Interaction of three-finger proteins from snake venoms and from mammalian brain with the cys-loop receptors and their models

With the use of surface plasmon resonance (SPR) it was shown that ws-Lynx1, a water-soluble analog of the three-finger membrane-bound protein Lynx1, that modulates the activity of brain nicotinic acetylcholine receptors (nAChRs), interacts with the acetylcholine-binding protein (AChBP) with high affinity, K_D = 62 nM. This result agrees with the earlier demonstrated competition of ws-Lynx1 with radioiodinated α-bungarotoxin for binding to AChBP. For the first time it was shown that ws-Lynx1 binds to GLIC, prokaryotic Cys-loop receptor (K_D = 1.3 μM). On the contrary, SPR revealed that α-cobratoxin, a three-finger protein from cobra venom, does not bind to GLIC. Obtained results indicate that SPR is a promising method for analysis of topography of ws-Lynx1 binding sites using its mutants and those of AChBP and GLIC.     

Possible involvement of neuronal nicotinic acetylcholine receptors in compensatory brain mechanisms at early stages of Parkinson’s disease

A role of nicotinic acetylcholine receptors (nAChR) in the development of Parkinson’s disease (PD) has been investigated using two mouse models corresponding to the presymptomatic stage and the early symptomatic stage of PD. Quantitative radioligand analysis of nAChR in the striatum and substantia nigra (SN) was performed using the radioactive derivatives of epibatidine, α-conotoxin MII, and α-bungarotoxin. These are selective ligands for different nAChR subtypes. The number of ligand-binding sites changed differently depending on their location in the brain, the stage of the disease and the receptor subtype. In the striatum epibatidine binding decreased by 66% and 70% at the presymptomatic and early symptomatic stages, respectively, while in SN epibatidine binding demonstrated a significant (160%) increase at the presymptomatic stage. The α-conotoxin MII binding to striatal dopaminergic axonal terminals at the presymptomatic stage decreased by 20% and at the symptomatic stage it demonstrated a further decrease. Striatal α-bungarotoxin binding increased at the presymptomatic stage and decreased at the early symptomatic stage. In SN, the level of α-bungarotoxin binding decreased at the presymptomatic stage and remained constant at the symptomatic stage. A significant decrease in the expression of Chrna4 and Chrna6 genes encoding α4 and α6 nAChR subunits was observed in SN at the early symptomatic stage, while a 13-fold increase in expression of the Chrna7 gene encoding the α7 nAChR subunit was detected at the presymptomatic stage. The data obtained on the altered mRNA levels or functional cholinergic receptors suggest possible involvement of nAChR in compensatory mechanisms at early PD stages.     

<Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry11Ba works synergistically with Cry4Aa but not with Cry11Aa for toxicity against mosquito <Emphasis Type="Italic">Culex pipiens</Emphasis> (Diptera: Culicidae) larvae

A 2,175-bp modified gene (cry11Ba-S1) encoding Cry11Ba from Bacillus thuringiensis subsp. jegathesan was designed and the recombinant protein was expressed as a fusion protein with glutathione S-transferase in Escherichia coli. The recombinant Cry11Ba was highly toxic against Culex pipiens mosquito larvae, being nine and 17 times more toxic than mosquitocidal Cry4Aa and Cry11Aa from Bacillus thuringiensis subsp. israelensis, respectively. Interestingly, a further increase in the toxicity of the recombinant Cry11Ba was achieved by mixing with Cry4Aa, but not with Cry11Aa. These findings suggested that Cry11Ba worked synergistically with Cry4Aa, but not with Cry11Aa, in exhibiting toxicity against C. pipiens larvae. On the other hand, the amount of Cry toxin bound to brush border membrane vesicles (BBMVs) did not significantly change between individual toxins and the toxin mixtures, suggesting that the increase in toxins binding to BBMVs was not a reason for the observed synergistic effect. It is generally accepted that synergism of toxins is a potentially powerful tool for enhancing insecticidal activity and managing Cry toxin resistance in mosquitoes. The mixture of Cry4Aa and Cry11Ba in order to increase toxicity would be very valuable in terms of mosquito control.     

Production of fluorescent and cytotoxic K28 killer toxin variants through high cell density fermentation of recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Background

Virus infected killer strains of the baker’s yeast Saccharomyces cerevisiae secrete protein toxins such as K28, K1, K2 and Klus which are lethal to sensitive yeast strains of the same or related species. K28 is somewhat unique as it represents an α/β heterodimeric protein of the A/B toxin family which, after having bound to the surface of sensitive target cells, is taken up by receptor-mediated endocytosis and transported through the secretory pathway in a retrograde manner. While the current knowledge on yeast killer toxins is largely based on genetic screens for yeast mutants with altered toxin sensitivity, in vivo imaging of cell surface binding and intracellular toxin transport is still largely hampered by a lack of fluorescently labelled and biologically active killer toxin variants.

Results

In this study, we succeeded for the first time in the heterologous K28 preprotoxin expression and production of fluorescent K28 variants in Pichia pastoris. Recombinant P. pastoris GS115 cells were shown to successfully process and secrete K28 variants fused to mCherry or mTFP by high cell density fermentation. The fluorescent K28 derivatives were obtained in high yield and possessed in vivo toxicity and specificity against sensitive yeast cells. In cell binding studies the resulting K28 variants caused strong fluorescence signals at the cell periphery due to toxin binding to primary K28 receptors within the yeast cell wall. Thereby, the β-subunit of K28 was confirmed to be the sole component required and sufficient for K28 cell wall binding.

Conclusion

Successful production of fluorescent killer toxin variants of S. cerevisiae by high cell density fermentation of recombinant, K28 expressing strains of P. pastoris now opens the possibility to study and monitor killer toxin cell surface binding, in particular in toxin resistant yeast mutants in which toxin resistance is caused by defects in toxin binding due to alterations in cell wall structure and composition. This novel approach might be easily transferable to other killer toxins from different yeast species and genera. Furthermore, the fluorescent toxin variants described here might likewise represent a powerful tool in future studies to visualize intracellular A/B toxin trafficking with the help of high resolution single molecule imaging techniques.

     

Porcine and bovine <Emphasis Type="Italic">Clostridium difficile</Emphasis> ribotype 078 isolates demonstrate similar growth and toxigenic properties

Clostridioides (C.) difficile are found in cows, pigs and poultry suggesting that this pathogen is present and more importantly animals could act as a reservoir, via food or environment, of human C. difficile infection. Molecular methods together with phenotypical characterisation bring integrated and important tools to describe diversity and nature of bacteria including C. difficile. Moreover, similar or identical C. difficile types are found in different farm animals. This study aimed to phenotypically characterise C. difficile isolates belonging to ribotype 078 and to identify differences such as growth and toxicity between porcine and bovine isolates. C. difficile isolates were assessed for the growth behaviour (turbidimetry), metabolic potential (Biolog AN) and toxin production (ELISA method) in vitro. The concentration of released either toxin A (TcdA) or toxin B (TcdB) varied greatly between the isolates tested; however, it did not differ between the porcine and bovine ribotypes. Also, the TcdA/TcdB ratio of the isolates did not show a difference either. The most common metabolised substrates were pyruvic acid followed by α-ketobutyric acid. The results show that both porcine and bovine C. difficile RT 078 share similar phenotypical characteristics including growth and production of toxins. The findings may help understand the virulence of C. difficile RT 078 in porcine and bovine species.     

Colon-Targeted Delivery of IgY Against <Emphasis Type="Italic">Clostridium difficile</Emphasis> Toxin A and B by Encapsulation in Chitosan-Ca Pectinate Microbeads

This study investigated the use of a newly developed chitosan-Ca pectinate microbead formulation for the colon-targeted delivery of anti-A/B toxin immunoglobulin of egg yolk (IgY) to inhibit toxin binding to colon mucosa cells. The effect of the three components (pectinate, calcium chloride, and chitosan) used for the microbead production was examined with the aim of identifying the optimal levels to improve drug encapsulation efficiency, swelling ratio, and cumulative IgY release rate. The optimized IgY-loaded bead component was pectin 5% (w/v), CaCl₂ 3% (w/v), and chitosan 0.5% (w/v). Formulated beads were spherical with 1.2-mm diameter, and the drug loading was 45%. An in vitro release study revealed that chitosan-Ca pectinate microbeads inhibited IgY release in the upper gastrointestinal tract and significantly improved the site-specific release of IgY in the colon. An in vivo rat study demonstrated that 72.6% of biologically active IgY was released specifically in the colon. These results demonstrated that anti-A/B toxin IgY-loaded chitosan-Ca pectinate oral microbeads improved IgY release behavior in vivo, which could be used as an effective oral delivery platform for the biological treatment of Clostridium difficile infection (CDI).     

Stability of fumonisin B<Subscript>1</Subscript>, deoxynivalenol,zearalenone, and T-2 toxin during processing of traditional Nigerian beer and spices

The stability of the Fusarium mycotoxins fumonisin B₁, deoxynivalenol, T-2 toxin, and zearalenone during processing of Nigerian traditional spices (dawadawa, okpehe, and ogiri) and beer (burukutu) using artificially contaminated raw materials was investigated. Results revealed the reduction of these toxins in all the final products. Boiling played a significant role (p?<?0.05) in Fusarium mycotoxin reduction in the traditional spices. The highest percentage reduction of deoxynivalenol (76%) and zearalenone (74%) was observed during okpehe processing (boiled for 12 h). Dehulling and fermentation further demonstrated a positive influence on the reduction of these toxins with a total reduction ranging from 85 to 98% for dawadawa, 86 to 100% for okpehe, and 57 to 81% for ogiri. This trend was also observed during the production of traditional beer (burukutu), with malting and brewing playing a major impact in observed reduction. In addition, other metabolites including deoxynivalenol-3-glucoside, 15-acetyl-deoxynivalenol, α-zearalenol, and β-zearalenol which were initially not present in the raw sorghum were detected in the final beer product at the following concentrations 26?±?11, 16?±?7.7, 22?±?18, and 31?±?16 μg/kg, respectively. HT-2 toxin was also detected at a concentration of 36?±?13 μg/kg along the processing chain (milled malted fraction) of the traditional beer. For the traditional spices, HT-2 toxin was detected (12 μg/kg) in ogiri. Although there was a reduction of mycotoxins during processing, appreciable concentrations of these toxins were still detected in the final products. Thus, the use of good quality raw materials significantly reduces mycotoxin contamination in final products.     

Comparison of Alleles at <Emphasis Type="Italic">Gli-2</Emphasis> Loci of Common Wheat by Means of Two-Dimensional Electrophoresis of Gliadin

Electrophoretic mobility (EM) and molecular weight (MW) of some allelic variants of α- and β-gliadins contrlled by Gli-2 loci were compared by means of two-dimensional (APAGE × SDS) electrophoresis. Comparison of α-gliadins of the alleles Gli-A2b and Gli-A2p, of β-gliadins of the Gli-B2b and Gli-B2c, and of β-gliadins of the Gli-D2b, Gli-D2c, Gli-D2j, and Gli-D2r indicated that a gliadin with lower EM had, as a rule, bigger MW which is known to depend on the length of the polyglutamine domain of gliadin of α-type. However, allelic variants of the α-gliadin encoded by Gli-D2b and Gli-D2e differ in EM but not in apparent MW. It might be caused by a substitution of some charged/uncharged aminoacids in the polypeptide of gliadin. Allele Gli-B2o which is very frequent in up-to-date common wheat germplasm originated probably by means of unequal crossingover. Some alleles at Gli-A2 is found to control completely different blocks of gliadins and therefore might come to common wheat from different genotypes of the polymorphic diploid donor of the A genome. The results indicate that the reason of the known more vast polymorphism of gliadins controlled by Gli-2 loci as compared with Gli-1 loci is the considerable difference of the structure, first, of Gli-1 and Gli-2 loci (Gli-2 loci have more expressed genes per locus) and, second, of genes encoding gliadins of α- and γ-types (α-gliadins are shown to contain a long polyglutamine sequences highly variable in their length).     

New weak toxins from the cobra venom

A protein with M 7485 Da containing five disulfide bonds was isolated from the venom of cobra Naja oxiana using various types of liquid chromatography. The complete amino acid sequence of the protein was determined by protein chemistry methods, which permitted us to assign it to the group of weak toxins. This is the first weak toxin isolated from the venom of N. oxiana. In a similar way, two new toxins with M 7628 and 7559 Da, which fall into the range of weak toxin masses, were isolated from the venom of the cobra N. kaouthia. The characterization of these proteins using Edman degradation and MALDI mass spectrometry has shown that one of these proteins is a novel weak toxin, and the other is the known weak toxin WTX with an oxidized methionine residue in position 9. Such a modification was detected in weak toxins for the first time. A study of the biological activity of the toxin from N. oxiana showed that, like other weak toxins, it can be bound by α7 and muscle-type nicotinic acetylcholine receptors.     

Expression of <Emphasis Type="Italic">GNA</Emphasis> and biting site-restricted <Emphasis Type="Italic">cry1Ac</Emphasis> in cotton; an efficient attribution to insect pest management strategies

Insect-resistant transgenic cotton has been commercialized for two decades. Most of the introduced cultivars express Bt gene(s) constitutively under the control of 35S promoter in whole-plant tissues. However, there have been other promoters considered by researchers to confine the toxin expression to targeted organ and tissues. We developed a triple-gene construct including GNA, cry1Ac and cp4 epsps genes. We attempted to confine cry1Ac expression to insect biting sites by cloning it to downstream of a wound-inducible promoter isolated from Asparagus officinalis (AoPR1). Moreover, to broaden the range of resistance, GNA was driven by the 35S promoter to target the sap-sucking insects like aphids which impose large losses in cotton production. To select the transformants in selection medium and for glyphosate tolerance, GNA and cry1Ac genes were accompanied with cp4 epsps gene. Two binary vectors harboring desired genes were constructed and utilized in the study (pGTGNAoC1AC and pGTGN35C1AC). Transformation of cultivar GSN-12 was carried out by employing Agrobacterium tumefaciens strain EHA105. Plantlets were primarily screened under glyphosate (N-phosphonomethyl glycine) selection pressure and subsequently subjected to molecular and biotoxicity assays. Introduction of cry1Ac and GNA to cotton plant conferred resistance to Spodoptera littoralis and Aphis gossypii Glover. Restriction of cry1Ac toxin protein to insect biting sites along with a plant lectin attributes significantly to insect pest management strategies.     

Molecular cloning and expression analysis of two <Emphasis Type="Italic">FAD2</Emphasis> genes from chia (<Emphasis Type="Italic">Salvia hispanica</Emphasis>)

FATTY ACID DESATURASE 2 (FAD2, EC 1.3.1.35), also known as delta-12 oleate desaturase, is a key enzyme for linoleic acid and α-linolenic acid biosynthesis. Chia (Salvia hispanica) seeds contain the highest known proportion of α-linolenic acid in any plant sources. In this study, two full-length FAD2 genes, named as ShFAD2-1 and ShFAD2-2, were isolated from S. hispanica based on RACE method. Both ShFAD2-1 and ShFAD2-2 proteins possess strong transmembrane helices, three histidine motifs and a C-terminal ER-located signal (YNNKL). Phylogenetic analysis showed that both ShFAD2-1 and ShFAD2-2 are grouped with constitutive plant FAD2s. Heterologous expression in Saccharomyces cerevisiae indicated that ShFAD2-1 and ShFAD2-2 genes both encode a bio-functional delta-12 oleate desaturase. ShFAD2-2 was mainly expressed in flowers and early-stage seeds while ShFAD2-1 expression was almost constitutive in different organs. qRT-PCR results demonstrated that ShFAD2-1 and ShFAD2-2 show a cold-induced and heat-repressed expression pattern, whereas they also were differentially up-regulated or repressed by other abiotic stresses. This is the first cloning and function characterization of FAD2 from S. hispanica, which can provide insights into molecular mechanism of high ALA traits of S. hispanica and enrich our understanding of the roles of FAD2 genes in various abiotic stresses.     

Regulatory activity of heterologous gene-activator <Emphasis Type="Italic">xlnR</Emphasis> of <Emphasis Type="Italic">Aspergillus niger</Emphasis> in <Emphasis Type="Italic">Penicillium canescens</Emphasis>

The gene encoding the xlnR xylanolytic activator of the heterologous fungus Aspergillus niger was incorporated into the Penicillium canescens genome. Integration of the xlnR gene resulted in the increase in a number of activities, i.e. endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-galactosidase, and feruloyl esterase, compared to the host P. canescens PCA 10 strain, while β-galactosidase, β-glucosidase, endoglucanase, and CMCase activities remained constant. Two different expression constructs were developed. The first consisted of the nucleotide sequence containing the mature P. canescens phytase gene under control of the axhA promoter region gene encoding A. niger (1,4)-β-D-arabinoxylan-arabinofuranohydrolase. The second construct combined the P. canescens phytase gene and the bgaS promoter region encoding homologous β-galactosidase. Both expression cassettes were transformed into P. canescens host strain containing xlnR. Phytase synthesis was observed only for strains with the bgaS promoter on arabinose-containing culture media. In conclusion, the bgaS and axhA promoters were regulated by different inducers and activators in the P. canescens strain containing a structural tandem of the axhA promoter and the gene of the xlnR xylanolytic activator.     

Role of Host Glycosphingolipids on <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> Adhesion

Binding of yeast forms to human lung fibroblast cultures was analyzed, aiming to better understand the initial steps of Paracoccidioides brasiliensis infection in humans. A significant P. brasiliensis adhesion was observed either to fibroblasts or to their Triton X-100 insoluble fraction, which contains extracellular matrix and membrane microdomains enriched in glycosphingolipids. Since human lung fibroblasts express at cell-surface gangliosides, such as GM1, GM2, and GM3, the role of these glycosphingolipids on P. brasiliensis adhesion was analyzed by different procedures. Anti-GM3 monoclonal antibody or cholera toxin subunit B (which binds specifically to GM1) reduced significantly fungal adhesion to fibroblast cells, by 35% and 33%, respectively. Direct binding of GM1 to yeast forms of P. brasiliensis was confirmed using cholera toxin subunit B conjugated to AlexaFluor^®488. It was also demonstrated that P. brasiliensis binds to polystyrene plates coated with galactosylceramide, lactosylceramide, trihexosylceramide, GD3, GM1, GM3, and GD1a, suggesting that glycosphingolipids presenting residues of beta-galactose or neuraminic acid at non-reducing end may act as adhesion molecules for P. brasiliensis. Conversely, no binding was detected when plates were adsorbed with glycosphingolipids that contain terminal residue of beta-N-acetylgalactosamine, such as globoside (Gb4), GM2, and asialo-GM2. In human fibroblast (WI-38 cells), GM3 and GM1 are associated with membrane rafts, which remain insoluble after treatment with Triton X-100 at 4°C. Taken together, these results strongly suggest that lung fibroblast gangliosides, GM3 and GM1, are involved in binding and/or infection by P. brasiliensis.     

Toxin Gene Contents and Activity of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strains Against Two Sugarcane Borer Species, <Emphasis Type="Italic">Diatraea saccharalis</Emphasis> (F.) and <Emphasis Type="Italic">D. flavipennella</Emphasis> (Box)

Bacillus thuringiensis (Berliner) bears essential characteristics in the control of insect pests, such as its unique mode of action, which confers specificity and selectivity. This study assessed cry gene contents from Bt strains and their entomotoxicity against Diatraea saccharalis (F.) and Diatraea flavipennella (Box) (Lepidoptera: Crambidae). Bioassays with Bt strains were performed against neonates to evaluate their lethal and sublethal activities and were further analyzed by PCR, using primers to identify toxin genes. For D. saccharalis and D. flavipennella, 16 and 18 strains showed over 30% larval mortality in the 7th day, respectively. The LC₅₀ values of strains for D. saccharalis varied from 0.08 × 10⁵ (LIIT-0105) to 4104 × 10⁵ (LIIT-2707) spores + crystals mL^?1. For D. flavipennella, the LC₅₀ values of strains varied from 0.40 × 10⁵ (LIIT-2707) to 542 × 10⁵ (LIIT-2109) spores + crystals mL^?1. For the LIIT-0105 strain, which was the most toxic to D. saccharalis, the genes cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C, cry1D, cry1F, cry1I, cry2Aa, cry2Ab, cry8, and cry9C were detected, whereas for the strain LIIT-2707, which was the most toxic to D. flavipennella, detected genes were cry1Aa, cry1Ab, cry1Ac, cry1B, cry1D, cry1F, cry1I, cry2Aa, cry2Ab, and cry9. The toxicity data and toxin gene content in these strains of Bt suggest a great variability of activity with potential to be used in the development of novel biopesticides or as source of resistance genes that can be expressed in plants to control pests.     

Molecular genetic characterization of the yeast <Emphasis Type="Italic">Lachancea kluyveri</Emphasis>

A comparative study of Lachancea kluyveri strains isolated in Europe, North America, Japan, and the Russian Far East was performed using restriction analysis, sequencing of non-coding rDNA regions, molecular karyotyping, and the phylogenetic analysis of the α-galactosidase MEL genes. This study showed a close genetic relatedness of these L. kluyveri strains. The chromosomal DNAs of the L. kluyveri strains were found to range in size from 980 to 3100 kb. The haploid number of chromosomes is equal to eight. The IGS2 restriction patterns and single nucleotide substitutions in the ITS1/ITS2 rDNA region correlate neither with geographic origin nor with the source of the strains. The L. kluyveri strains isolated from different sources have a high degree of homology (79–100%) of their MEL genes. The phylogenetic analysis of all of the known α-galactosidases in the “Saccharomyces” clade showed that the MEL genes of the yeasts L. kluyveri, L. cidri, Saccharomyces cerevisiae, S. paradoxus, S. bayanus, and S. mikatae are species specific.     

Horizontal transfer of the C-termini of <Emphasis Type="Italic">tccC</Emphasis> genes in <Emphasis Type="Italic">Photorhabdus</Emphasis> and <Emphasis Type="Italic">Xenorhabdus</Emphasis>

The high molecular weight insecticidal toxin complexes (Tcs), including four toxin-complex loci (tca, tcb, tcc and tcd), were first identified in Photorhabdus luminescens W14. Each member of tca, tcb or tcc is required for oral toxicity of Tcs. However, the sequence sources of the C-termini of tccC3, tccC4, tccC6 and tccC7 are unknown. Here, we performed a whole genome survey to identify the orthologs of Tc genes, and found 165 such genes in 14 bacterial genomes, including 40 genes homologous to tccC1-7 in P. luminescens TT01. The sequence sources of the C-termini of tccC2-6 were determined by sequence analysis. Further phylogenetic investigations suggested that the C-termini of 6 tccC genes experienced horizontal gene transfer events.     

Stability,oviposition attraction,and larvicidal activity of binary toxin from <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bacillus sphaericus produces a two-chain binary toxin composed of BinA (42 kDa) and BinB (51 kDa), which are deposited as parasporal crystals during sporulation. The toxin is highly active against Culex larvae and Aedes and Anopheles mosquitoes, which are the principal vectors for the transmission of malaria, yellow fever, encephalitis, and dengue. The use of B. sphaericus and Bacillus thuringiensis in mosquito control programs is limited by their sedimentation in still water. In this study, the binA and binB genes were cloned and the recombinant BinAB protein was expressed in three strains of Escherichia coli. These recombinant strains were used in a toxicity assay against Culex quinquefasciatus larvae. The highest expression level was achieved when both proteins were expressed in a single operon construct. The BinAB protein expressed in the E. coli Arctic strain showed higher larvicidal activity than either of the recombinant proteins from the E. coli Ril or pLysS strains. Furthermore, it had the highest oviposition attraction (49.1%, P?相似文献   

Synthesis of oligosaccharide fragments of mannan from <Emphasis Type="Italic">Candida albicans</Emphasis> cell wall and their BSA conjugates

3-Aminopropyl glycosides of α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranose, α-D-mannopyranosyl-(1→3)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranose, and α-D-mannopyranosyl-(1→2)-[α-D-mannopyranosyl-(1→3)]-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranose were efficiently synthesized starting from ethyl 2-O-acetyl(benzoyl)-3,4,6-tri-O-benzyl-1-thio-α-D-mannopyranoside, ethyl 4,6-di-O-benzyl-2-O-benzoyl-1-thio-α-D-mannopyranoside, ethyl 4,6-di-O-benzyl-2,3-di-O-benzoyl-1-thio-α-D-mannopyranoside, and 2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl bromide. The oligosaccharide chains synthesized correspond to the three structural types of side chains of mannan from Candida albicans cell wall. A conjugate of the third pentasaccharide with bovine serum albumin was prepared using the squarate method.     

Biotic factors in induced defence revisited: cell aggregate formation in the toxic cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> PCC 7806 is triggered by spent <Emphasis Type="Italic">Daphnia</Emphasis> medium and disrupted cells

Bioassays with the toxic cyanobacterium Microcystis aeruginosa PCC 7806, its non-toxic mutant ΔmcyB, and Daphnia magna as grazer were used to evaluate biotic factors in induced defence, in particular cyanobacterial and grazer-released info-chemicals. Three main questions were addressed in this study: Does Daphnia grazing lead to a loss of cyanobaterial biomass? Is the survival time of Daphnia shorter in a culture of the toxic cyanobacterium? Does direct grazing or the presence of spent Daphnia medium or a high number of disrupted toxic Microcystis cells in the assays lead to an increase in the cellular microcystin content in the remaining intact cells? The biovolume (growth) as well as size and abundance of Microcystis aggregates were determined by particle analysis, while the survival time of Daphnia individuals was recorded by daily observation and counting, with the relative concentration of cell-bound microcystin-LR, was measured by HPLC analysis. Compared to some recent studies in the field of induced defence, in this study, evidence was found for a direct grazing effect, i.e. the loss of biovolume in the toxic culture. In addition, Daphnia magna ingested more non-toxic than toxic cells, and survived longer with non-toxic cells. In terms of increased cell-bound toxin concentration as a means of defence reported in some studies, a higher cell-bound microcystin-LR content was not measured in this study in any of the treatments (P > 0.05). Under low light conditions with impaired growth of Microcystis, and the presence of a high number of particles with less than 1-μm diameter (possibly heterotrophic bacteria), Daphnia medium was associated with a strong reduction in cell-bound toxin concentration (P < 0.05). This study showed no increased cell aggregation under direct grazing (P > 0.05), but increased aggregation with spent Daphnia medium under high light conditions (P < 0.05). Further, the addition of cell-free extract from disrupted toxic Microcystis cells strongly increased the aggregation of the intact cells under low light (P < 0.05). These findings are discussed with the possible role of microcystin and other infochemicals in the expression of proteins and morphology changes in Microcystis.