observed in triaxenic cultures with Picea abies Karst. seedlings but only in the vicinity of the mycorrhizal root tips. Both mycorrhizas similarly increased the endogenous plant resistance against the infection of C. destructans. This suggests that direct pathogen-symbiont interactions are an important factor for population dynamics in the mycorrhizo sphere. Moreover, endogenous plant resistance constitutes one of the key factors for an effective defence against pathogenic fungi.     

Dichelobacter nodosus, Fusobacterium necrophorum and the epidemiology of footrot

Footrot is a debilitating disease of sheep resulting in lameness, production losses and suffering. To study the basic bacteriology of the disease, a survey was initiated across commercial farms and non-commercial research flocks to compare the bacteriology of symptomatic footrot infected sheep with healthy asymptomatic sheep. Of the 80 farmers initially contacted, 14 collected hoof swabs and returned the swabs by post. Following DNA extraction, species-specific PCR was used to identify if Dichelobacter nodosus (D. nodosus) or Fusobacterium necrophorum (F. necrophorum) species were present on each swab. Of the 42 swabs taken from symptomatic footrot infected sheep, 17 were positive for both F. necrophorum and D. nodosus, two were positive for F. necrophorum only, two for D. nodosus only and 23 swabs were negative for both F. necrophorum and D. nod osus. Of the 50 swabs received from healthy asymptomatic sheep, one was positive for F. necrophorum only and 49 were negative for both D. nodosus and F. necrophorum. This suggests that both F. necrophorum and D. nodosus are linked to footrot in the field in a pastoral farming system. If these bacteria are linked together and collectively cause footrot, this may need to be considered when managing a footrot outbreak, or maintaining a quarantine.     

Characterization of SSEA-1 glycolipids from the brain of a patient with fucosidosis

Neutral glycolipids from the brain of a patient with Fucosidosis were analyzed and two complex glycolipids containing five and eight sugars were isolated from the cortical grey matter. These two glycolipids reacted with antibodies recognizing the SSEA-1 [Le^x(X)] carbohydrate determinant. SSEA-1 glycolipids are normally expressed in human embryonic brain but are found in only small amounts in postnatal human brain. The accumulation of the two SSEA-1 glycolipids in Fucosidosis brain thus represents a defect which affects the normal developmentally regulated decrease in postnatal, expression of these glycolipids, and may be a contributing factor in the abnormal brain development associated with the disease. Chemical characterization of the two isolated glycolipids by gas chromatographic and mass spectrometric analyses has identified the two glycolipids as lacto-N-fucopentaosylceramide (III) and difucosyl-neolactonorhexaosylceramide.Abbreviations DCl direct chemical ionization - FAB tastatiom bombardment - GC gas chromatography - GSLs glycosphingolipids - MS mass spectrometry - SSEA-1 stage specific embryonic antigen-1 - TLC thin layer chromatographys     

Binding and activity ofBacillus thuringiensis delta-endotoxin to invertebrate cells

Fluorescein isothiocyanate was used as a label to detect delta-endotoxin ofBacillus thuringiensis subsp.thuringiensis andisraelensis in binding studies with different in vitro cell systems. Protoxin of the subspeciesthuringiensis could be labelled directly whereas the activated toxin had to be traced indirectly with labelled antibodies. Both protoxin and activated toxin bound to primary midgut cell cultures ofPieris brassicae larvae as well as to cells of an established culture ofDrosophila melanogaster. No binding with either toxin form could be observed with hemocytes ofP. brassicae. Biological activity as shown by the trypan blue viability assay was obtained only with the activated toxin against the midgut cells. Toxin of the subspeciesisraelensis reacted very unspecifically. Binding followed by rapid destruction was obtained with all the tested cultures.Abbreviation FITC fluorescein isothiocyanate     

Evaluation of glycoconjugates on the K562 cell surface by means of lectin binding — comparison with human erythrocytes

The binding of five radiolabelled lectins (Vicia graminea, peanut,Phaseolus vulgaris isolectins E-PHA and L-PHA,Evonymus europaeus) to untreated and desialylated K562 cells and human erythrocytes was compared. The number of glycophorin A receptors recognized on the K562 cells by anti-blood group NV. graminea lectin was comparable to that found on the MN or NN erythrocyte surface. However, K562 cells had a severalfold higher number of oligosaccharide chains (presumablyO-glycosidic) which after desialylation became high-affinity receptors for peanut agglutinin, and of complex typeN-glycosidic chains available for the reaction with E-PHA and also with L-PHA (the latter lectin was not bound to erythrocytes). Moreover, K562 cells not treated with neuraminidase had a significant amount of extremely low affinity receptors for peanut agglutinin, whereas binding of this lectin to untreated erythrocytes was undetectable. On the other hand, the untreated K562 cells did not bind anti-blood group B and HE. europaeus lectin, but a small amount of binding by the desialylated cells was observed. Some other differences observed in the mode of lectin binding to K562 cells and erythrocytes are discussed.     

Virulence and antibiotic susceptibility of Aeromonas spp. isolated from drinking water

Aeromonas isolates from tap water, mineral water, and artesian well water were investigated for their ability to produce different potential virulence factors or markers such as hemolysins, cytotoxins, phospholipase, DNase, hydrophobicity and their ability to adhere to epithelial cells and to abiotic surfaces. The susceptibility to antibiotics of Aeromonas isolates was also examined. Majority of the isolates displayed hemolytic activity against sheep erythrocytes, while only 7 of the 23 Aeromonas strains displayed DNase activity and 4 of the 23 Aeromonas strains tested were regarded as positive for phospholipase production. Most of the isolates showed cytotoxic activities in culture filtrate dilutions at titer of 1/8 or lower. No general relation between the strain isolated and the ability to interact with epithelial cells could be established. Using the bacterial adherence to hydrocarbons method, most of the strains were classified as highly hydrophilic. All five Aeromonas jandaei strains isolates, 9 of the 12 Aeromonas sp strains and four of the five Aeromonas hydrophila were multidrug resistant. The most active antimicrobial was ciprofloxacin (susceptible in 100% of the isolates), and the least active antibiotic was ampicillin (resistance in 92% of the isolates). The majority of the isolates tested were not killed by chlorine at 1.2 mg/l. Whether the high tolerance to chlorine of Aeromonas isolates can be linked to greater virulence is not know.     

Immunological analysis of hemoglobin transition during metamorphosis of normal and isogenicXenopus

Summary Antisera against larval and adultXenopus hemoglobins as well as adult human hemoglobin showed no cross-reaction when tested by immunodiffusion against each heterologous antigen. In this test hemoglobin of a single animal produced two precipitation lines for larvae, but only one for adult stages. Immunoelectrophoresis also revealed more complex precipitation patterns for larval than for adult hemoglobins. Hemoglobin of the isogenic hybrid cloneXenopus laevis/X. gilli also reacted with antisera against normalXenopus hemoglobin.Quantitation of hemoglobins, analyzed by radial immunodiffusion showed fewer than 1% of adult hemoglobin in red cells of larvae, but 30% at completion of metamorphosis. Two weeks later adult hemoglobin attained over 90%, and in red cells of adultXenopus an average of 1% larval hemoglobin were detected.The relatively short transition period suggests that the loss of larval hemoglobin may be due to the elimination of larval red cells, and that the increase in adult hemoglobin may be indicative of a new cell line.     

Emulsions of oil from Adenanthera pavonina L. seeds and their protective effect

In our previous study, we developed very stable formulations of submicron oil-in-water emulsions from Adenanthera pavonina L. (family Leguminosae, subfamily Mimosoideae) seed oil, stabilised with soybean lecithin (SPC). Continuing our research, we introduced an additional co-emulsifier, Tween 80, to those formulations in order to decrease the size of the emulsion particles and improve their stability. Formulations with a mean particle size ranging from 43.6 to 306.5 nm and a negative surface charge from −45.3 to −28.5 mV were obtained. Our stability experiments also revealed that most of the tested formulations had a very good degree of stability over a 3-month storage period, both at 4°C and at room temperature. Since many intravenous injectable drugs exhibit lytic activity against erythrocytes, we examined this activity for the emulsion form of cardol, a natural compound with already proven hemolytic properties. The incorporation of this agent into the emulsion caused an evident decrease in hemolytic activity (97–99%). This highly protective effect, observed against sheep erythrocytes, was independent of both the composition and the particle size of the emulsions used. Our studies suggest that nonionic surfactant/phospholipid-based emulsions containing this edible oil of A. pavonina L. may be useful as an alternative formulation matrix for pharmaceutical, nutritional or cosmetic applications of otherwise membrane-acting components.