Cell surface hydrophobicity ofBifidobacterium bifidum subsp.pennsylvanicum

The possible role of lipoteichoic acid with respect to cell surface properties ofBifidobacterium bifidum subsp.pennsylvanicum was studied. Standard suspensions of bacteria were mixed with octane or xylene.B. bifidum subsp.pennsylvanicum was shown to possess a strongly hydrophobic cell surface. Hydrophobicity of the bacteria could be reduced by treatment with trypsin, pepsin (at pH 4.5), HCl and penicillin. The latter treatment resulted in an increased excretion of lipoteichoic acid. Albumin was capable of inhibiting the adherence to octane when it was present in the assay buffer. The data suggest that both protein and lipoteichoic acid may be involved in cell surface hydrophobicity. A great divergence in cell surface properties was observed within the genusBifidobacterium.     

Expression of fungal phytase on the cell surface ofSaccharomyces cerevisiae

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals, and reduces the phosphorus pollution of animal waste. We have engineered the cell surface of the yeast,Saccharomyces cerevisiae by anchoring active fungal phytase on its cell wall, in order to apply it as a dietary supplement containing bioconversional functions in animal foods and a whole cell bio-catalyst for the treatment of waste. The phytase gene (phyA) ofAspergillus niger with a signal peptide of rice amylase 1A (Ramy1A) was fused with the gene encoding the C-terminal half (320 amino acid residues from the C-terminus) of yeast α-agglutinin, a protein which is involved in mating and is covalently anchored to the cell wall. The resulting fusion construct was introduced intoS. cerevisiae and expressed under the control of the constitutive glyceraldehydes-3-phosphate dehydrogenase (GPD) promoter. Phytase plate assay revealed that the surface-engineered cell exhibited a catalytically active opaque zone which was restricted to the margin of the colony. Additionally, the phytase activity was detected in the cell fraction, but was not detected in the culture medium when it was grown in liquid. These results indicate that the phytase was successfully anchored to the cell surface of yeast and was displayed as its active form. The amount of recombinant phytase on the surface of yeast cells was estimated to be 16,000 molecules per cell.     

Adherence ofStaphylococcus epidermidis to pharyngeal epithelial cells mediated by lipoteichoic acid

Prior treatment of pharyngeal epithelial cells (PEC) with lipoteichoic acid (LTA) derived fromStaphylococcus epidermidis produced a marked inhibition of adherence of the homologous strain and two heterologous strains. The inhibition was dose dependent and saturable with 100 µg/ml of LTA. However, pretreatment of PEC with deacylated LTA did not block the adherence of the three strains tested. A similar but less marked blocking effect on the adherence ofS. epidermidis to PEC was also observed with LTAs derived fromS. aureus andStreptococcus pyogenes. On treatment of bacteria with substances capable of binding to LTA, such as polyclonal mouse anti-LTA antibodies or with human albumin, a marked inhibition of bacterial adherence was observed. Immunofluorescence studies showed that anti-LTA antiserum bound readily to the surface of bacterial cells. These findings provide clear evidence that the lipid component of LTA located on the bacterial surface is centrally involved in the adherence ofS. epidermidis to human mucosal cells.     

Screening of a specific monoclonal antibody against and detection ofListeria monocytogenes whole cells using a surface plasmon resonance biosensor

In this study, a specific monoclonal antibody againstListeria monocytogenes was screened using an SPR biosensor Monoclonal antibodies were bound to protein L, after which theL. monocytogenes cells were subjected to an affinity assay. Protein L was immobilized on a carboxymethyl dextran (CM-Dex) surface via an amine coupling method and utilized repeatedly by regeneration. The monoclonal antibody, ‘A18’, was selected and employed for the high-sensitivity detection ofL. monocytogenes. Under optimized conditions, 10³ cells/ml or 50 cells were detected by the SPR biosensor.     

Morphological study of cytotoxicity produced by PSK-induced polymorphonuclear leukocytes (PMNs) andNocardia rubra cell wall skeleton

The morphologic changes in PMNs induced by an i.p. injection of PSK, a polysaccharide from the mycelia ofCoriolus versicolor, and tumor cells undergoing cell death, were evaluated by immunohistochemical staining and electron microscopy. Male C3H/He mice, 8–10-weeks old, received an i.p. injection of 125 mg/kg of PSK. Their PMNs were obtained 6 h after the PSK injection by peritoneal lavage. N-CWS (Nocardia rubra cell wall skeleton) was added at the start of the chromium release assay using the MM46 mammary carcinoma cell line, which is syngeneic to C3H/He mice, as target cells. During the cytotoxic assay, the cells were fixed at various time points. The MM46 cells expressed ICAM-1 while the PMNs expressed both ICAM-1 and LFA-1 as determined by immunohistochemical staining and immunoelectron microscopy using anti-ICAM-1 and anti-LFA-1 antibodies. PMNs with ruffle-like microvilli adhered to the MM46 tumor cells 30 min after the addition of N-CWS. Immunoelectron microscopic findings suggested that the adhesion molecules were LFA-1 on the PMNs and ICAM-1 on the MM46 tumor cells, but cell fusion between the PMNs and tumor cells was not observed. The MM46 tumor cells gradually lost their microvilli, which showed cell damage, and died 6–7 h after the addition of the N-CWS. This time course of tumor cell death is compatible with the results of the cytotoxic assay. Pretreatment of PMNs by anti-LFA-1 antibody suppressed % lysis of MM46 tumor cells from 90 % to 10 %(p<0.01). These data suggest that adhesion molecule on the surface of PMNs such as LFA-1 might play an important role on signal transduction of these PMNs cytotoxic function in this experimental system.     

Investigating the candidacy of a lipoteichoic acid-based glycoconjugate as a vaccine to combat Clostridium difficile infection

A lipoteichoic acid has recently been shown to be conserved in the majority of strains from Clostridium difficile and as such is being considered as a possible vaccine antigen. In this study we examine the candidacy of the conserved lipoteichoic acid by demonstrating that it is possible to elicit antibodies against C. difficile strains following immunisation of rabbits and mice with glycoconjugates elaborating the conserved lipoteichoic acid antigen. The present study describes a conjugation strategy that utilises an amino functionality, present at approximately 33 % substitution of the N-acetyl-glucosamine residues within the LTA polymer repeating unit, as the attachment point for conjugation. A maleimide-thiol linker strategy with the maleimide linker on the carboxyl residues of the carrier protein and the thiol linker on the carbohydrate was employed. Immunisation derived antisera from rabbits and mice, recognised all strains of C. difficile vegetative cells examined, despite an immune response to the linkers also being observed. These sera recognised live cells in an immunofluorescence assay and were also able to recognise the spore form of the bacterium. This study has illustrated that the LTA polymer is a highly conserved surface polymer of C. difficile that is easily accessible to the immune system and as such merits consideration as a vaccine antigen to combat C. difficile infection.     

Further studies on the surface charge of various strains ofTrichomonas vaginalis andTritrichomonas foetus

The surface charge of three strains ofTrichomonas vaginalis and five strains ofTritrichomonas foetus was determined by direct measurement of the mean cellular electrophoretic mobility (EPM) of cells suspended in solutions of different ionic strength and pH. No differences were observed in the mean EPM among the two species, although significant differences among the strains exist. Strains that are more pathogenic to mouse, as measured using the subcutaneous assay, had a surface more negative. Treatment of the parasites with trypsin or neuraminidase reduced significantly their mean EPM and increased their isoelectric point.Tritrichomonas foetus was more sensitive to the enzyme treatment thanT. vaginalis. Enzyme-treated cells recovered their normal EPM if, after enzyme treatment, they were incubated in fresh culture medium. The recovery process of trypsintreated cells was inhibited 10–20% by addition of inhibitors of either protein synthesis (puromycin) orN-glycosylation of proteins (tunicamycin) to the incubation medium, suggesting that a cytoplasmic pool of sialoglycoproteins may exist. The recovering of the EPM ofT. foetus andT. vaginalis previously treated with neuraminidase was inhibited by puromycin or tunicamycin about 40–50% and 17–30%, respectively. These observations suggest that sialoglycolipids exist on the surface of both parasite species, and that they contribute more to the surface charge ofT. vaginalis than to that ofT. foetus.     

Zur Lebensgeschichte der DiatomeeAchnanthes linearis und Bemerkungen über andereAchnanthes-Arten

Achnanthes linearis produces two auxospores from one cell pair by allogamic fusion of migratory and stationary gametes. Unusually little copulation jelly is produced. Pairing occurs by chance in regard to the heteropoly of the transapical axis. The orientation of the primary cells in relation to the substratum and to the mother cells is constant and characteristic. Variation of cell size is much larger than hitherto known; correspondingly the diagnosis is to be completed.—The position and orientation of vegetative cells and partner cells is strongly determined by the relief of the leaves ofFontinalis on which they are fixed with their raphe valve. The same is true for the cells ofA. minutissima which usually are fixed at one cell pole only, but exceptionally at both poles; this means that morphological heteropoly of apical axis is lacking, and that the function of the second holdfast-jelly producing structure usually is suppressed. The cells of both species normally creep by means of the raphe mechanism; the direction is very independent of the relief of theFontinalis leaves. After cell division the moving down of the upper daughter cell follows a different mechanism.A. linearis is the first to settle onFontinalis leaves, other epiphytes come much later.

     

Antibodies to flowering-plant sperm

Summary The mechanisms by which sperm cells recognize and fuse with the egg and central cell during double fertilization in flowering plants are unknown. To identify membrane surface molecules that might function in fertilization, we immunized mice with isolated sperm ofBrassica campestris and screened the polyclonal sera and monoclonal hybridoma supernatants by immunocytochemistry for binding to isolated sperm cells. We identified three cell lines producing hybridoma supernatants which bind to sperm cell surfaces inB. campestris and further analyzed the properties of one of these, BRSP1. The molecular mass of the epitope to BRSP1 was 54 kDa, and was not glycosylated. Although the antibodies were immunoglobulin M, neither removal of carbohydrates nor competition with antibodies which recognize arabinogalactan decreased binding. BRSP1 recognized sperm ofPlumbago zeylanica, Nicotiana tabacum. Arabidopsis thaliana, andEruca vesicaria and generative cells ofLilium longiflorum and ofNarcissus tazetta but did not recognize sperm ofHelianthus annuus, Gerbera jamesonii, orZea mays. Antibodies to plant sperm allow us to probe sperm membranes for functional components.     

The influence of post-filtration washing on the in vitro assay ofCandida albicans adherence to human buccal epithelial cells

A simple in vitro assay technique was used to determine the effect of post-filtration washing on the adherence ofC. albicans (NCPF 3736) to human buccal epithelial cells (BEC). Washing was carried out with a range of volumes of phosphate buffered saline (PBS), viz. 0, 5, 10 and 20 ml, at a standard flow rate. Both the number ofC. albicans adherent to BEC and the percentage of BEC with adherentC. albicans were significantly decreased (p<0.001 for each of these measures) after washing with 5 ml PBS. Further increases in the volume of PBS did not significantly decrease either measure of adherence. These data indicate that only a small volume of PBS, 5 ml, is required to achieve the removal of non-adherentC. albicans from the surface of BEC. The result of the adherence assay is not significantly affected by increasing the volume of PBS used. It is concluded that considerable savings in time may be made through using only a small (5 ml) volume of washing buffer at a standard flow rate.Abbreviations BEC buccal epithelial cells - PBS phosphate buffered saline - MEM Eagle's minimum essential medium - NCPF National collection of pathogenic fungi     

Improved ultrastructural preservation ofPetunia andBrassica ovules and embryo sacs by high pressure freezing and freeze substitution

Summary In order to improve the ultrastructural preservation of the female gametophyte ofPetunia x hybrida andBrassica napus we tested several cryofixation techniques and compared the results with those of conventional chemical fixation methods. Ovules fixed with glutaraldehyde and osmium tetroxide in the presence or absence of potassium ferrocyanide showed poor cell morphological and ultrastructural preservation. In ovules cryo-fixed by plunging into liquid propane, the cell morphology was well preserved. However, at the ultrastructural level structure-distorting ice crystals were detected in all tissues. Due to the large size of the ovules, cryofixation by plunging in liquid propane is not adequate for ultrastructural studies. In contrast,P. x hybrida andB. napus ovules cryo-fixed by high pressure freezing showed improved cell morphological as well as ultrastructural preservation of the embryo sac and the surrounding integumentary tissues. The contrast of the cellular membranes after freeze substitution with 2% osmium tetroxide and 0.1% uranyl acetate in dry acetone was high. At the ultrastructural level, the most prominent improvements were: straight plasma membranes which were appressed to the cell walls; turgid appearing organelles with smooth surface contours; minimal extraction of cytoplasmic and extracellular substances. In contrast to the chemically fixed ovules, in high pressure frozen ovules numerous microtubules and multivesicular bodies could be distinguished.     

Effect of acetylsalicylic acid on secondary metabolism ofCatharanthus roseus tumor suspension cultures

Summary Addition of various concentrations (0.5–20 mM) of acetylsalicylic acid (ASA) to tumor lines ofCatharanthus roseus cultivatedin vitro and requiring corn starch as carbon source, produced remarkable effects on secondary metabolite production. An increase of 505% total alkaloids per culture (cells plus liquid medium), 1587% total phenolics (liquid medium), 612% total furanocoumarins (liquid medium) and 1476% total anthocyanins (liquid medium) was detected. 1 mM ASA in combination with other elicitors, such as homogenates ofAspergillus fumigatus or trans-cinnamic acid, did not further increase the metabolite content substantially. The results suggest that ASA could act as a new biotic elicitor of metabolite production inC. roseus cell suspension culture.     

A sensitive GLC-method for component sugars andO-glycosidic linkage monosaccharides of cartilage proteoglycans

A method is described for the measurement ofN-acetylgalactosamine,N-acetylglucosamine, galactose, mannose and xylose present in the different carbohydrate chains of cartilage proteoglycans (PG). Bovine articular cartilage PG samples corresponding to the minimum of 1 nmol of each monosaccharide were reproducibly quantified following hydrolysis with 2 M HCl and derivatization into alditol acetates. An on-column injection mode and an OV-1701 fused silica capillary column were used for chromatography.Alkaline borohydride treatment of the PG was exploited to reduce the acid labile xylose in the base of the chondroitin sulphate chain into more stable xylitol, allowing the assay of chondroitin sulfate chain length as anN-acetylgalactosamine/xylose ratio. A novel procedure is described for the measurement of the galactosaminitol evolving from the protein linkage of oligosaccharides and of keratan sulphate.     

Changes in the chemical composition of carbohydrates and proteins in surface water during a bloom ofMicrocystis in Lake Suwa

Carbohydrates and proteins in surface water during a bloom ofMictrocystis, which is the dominant summer phytoplankton in Lake Suwa, were analyzed in order to evaluate the function ofMicrocystis in organic matter metabolism. Glucose was the predominant sugar constituent of the cellular carbohydrate fraction and decreased in quantity from inside towards the outside of the cell through the slime layer. Other constituent sugars, on the other hand, were present in larger proportions in the lake water. Although the sugar composition of the cells did not change in July and August, during the first period of theMicrocystis bloom, it changed appreciably in September when the water temperature decreased below 20°C accompanied by the decrease in solar radiation and a marked change in nutrient concentration. It appears that the sugar composition of the cells may change in response to some environmental stresses. In addition, a temporal change in the sugar composition was found, particularly in the fraction containing the slime extracted by shaking. Among the constituent amino acids of the cells, the percentage of arginine, aspartic acid and leucine decreased from inside toward the outside of the cell, while glutamic acid, threonine, serine and glycine showed an opposite trend. In contrast to the carbohydrates, the percentage composition of each amino acid varied little throughout the period of the bloom.     

Production and harvesting of ionically wall-bound extensin from living cell suspension cultures

Cell-suspension cultures ofSpinacia andRosa accumulated a cell wall protein, extensin, in a form that was amenable to leaching from the surface of the living cells by a brief treatment with non-toxic salts. Cultures ofLycopersicon, Capsicum, Acer andFestuca did not accumulate this class of extensin. InSpinacia andRosa, optimum yields of leachable extensin were achieved from young cultures, in media at relatively low pH, by leaching with 0.1 M CaCl₂. Older cultures, pH values >6.5, and LaCl₃ or higher concentrations of CaCl₂ were less effective.Abbreviation TCA trichloroacetic acid     

Identification of a novel lectin inStreptococcus pyogenes and its possible role in bacterial adherence to pharyngeal cells

During investigation of the interaction of human lactoferrin (HLf) with variou bacteria, it was found that inStreptococcus pyogenes, HLf binding occurred to agar-rather than broth-grown cells irrespective of the nutrients used. Furthermore, binding of HLf to broth-grown, heat-killed bacteria was induced by overnight incubation on agar media or short-time exposure of the cells to water-soluble agar extract. The binding pattern was revealed in most of 92S. pyogenes strains representing various M-or T-types with no apparent type variation. The component thus bridging the attachment of HLf to the streptococcal cell surface was recovered in extracts of agar-grown cells and isolated by affinity chromatography on HLf-sepharose. By gel filtration in the presence of radiolabeled HLf, this component exhibited similar elution position as crude water-soluble agar extract. Chemical analysis identified the active HLf-binding agar component to be a galactose-rich polysaccharide (GRP). Further binding tests showed that the interaction between streptococci and GRP was stable in the presence of high molar NaCl, KSCN, or urea and was unaffected by various serum or matrix proteins or by streptococcal lipoteichoic acid; however, a moderate inhibition by heparin or bovine mucin was observed. Studies on isogenic mutants ofS. pyogenes did not support the involvement of M-protein or the hyaluronate capsule in the binding of GRP. SDS-PAGE and Western blot analyses revealed a GRP-binding protein of approximately 70 kDa in the cell-wall extracts of two strains ofS. pyogenes, types M19 and M55. Finally, the adherence of (broth-grown)³H-thymidine-labeledS. pyogenes, type M19, to the pharyngeal epithelial cell line DT-562 or to normal tonsillar epithelial cells was inhibited by GRP in a dose-related manner. We thus propose that the streptococcal GRP-binding component may represent a novel surface lectin acting as a mucosal adhesin forS. pyogenes, in accordance with previous data indicating that galactosecontaining sugar moieties may serve as ligands for the adherence of streptococci to pharyngeal cells. Our results also indicate that GRP-like components such as mucin or heparin might act to block epithelial adherence ofS. pyogenes at the mucosal level.     

Cyclic kinetics and mathematical expression of the primary immune response to soluble antigen

A mathematical expression of the accumulation of the plasma cells in the spleen of CBA mice immunized intraperitoneally is presented. The dependence of the plasma cell reaction in the spleen on the kinetics of antigen concentration in the blood was confirmed. For the transition from antigen to plasma cells, index A was proposed. The mean values of index A were used for comparison of the calculated and experimental values of the plasma cell reaction and the recorded differences were not great. In a similar way, index A was used for prediction of plasma cell accumulation in the spleen of animals, immunized with a mixture of two soluble antigens — capsular antigen ofPasteurella pestis and complete antigen ofFrancisella tularensis. The calculated values of plasma cell reaction corresponded to experimental values.     

Metabolic discrimination of sucrose starvation from<Emphasis Type="Italic">Arabidopsis</Emphasis> cell suspension by<Superscript>1</Superscript>H NMR based metabolomics

Whole cell extracts ofArabidopsis cell cultures maintained on various sucrose concentrations (0,3, and 6%) were analyzed by¹H NMR spectroscopy to determine the comprehensive metabolic change in these cultures during sucrose starvation. The amount of sucrose, glucose, and fructose in the cells decreased to almost nothing after 12 h of culture in medium without sucrose. In contrast, the total free amino acid content of the cells increased as the culture proceeded. Among the free amino acids, phenylalanine and malic acid increased the most, followed by asparagine and alanine, whereas glutamic acid did not change significantly. These results are in agreement with previous studies using HPLC.¹H NMR spectroscopy enabled measurement of changes in the sugar and free amino acid content of whole cell extracts without fractionation and complicated sample preparation. These results indicate that comprehensive metabolic changes in the cells can be determined by a simple, rapid method using whole cell extracts and¹H NMR spectroscopy.     

Some properties of thePseudomonas fluorescens adhesin and antiadhesin

Some properties of the adhesion-modifying factors ofPseudomonas fluorescens are described. Adhesin, which promotes the adhesion ofP. fluorescens cells, is a hydrophobic compound of a protein nature with a molecular mass of more than 10 kDa located either at the cell surface or in the medium. Antiadhesin, which suppresses the adhesion ofP. fluorescens cells, is a thermolabile hydrophobic compound of a nonprotein nature with a molecular mass of less than 3 kDa. Heating makes antiadhesin hydrophilic. The role of adhesin and antiadhesin in the adhesion and adaptation ofP. fluorescens cells is discussed.     

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