Determination of the cell adhesion specificity of Streptococcus suis with the complete set of monodeoxy analogues of globotriose

Streptococcus suis causes meningitis and other serious infections in pigs and humans, and binds to host cell globotriosylceramide. In order to determine the essential hydroxyls involved in binding, the complete set of monodeoxy derivatives of the receptor trisaccharide Gal1-Gal1-4Glc were tested as inhibitors of bacterial hemagglutination. Removal of the 4-, 6, 2 or 3-hydroxyls abolished inhibitory activity, which indicated that they were critically involved in binding. The same results were obtained using synthetic lipid-linked monodeoxy derivatives of the trisaccharides in a thin-layer overlay assay. The P_N and P_O subtypes of the S. suis adhesin showed similar binding patterns. The hydroxyls of the glucose moiety were not critical for binding, although the adhesin binds better to the trisaccharide Gal1-4Gal1-4Glc than the disaccharide Gal1-4Gal.     

Synthesis of the penicillin precursor, δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (ACV), by an immobilized enzyme preparation

Summary -(l--Aminoadipyl)-l-cysteinyl-d-valine (ACV) synthetase activity has been partially-purified from cell-free extracts of Streptomyces clavuligerus by ammonium sulfate precipitation. The salt precipitated enzyme was immobilized on an anion exchange resin and synthesis of ACV was observed by exposing the immobilized enzyme preparation to a reaction mixture containing l--aminoadipic acid, l-valine and l-cysteine in the presence of appropriate cofactors. Reaction mixtures containing l--aminobutyric acid(aB) in place of l-valine synthesized the ACV analog ACaB. Immobilized ACV synthetase can be reused, and after six cycles of reaction, 28.9% of original activity remains.     

Identification of rate-limiting steps in cephalosporin C biosynthesis in Cephalosporium acremonium: a theoretical analysis

Summary A kinetic model describing the biosynthesis of celphalosporin C in Cephalosporium acremonium has been developed to identify the rate-limiting step(s). Using this model and in-vitro kinetic data of the biosynthetic enzymes, the production kinetics of cephalosporin C were examined theoretically. The predicted time profile of the specific production rate during batch culture is in good agreement with that of experimental results published previously. Sensitivity analysis indicates that -(l--aminoadipyl)-l-cysteinyl-d-valine (ACV) synthetase is the rate-limiting enzyme. Our analysis also predicts that increasing ACV synthetase enhances the production rate initially until expandase/hydroxylase becomes rate-limiting. Furthermore, increasing expandase/hydroxylase reduces the accumulation of penicillin N, and thus, enhances the production of cephalosporin C. Based on our analysis, amplifying both ACV synthetase and expandase/hydroxylase concurrently should enhance the production rate to a great extent.Correspondence to: W. S. Hu     

Regulation of antibiotic production by iron and oxygen during defined medium fermentations of Streptomyces clavuligerus

Summary When grown in a chemically defined medium, Streptomyces clavuligerus excreted cephamycin C, in addition to other components, throughout most of the growth phase. Ferrous iron and oxygen are required for the biosynthesis of this antibiotic and the concentration of these cofactors was manipulated to maximize cephamycin C production. The iron content of the chemically defined medium was shown to be sub-optimal for antibiotic production and the addition of 130 g/ml ferrous iron almost doubled the cephamycin C levels to 200 g/ml. When dissolved oxygen was maintained at saturation levels, only 60–80 g/ml cephamycin C was produced, and the intermediate penicillin N accumulated to high levels (50 g/ml). This suggests that the high concentration of dissolved oxygen had a greater effect on the enzymes catalysing the conversion of penicillin N to cephamycin C, than on those involved in the earlier steps of the pathway leading to the formation of penicillin N.     

A comparison and evaluation of some phytosociological techniques

Summary In three areas of vegetation (dune, mountain heath and salt marsh) the following phytosociological techniques have been tested and compared, using the same data: the Braun-Blanquet method; association and inverse analysis ofWilliams &Lambert; cluster analysis (agglomerative classification) based on different coefficients of similarity; and ordination (principal components analysis performed on matrices of different coefficients).The Braun-Blanquet method is considered to combine several advantages of the other methods and to be most economical in terms of efficiency (ratio of time input to information emerging).

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Zusammenfassung Auf drei verschiedenen Vegetationsflächen (Bergheide, Küstendünen und Salzwiesen) sind die folgenden pflanzensoziologischen Methoden geprüft und verglichen worden: die Methode von Braun-Blanquet; association analysis vonWilliams &Lambert (1959, 1961); Ordination (principal components analysis); und cluster analysis (Sokal &Sneath, 1963). Die letzteren beiden wurden mit verschiedenen Ähnlichkeitskoeffizienten geprüft.Auf Grund solchen Erfahrungen, zeigte sich die Braun-Blanquetische Methode leistungsfähiger als die anderen Methoden (d.h. optimale Einsicht in der Vegetation pro Arbeitsstunde). Sie vereinigte viele Vorteile der anderen Methoden.

     

Polymorphisms and diversity of T-cell receptor-γ proteins expressed in mouse spleen

Bulk populations of T-cell receptor (Tcr) -expressing splenocytes from different inbred strains of mice were examined for the diversity of Tcr proteins. Immunoprecipitations with anti-C1/2, anti-C4, and anti-V1 sera demonstrated that splenocytes from B10.BR, C57BL/6, and C57L strains of mice expressed the same array of Tcr proteins, namely V1-C2, V1-C4, and V2-C1, although the Tcr heterodimers observed for each of these strains were biochemically distinct. Examination of bulk splenic Tcr heterodimers from several other inbred strains of mice demonstrated that each of the strains could be categorized into one of three basic phenotypes. For several reasons, the differences observed between the strains appeared to be solely dependent on polymorphisms of the Tcrg loci. First, F₁ mice co-expressed both parental Tcr phenotypes. Second, the distinguishing polymorphism between mice of phenotype 1 and phenotypes 2 or 3 was due to the presence of an N-linked glycosylation site within the Tcrg-C1 gene segment, previously described for BALB.B and C57BL/6 Tcrg-C1 genes. Finally, the V1-C4 polymorphism between mice of phenotype 3 and phenotypes 1 or 2 was due to differences in core protein size. Furthermore, the three defined Tcr chains were expressed independently of the major histocompatibility complex (MHC) haplotype. Although no striking qualitative differences in Tcr heterodimers were observed between strains (including those with autoimmune disorders), a quantitative difference in the relative amount of C4-encoded proteins was observed on Tcr splenocytes from both newborn euthymic and adult athymic mice when compared to adult Tcr splenocytes from euthymic mice. These results demonstrate that genetic polymorphisms exist among different mouse strains and suggest that selective developmental pressures may govern Tcr expression. Offprint requests to: J. A. Bluestone     

Heterogeneous binding and killing behaviour of human γ/δ-TCR+ lymphokine-activated killer cells against K562 and Daudi cells

Effector-target conjugates, formed by coincubation of lymphokine-activated killer (LAK) cells with either K562 or Daudi cells, were separated from single cells by Percoll sedimentation. The occurrence of various CD molecules (CD3, CD56, CD57, CD16, /-TCR) was compared in both fractions. Only LAK cells expressing the / T cell receptor (TCR) were found in a significantly increased percentage in fractions containing conjugates indicating that /-TCR⁺ LAK cells were preferably bound to target cells at the time of separation. In order to determine whether /-TCR⁺ LAK cells also show a preferred killing activity against the targets, cultures enriched with or depleted of /-TCR⁺ cells were established. Against K562 cells, /-TCR⁺-enriched cultures showed a greatly reduced killing activity compared to LAK bulk cultures or cultures depleted of /-TCR⁺ cells. Using Daudi cells as targets the enriched fraction revealed a slightly increased killing activity compared to bulk cultures or depleted fractions. Preincubation of /-TCR⁺ LAK cells with anti-/ or anti-CD3 mAb resulted in a distinct increase of the killing activity against K562 cells, but in only a slightly enhanced activity against Daudi cells. It is postulated that /-TCR⁺ LAK cells use the same adhesion mechanism for both targets but that only Daudi cells express a specific ligand for the /-TCR. Occupation of the /-TCR/CD3 complex by mAb, however, seems to substitute for the absent epitope on K562 cells by eliciting stimulatory signals in /-TCR⁺ LAK cells which, in combination with the binding stimulus, trigger cytolytic activity.This work was supported by the Hartmann-Müller Foundation, Zürich     

Selection of microorganisms which produce raw-starch degrading enzymes

Summary Microorganisms which produce strong raw-starch degrading enzymes were isolated from soil using a medium containing a unique carbon source, -amylase resistant starch (-RS), which is insoluble in water and hardly digested with Bacillus amyloliquefaciens -amylase. Among the isolates, three strains showing high activities were characterized. Two of them, K-27 (fungus) and K-28 (yeast), produced -amylase and glucoamylase, and the final product from starch was only glucose. The third strain, K-2, was a bacterium and produced -amylase, which produced glucose and malto-oligosaccharides from starch. The enzyme preparation of these strains degraded raw corn starch rapidly.     

The supramolecular organization of red algal cell membranes and their participation in the biosynthesis and secretion of extracellular polysaccharides: a review

Summary The relationship between the supramolecular organization of red algal cell membranes and the biosynthesis and secretion of the cell wall skeletal and matrix polysaccharides is reviewed. Freeze-fracture studies have revealed that organized macromolecular structures — linear terminal complexes and tetrads — are present on the plasma membrane and on membranes of the endomembrane system. The linear terminal complexes seem to be involved in the biosynthesis, assembly, and orientation of the cellulose microfibrils and the tetrads in the synthesis of the matrix polysaccharides. It is shown how the research on the supramolecular organization of cell membranes has increased the knowledge on the biosynthesis and secretion of the extracellular crystalline and non-crystalline polysaccharides in red algae. In this review, the progress to date is discussed.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Genetic transformation in two potato cultivars with T-DNA from disarmed Agrobacterium

Summary Derivatives of potato (Solanum tuberosum cv.'s Maris Bard and Desiree) transformed with disarmed T-DNA from genetically engineered Agrobacterium tumefaciens strains were isolated. The transformed plants were recovered from shoot-forming tumours induced by infection of wounds with mixedcultures of shoot-inducing A. tumefaciens strains T37 and either Agrobacterium strain LBA1834(pRAL1834), (Hille et al. 1983) or LBA4404(pBIN6; pRAL4404), (Bevan 1984). Two small-scale feasibility experiments gave at least four Maris Bard plants transformed with pRAL1834 T-DNA and two Desiree plants with pBIN6 T-DNA. The transformed Maris Bard plants were morphologically abnormal and highly aneuploid. This was probably an unfortunate side-effect of a tissue culture-step introduced to promote the efficiency of shoot regeneration. The transformed Desiree plants, in contrast, were isolated without promoting additional shoot-growth. They were morphologically normal, contained 47 and the euploid 48 chromosomes per cell respectively and had improved growth on media containing kanamycin.     

Amino acid esters of 9-(2′,3′-dihydroxypropyl-1′)-adenine are the specific inhibitors of protein synthesis on ribosomes

Peptide acceptor properties of phenylalanine and glycine esters of 9-(2,3-dihydroxypropyl-1)-adenine and 1-(2,3-dihydroxypropyl-1)-4-thiouracyl were investigated. All these esters appeared to be powerful inhibitors of polyphenylalanine synthesis in E. coli MRE-600 ribosomes charged with poly U. Like puromycin, esters of adenine derivatives accepted the AcPhe residue from Ac-[¹⁴C] Phe-tRNA in a ribosomal system charged with poly U. However, peptidyl esters of 9-(2,3-dihydroxypropyl-1)-adenine remained bound with ribosomes. The structure of the peptide esters synthesized was ascertained after dissociation of ribosomes into subparticles by direct comparison with the synthetic specimens.Abbreviations AcPhe acetyl-l-phenylalanine - HP-Ade 9-(2,3-dihydroxypropyl-1)-adenine - Phe-HP-Ade and Gly-HP-Ade l-phenylalanine and glycine esters of HP-Ade - Phe-HP-TUra l-phenylalanine ester 1-(2,3-dihydroxypropyl-1)-4-thiouracyl - AcPhePhe-HP-Ade and AcPheGly-HP-Ade acetyl-l-diphenylalanine and acetyl-l-phenylalanylglycine esters of HP-Ade respectively - AcPhe-puromycin acetyl-l-phenylalanyl-puromycin     

Phosphate repression of cephamycin and clavulanic acid production by Streptomyces clavuligerus

Summary Production of cephamycin and clavulanic acid by Streptomyces clavuligerus is controlled by the phosphate concentration. Phosphate represses the biosynthesis of cephamycin synthetase, expandase and clavulanic acid synthetase. In the presence of 2 mM phosphate, the specific activities of expandase, cephamycin synthetase and clavulanic acid synthetase were higher than in the presence of 75 mM phosphate. The specific activity of cephamycin synthetase is maximal with an initial phosphate concentration of 10 mM, whereas the specific activity of expandase is maximal with 1 mM phosphate. A correlation between cephamycin synthetase specific activity and expandase specific activity was established at phosphate concentrations higher than 10 mM. This shows that the expandase is an important enzyme in the mechanism by which the phosphate concentration affects the biosynthesis of cephamycin.     

Interactions of γ-L-glutamyltaurine with excitatory aminoacidergic neurotransmission

The in vitro effects of -L-glutamyltaurine on different stages of excitatory aminoacidergic neurotransmission were tested with -D-glutamyltaurine as reference. -L-Glutamyltaurine enhanced the K⁺-stimulated release of [³H]glutamate from cerebral cortical slices (25% at 0.1 mM) and slightly inhibited the uptake by crude brain synaptosomal preparations (about 10% at 1 mM). -L-Glutamyltaurine was also a weak displacer of glutamate and its agonists from their binding sites in brain synaptic membrane preparations, being, however, less selective to quisqualate (QA) sites than -D-glutamyltaurine. The basal influx of Ca²⁺ into cultured cerebellar granular cells was not affected by 1 mM -L-glutamyltaurine, but the glutamate- and its agonist-activated influx was significantly inhibited in low-Mg²⁺ (0.1 mM) and Mg²⁺-free media. The glutamate-evoked increase in free intracellular Ca²⁺ and the kainate-activated formation of cGMP in cerebellar slices were both markedly inhibited by 0.1 mM -L-giutamyltaurine. We propose that -L-glutamyltaurine may act as endogenous modulator in excitatory aminoacidergic neurotransmission.     

Regulation of α-aminoadipyl-cysteinyl-valine,isopenicillin N synthetase,isopenicillin N isomerase and deacetoxycephalosporin C synthetase by nitrogen sources in Streptomyces lactamdurans

Summary Ammonium and asparagine produced a concentration-dependent reduction of cephamycin C biosynthesis by Streptomyces lactamdurans. Addition of ammonium salts at 1 mM concentration reduced cephamycin biosynthesis by resting cells of S. lactamdurans, whereas concentrations of asparagine above 10 mM were required to get the same effect. High ammonium concentrations decreased glutamine synthetase activity in cell extracts of S. lactamdurans in parallel to the reduction of antibiotic biosynthesis. Ammonium supplementation decreased the pool of glutamic acid and glutamine whereas the intracellular content of ammonium, alanine, and phosphoserine increased significantly. The pool of the tripeptide (l--aminoadipyl)-l-cysteinyl-d-valine, an intermediate in cephamycin biosynthesis, was greatly reduced in ammonium-supplemented cultures. Isopenicillin N synthetase, that converts the tripeptide (l--aminoadipyl)-l-cysteinyl-d-valine to isopenicillin N, isopenicillin N isomerase (that isomerises isopenicillin N to penicillin N) and deacetoxycephalosporin C synthetase (converting penicillin N into deacetoxycephalosporin C) were also reduced in ammonium-supplemented cultures. However, the activities of these enzymes were not inhibited in vitro by 40 mM ammonium, suggesting that the enzymes were repressed but not inhibited by ammonium in vivo.     

Synthesis of FimH receptor-active manno-oligosaccharides by reverse hydrolysis using α-mannosidases from Penicillium citrinum,Aspergillus phoenicis and almond

Recombinant Penicillium citrinum -1,2-mannosidase, expressed in Aspergillus oryzae, was employed to carry out regioselective synthesis of -d-mannopyranosyl-(12)-d-mannose. Yields (w/w) of 16.68% disaccharide, 3.07% trisaccharide and 0.48% tetrasaccharide were obtained, with 12 linkages present at 98.5% of the total linkages formed. Non-specific -mannosidase from almond was highly efficient in reverse hydrolysis and oligosaccharide yields of 45–50% were achieved. The products of the almond mannosidase were a mixture of disaccharides (30.75%, w/w), trisaccharides (12.26%, w/w) and tetrasaccharides (1.89%, w/w) with 12, 13 and 16 isomers. -1,2-linkage specific mannosidase from P. citrinum and -1,6-linkage-specific mannosidase from Aspergillus phoenicis were used in combination to hydrolyse the respective linkages from the mixture of isomers, resulting in -d-mannopyranosyl-(13)-d-mannose in 86.4% purity. The synthesised oligosaccharides can potentially inhibit the adhesion of pathogens by acting as "decoys" of receptors of type-1 fimbriae carried by enterobacteria.     

Non-radioactive assay and HPLC analysis of cephalosporin C 7α-methoxylation by cell-free extracts of Streptomyces clavuligerus

Summary Escherichia coli W3110 was found to be 50 times more sensitive to cephamycin C than to cephalosporin C, and also markedly more sensitive to 7-methoxycephalosporin C than to 7-hydroxycephalosporin C. Accordingly, this organism could be used to establish a bioassay of cephalosporin C 7-methoxylation by a Streptomyces clavuligerus cell-free extract. The bioassay results were complemented by HPLC analysis. Modification of the mobile phase from 100 mm NaH₂PO₄, pH 4.2, to 200 mm, pH 4.0, improved HPLC resolution such that an unidentified peak could be separated from the 7-hydroxycephalosporin C peak, and the formation of this intermediate as well as the product, 7-methoxycephalosporin C, could be directly measured in the cell-free reaction system. Offprint requests to: A. L. Demain     

Calcium control of differentiation in Phytophthora palmivora

A method has been developed for the preparation of zoospores from Phytophthora palmivora which allows the ionic composition of the suspension medium to be closely controlled. Sub-micromolar concentrations of calcium ions have been shown to play a key role in maintaining the zoospore state and in the transition to the cyst stage. Restriction of free Ca²⁺ to between 0.2 and 1 M resulted in zoospores which could be maintained for several hours before they finally encysted and germinated. When exposed to citrus-pectin, or 3 mM SrCl₂, or to vigorous shaking, these zoospores underwent rapid synchronous encystment. At free Ca²⁺ concentrations below 0.1 M, zoospores lysed slowly. If exposed to inducers of encystment before lysis had occurred, the zoospores failed to respond to pectin or to vigorous shaking. However, they did differentiate in response to SrCl₂ addition. Provided the free Ca²⁺ was maintained between 0.02 and 0.2 M, zoospores survived gentle centrifugation, a procedure which previously had resulted in encystment.Abbreviations IM (ion-mix) release medium containing 100 M KCl, 10 M CaCl₂, and 10 M MgCl₂     

Disruption of the Gi2α locus in embryonic stem cells and mice: a modified hit and run strategy with detection by a PCR dependent on gap repair

We have used an insertion vector-based approach to target the G_i2 gene in AB-1 embryonic stem cells. 105 bp located 0.8–0.9 kb upstream of a disrupting Neo marker in exon 3 were deleted and replaced with an engineeredNot I site, that served to linearize the vector. The 105 bp deletion served as a primer annealing site in a polymerase chain reaction (PCR) designed to detect the gap repair associated with homologous recombination. Both target conversion and vector insertion events were obtained (hit step). Clones that had inserted the entire targeting vector were taken into FIAU (1-[2-deoxy, 2-fluoro--d-arabinofuranosyl]-5-ioduracil) counterselection to select against a thymidine kinase (TK) marker flanking the homologous genomic sequences and thus for cells that had excised the plasmid and the TK marker by intrachromosomal recombination (run step). Additional selection in G418 reduced the number of drug-resistant colonies at least five-fold. Thus, the Neo marker disrupting the homologous sequences allows for a more specific selection of the desired intrachromosomal recombination event in tissue culture. This modified hit and run strategy represents a novel approach for vector design and the use of the polymerase chain reaction to detect targeting. It may be particularly useful for targeting genes that display a low frequency of homologous recombination. Germ line transmission of the mutated G_i2 allele is also demonstrated.     

Production of β-fructofuranosidase byAspergillus japonicus

A newly isolated strain, MU-2, which produces very high -fructofuranosidase activity, was identified asAspergillus japonicus. For enzyme production by the strain, sucrose at 20% (w/v) was the best carbon source and yeast extract at 1.5 to 3% (w/v) the best nitrogen source. Total enzymatic activity and cell growth were at maximum after 48 h, at 1.57×10⁴ U/flask and 0.81 g dry cells/flask, respectively. The optimum pH value of the enzymatic reaction was between 5.0 and 5.5 and the optimum temperature 60 to 65°C. The enzyme produced 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose by fructosyl-transferring activity. The strain was found to be very useful for industrial production of -fructofuranosidase.     

Comparative structural analysis of snail galactans by a radioimmunoassay to elucidate species-specific determinants

Summary Galactans, the storage polysaccharides in the perivitelline fluid of many snails showed a high degree of species-specificity as revealed by quantitative precipitin formations with lectins, polyclonal antisera, myeloma proteins as well as by the reactivity with the enzyme galactose oxidase. However, their chemical compositions were remarkably similar since thed-Gal residues were all linked 13 and 16 glycosidically.The specificity seemed to be related to the different degrees of branching in the various galactans but could also be due to some other minor constituents in some galactans such asl-galactose or phosphate.In this study a Radioimmunoassay was developed using the galactan of the snailLymnaea stagnalis to elucidate those differences which were only related to a unique distribution of the 13 and 16 linkages, since this galactan was composed exclusively ofd-galactose residues. The galactan was labeled by sequential oxidation with galactose oxidase and reduction with tritiated sodium borohydride. Inhibition of the binding of the labeled galactan to insolubilized antibodies was investigated by galactans of different species, their chemically modified products, andd-galactose-composed oligosaccharides of unambiguously identified structures.Inhibition byLymnaea stagnalis galactan was about 45 000 times that ofHelix pomatia galactan. The most complementary oligosaccharide found was -d-Gal13[-d-Gal16]-d-Gal11l-Gro, being about 200 times more effective thand-Gal. However, a fraction with molecular weights between 700 and 1000 isolated from the partially hydrolized galactan was still seven times more effective. From the reactivity of the antiserum with the different oligosaccharides tested the following structure was inferred which most likely represented the complete determinant recognized by the antiserum: -d-Gal13[-d-Gal16]-d-Gal16[-d-Gal13]-d-Gal1. This determinant seemed to be most common inLymnaea stagnalis galactan and its frequency of occurrence appears to correspond to the inhibitory potency of other snail galactans.