NovelN-linked oligo-mannose type oligosaccharides containing an α-d-galactofuranosyl linkage found in α-d-galactosidase fromAspergillus niger

Structures of oligosaccharides fromAspergillus niger -d-galactosidase [EC 3.2.1.22] were studied. Purified -d-galactosidase was treated withN-glycosidase F, and six kinds of oligosaccharides were isolated by gel chromatography and anion-exchange chromatography. The structures of the oligosaccharides were determined by¹H-NMR and compositional analysis to be Man₅GlcNAc₂, Man₆GlcNAc₂, Man₉GlcNAc₂, GlcMan₉GlcNAc₂, GalMan₄GlcNAc₂ and GalMan₅GlcNAc₂. From mild acid hydrolysis, methylation analysis and ROESY spectral analysis, it was ascertained that the galactosyl residue in two oligosaccharides was in the furanose form and was bound to mannose at the nonreducing end with an 1–2 linkage (GalfMan₄GlcNAc₂ and GalfMan₅GlcNAc₂).     

Identification of a locality in snake venomα-neurotoxins with a singificant compositional similarity to marine snailα-conotoxins: Implications for evolution and structure/activity

Summary -neurotoxins from elapid snake venoms and-conotoxins from marine snails bind specifically and with high affinity to nicotinic cholinoceptors. Although both types of toxin are polypeptides, there is more than a fourfold difference in size between the two and no clear sequence homology is evident. A systematic computer search of the three-dimensional structure of erabutoxin b (an-neurotoxin from the false sea snakeLaticauda semifasciata) was performed to identify the locality that most closely matched the amino acid compositions of the smaller-conotoxins (from the marine snailsConus magus andConus geographus). The area of greatest similarity centered on residue position 25 of erabutoxin b, a locale that is conserved throughout the snake-neurotoxins and their homologues. Six Proteins unrelated to erabutoxin b were compared to the-conotoxins to show that the extent of the erabutoxin b/-conotoxin match was too high to be coincidental. Homologues of erabutoxin b, namely-cobratoxin fromNaja naja siamensis and cytotoxin V^II4 fromNaja mossambica mossambica, were also analyzed. The extent of the matching with the-conotoxins decreased in the series erabutoxin b>-cobratoxin>cytotoxin V^II4, and this also relates the order of similarity to the pharmacological properties of the-conotoxins.The-conotoxin-like area of the snake-neurotoxins is peripheral to the site previously considered important for binding to the cholinoceptor, even though it seems to represent the focus of evolutionary convergence between the two types of neurotoxin. The area of resemblance does, however, have strong associations with the conformational behavior of the snake toxins. Hence, the outcome of this study has important consequences for the current ideas on snake-neurotoxin structure/activity relationships and the evolutionary origins of neurotoxicity.     

Bacteriological tests as an aid in the management of dental caries in Iceland

The activities of three natural coumarins, xanthotoxin and bergapten (fromAmmi majus, Umbelliferae) and psoralene (fromFicus cycomorus, Moraceae), were tested against mycelial growth and aflatoxin production of a toxigenic strain ofAspergillus flavus grown in a rice/corn steep liquor medium. Two other natural chromones, khellin and visnagin (fromAmmi visnaga) were also compared. Complete inhibition of aflatoxin release occurred with either xanthotoxin or khellin at 5 mM. The other three compounds also at 5 mM reduced aflatoxin to 12 to 16% of its original concentration. The mould growth was only slightly inhibited by all the compounds used.     

In vitro physiological approach to classification ofFrankia isolates of ‘the Alnus group’, based on urease,protease and β-glucosidase activities

Summary Most of theFrankia strains isolated fromAlnus andMyrica species are morphologically almost indistinguishable, when grown under standard culture conditions. They form similar vegetative hyphae while sporangia are produced in variable amounts from strain to strain.Physiological reactions were assessed in order to compare 20 strains isolated from various species ofAlnus and one species ofMyrica in Europe and North America. Among invariant negative or positive characteristics, differences in urease, protease and -glucosidase activities appeared to be of significant value.     

Two new 17 α-hydroxyprogesterone transformation products fromNocardia DSM 43298

Two new products: 9,10-seco-1,3,5(10)-pregnatrien-3,17 a-dihydroxy-9,20-dione and 9,10-seco-1,3,5(10)-pregnatrien-3,17a,20-triol-9-one, have been isolated as 17 -hydroxyprogesterone transformation products fromNocardia DSM 43298. The structures were established by spectroscopic methods.This paper is dedicated to Dr Klaus Kieslich.     

Properties of theβ-glucosidase fromCellulomonas flavigena and fromEscherichia coli harboring the recombinant plasmid pJS3

Summary Plasmid-coded -glucosidase produced byEscherichia coli was characterized and compared to the enzyme produced byCellulomonas flavigena. Cell-free extracts, non-denaturing PAGE and 5-bromo-4-chloro-3-indolyl--d-glucopyranoside (X-glu) as substrate were used to compare both enzymes. The -glucosidase was assayed for cellobiose andp-nitrophenyl-glucopyranoside (PNPG). Cellobiose hydrolysis was performed at 50°C for the enzyme fromC. flavigena and at 37°C for that fromE. coli pJS3, both with an optimal pH of 6.5. For PNPG hydrolysis, the optimal conditions were pH 5.5 and 37°C for both cell extracts. Most of the -glucosidase activity was intracellular. When cultures ofC. flavigena were grown with cellobiose or carboxymethylcellulose (CMC) as inducers, the expression of -glucosidase was increased considerably.E. coli pJS3 produces a cellobiase which hydrolyzes cellobiose and PNPG. TheK _m values for cellobiose and PNPG indicated that the -glucosidase activity ofC. flavigena had a higher affinity for cellobiose as substrate, whereas the -glucosidase fromE. coli pJS3 showed higher affinity for PNPG.     

Specificity towards oligomannoside and hybrid type glycans of the endo-β-N-acetylglucosaminidase B from the basidiomy ceteSporotrichum dimorphosporum

We have previously shown that an endo--N-acetylglucosaminidase (EC 3.2.1.96) named Endo B, isolated from culture filtrates of the basidiomyceteSporotrichum dimorphosporum cleaves asialo-, and to some extent, monosialylated bi-antennary glycans of theN-acetyllactosamine type linked to the asparagine residue of peptide or protein moieties [Bouquelet S, Strecker G, Montreuil J, Spik G (1980) Biochimie 62:43–49]. In the present paper, the substrate specificity of the enzyme towards oligomannoside and hybrid type glycans has been analyzed. The results obtained indicate that ovalbumin glycopeptides containing four to seven mannose residues and bovine lactotransferrin glycopeptides containing four to nine mannose residues were completely hydrolyzed by the enzyme. The degree of cleavage was variable among hybrid type structures, since glycopeptides containing the following glycans: (Gal)₁(GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₄(GlcNAc)₂ were not hydrolyzed by the enzyme while the percentage of hydrolysis of a glycopeptide containing (GlcNAc)₂(Man)₅(GlcNAc)₂ glycan reached 90%. The bovine lactotransferrin was partially deglycosylated (40%) in the absence of non-ionic detergent while native ovalbumin glycoprotein was not hydrolyzed by the enzyme.The oligomannoside-and theN-acetyllactosamine-type degrading activities present in the culture filtrates were not separated at any step of the purification procedure. Both activities were eluted as a single component with an apparent molecular mass of 89 kDa suggesting that they are located on the same enzyme molecule.Endo B represents a powerful tool for removing oligomannoside-andN-acetyllactosamine-type glycans fromN-glycopeptides andN-glycoproteins. Moreover, advantages in the use of Endo B in a soluble form as well as in an immobilized form result in its high activity and in its stability to heat denaturation and storage.Abbreviations Gal d-galactose - Man d-mannose - GlcNAc N-acetyl-d-glucosamine - Con A concanavalin A - Asn asparagine - GLC gas liquid chromatography - TLC thin layer chromatography - Endo endo--N-acetylglucosaminidase - Endo B endo--N-acetylglucosaminidase isolated fromSporotrichum dimorphosporum - PBE polybuffer exchanger - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

β-1-3- and β-1-4-glucan synthesis by membrane fractions from the fungus Saprolegnia

The -glucan synthetase activity of the fungus Saprolegnia monoica was assayed by supplying UDP-glucose to membrane fractions of mycelial homogenate. The analysis of glucan products by hydrolysis with various -glucanases and by chromatography show that both -1-3- and -1-4-linkages are formed at high substrate concentrations. In the absence of MgCl₂, -1-3-linked glucans are mainly produced. By increasing MgCl₂ concentrations the total synthesis activity and -1-3-linkages production are reduced. At low substrate concentrations in the presence of MgCl₂, -1-4-linked glucans are the only polysaccharide synthesized. Electron microscopy of radioactive products, synthesized by original membrane fractions or by membrane fractions isolated from continuous sucrose density gradients, shows microfibrils when the assays are conducted at high substrate concentrations in the absence of MgCl₂.Abbreviations G.S. I glucan synthetase I - G.S. II glucan synthetase II - Dol. P dolichol phosphate     

Subdomain organization ofBacillus thuringiensis entomocidal proteins'' N-terminal domains

N-Terminal domain (65 kD) of -endotoxin produced byBacillus thuringiensis ssp.alesti, as shown by limited proteolysis, consists of two subdomains of molecular mass 30 and 33 kD that correspond, respectively, to conservative and variable regions of the -endotoxin primary structure. Furthermore, proteolysis of these subdomains leads to their conversion into at least two fragments of molecular mass 10 kD stable to proteinase action. Such a pattern of molecular organization appears to be common for several structurally related -endotoxins that belong to thekurstaki group. Entomicidal protein produced by ssp.israelensis (70 kD), which differs strongly fromalesti and otherkurstaki group -endotoxins, retains a similar type of molecular organization and consists of two subdomains with molecular mass of 35 kD. Apparently, the characteristic pattern of the -endotoxins' molecular structure reflects separation of functions (e.g., host recognition and toxicityper se) between domains and subdomains of these proteins.     

Effects of pH,Ca and Al on the exudation from clover seedlings of compounds that induce the expression of nodulation genes inRhizobium trifolii

The expression of nodulation genes inR. trifolii is induced by flavone compounds present in clover root exudates. In the present experiments a bioassay with an indicator strain ofR. trifolii, which contained thelacZ gene fromEscherichia coli fused to theR. trifolii nodA gene, was used to measure the level ofnod gene expression inR. trifolii. Compounds that stimulatednodA gene expression were shown to be present in exudates of white clover (Trifolium repens L.) and nine cultivars of subterranan clover (T. subterraneum L.) seedling sgrown at a range of pH between pH 3.0 and pH 8.0. Thenod gene-induction activity of exudates was, however, reduced when seedlings of all clover species were grown at pH>7.0 and at pH<4.0 and pH<5.0 for white clover and subterranean clover respectively. No major differences were apparent in the activity of exudates from seedlings of the various cultivars of subterranean clover.Nod gene-induction activity of exudates was shown to increase markedly with seedling age. The presence of Ca at concentrations up to 10 mM in seedling culture solutions also resulted in marked increases in thenod gene-induction activity of seedling exudates. Increases in activity due to the presence of Ca were most apparent at low pH where between 5 and 10-fold increases were observed for white clover and subterranean clover respectively. Conversely, the presence of Al at concentrations up to 60 M in seedling culture solutions had no effect on thenod gene-induction activity of seedling exudates.The observations that both low pH and Ca concentrations affected thenod gene-induction activity of seedling exudates suggested that the net presence of stimulatory flavones in root exudates was an important contributing factor to the acid-sensitive step in nodule formation.     

Purification,properties and primary structure of H2-formingN 5,N10-methylenetetrahydromethanopterin dehydrogenase fromMethanococcus thermolithotrophicus

H₂-FormingN ⁵,N¹⁰-methylenetetrahydromethanopterin dehydrogenase (Hmd) is a novel type of hydrogenase found in methanogenic Achaea that contains neither nickel nor iron-sulfur clusters. The enzyme has previously been characterized fromMethanobacterium thermoautotrophicum and fromMethanopyrus kandleri. We report here on the purification and properties of the enzyme fromMethanococcus thermolithotrophicus. Thehmd gene was cloned and sequenced. The results indicate that the enzyme fromMc. thermolithotrophicus is functionally and structurally closely related to the H₂-forming methylene tetrahydromethanopterin dehydrogenase fromMb. thermoautotrophicum andMp. kandleri. From amino acid sequence comparisons of the three enzymes, a phylogenetic tree was deduced that shows branching orders similar to those derived from sequence comparisons of the 16S rRNA of the orders Methanococcales, Methanobacteriales, and Methanopyrales.Abbreviations H ₂ Forming dehydrogenase orHmd - H₂-FormingN ⁵,N¹⁰ methylene tetrahydromethanopterin dehydrogenase - H ₄MPT Tetrahydromethanopterin - CH ₂=H₄MPT N⁵,N¹⁰ Methylene tetrahydromethanopterin - CHH ₄MPT⁺ N⁵,N¹⁰ Methenyltetrahydromethanopterin - MALDI-TOF-MS Matrix-assisted laser desorption     

Immunological homologues of theArabidopsis thaliana β1 tubulin are polyglutamylated inNicotiana tabacum

Summary Peptide-specific antibody AAB1, raised to the C-terminal 13 amino acids ofArabidopsis thaliana 1 tubulin, identifies a single electrophoretically separable -tubulin on 2-D-gel Western blots of total protein extracts fromA. thaliana seedlings. We show that AAB1 crossreacts with two of the eight polyglutamylated -tubulin isoforms present in purifiedNicotiana tabacum tubulin fractionated by high-resolution isoelectric focussing. Immunolocalisation studies using AAB1 revealed that the twoN. tabacum polyglutamylated 1-tubulin isoforms are utilised in all four plant microtubule arrays (the interphase cortical array, the preprophase band, the spindle and the phragmoplast) indicating that there is no apparent subcellular sorting of these isotypes.Abbreviations AAB1 Anti-Ambidopsis thaliana 1-tubulin antibody - HRIF high-resolution isoelectric focussing     

<Emphasis Type="Italic">Penicillium frequentans</Emphasis> isolated from<Emphasis Type="Italic"> Picea glehnii</Emphasis> seedling roots as a possible biological control agent against damping-off

Picea glehnii seedlings are affected by damping-off fungi in nurseries. The aims of this study were (1) to isolate fungi grown in the seedling rhizosphere in forest soil of P. glehnii, (2) to select fungi that produce antifungal compounds against Pythium vexans, and (3) to examine whether or not selected fungi can protect seedlings from P. vexans. Penicillium frequentans from Picea glehnii seedling roots produced antibiotic penicillic acid. Penicillic acid did not cause significant phytotoxicity to the seedlings. Penicillium frequentans increased the average percentage of surviving seedlings when inoculated together with Pythium vexans, but the increase was not significant. Vigorous mycelial growth of P. frequentans around seedling roots seems to be one of the mechanisms for protection, but the amount of penicillic acid was too low to show antifungal activity in the seedling rhizosphere.     

Polymorphism and chromosomal location of endogenous α-amylase inhibitor genes in common wheat

Summary Polymorphism of an endogenous -amylase inhibitor in wheat was studied using iso-electric focusing followed by monoclonal antibody — based immunoblotting. Ten isoforms of the inhibitor detected in common wheat and its wild counterparts were assigned to five homoeologous loci. Three -amylase inhibitor loci (Isa-1) were identified in common wheat and located on the long arms of chromosomes 2A, 2B and 2D. In a sample of 27 bread wheats, eight durum wheats, and 12 diploid wheat relatives, amphiploids and triticales, a high resolution isoelectric-focusing separation demonstrated two active and one null allele at the Isa-A1, two alleles at the Isa-B1, one allele at the Isa-D1, four alleles at the Isa-S1, and one allele at the Isa-G1 locus. The most frequent electrophoretic pattern of common wheat cultivars consisted of two isoforms, encoded respectively by the Isa-B1b, Isa-D1 a alleles and the Isa-Alnull allele. All the durum wheats had only one inhibitor form controlled by allele Isa-B1b, which was accompanied by the null allele at the Isa-A1 locus.Contribution No. 210 of the Food Science Department, University of Manitoba     

Biosynthesis of heparin

The formation of labeled heparin-precursor polysaccharide (N-acetylheparosan) from the nucleotide sugars, UDP-[¹⁴C]glucuronic acid and UDP-N-acetylglucosamine, in a mouse mastocytoma microsomal fraction was abolished by the addition of 1% Triton X-100. In contrast, the detergent-treated microsomal preparation retained the ability to convert such preformed polysaccharide into sulfated products during incubation with 3-phosphoadenylylsulfate (PAPS). However, as shown by ion-exchange chromatography of these products, the detegent treatment changed the kinetics of sulfation from the rapid, repetivive process characteristic of the unperturbed system to a slow, progressive sulfation, which involved all polysarccharide molecules simultaneously and yielded, ultimately, a more highly sulfated product. The detergent effect was attributed to solubilization of sulfotransferases from the microsomal membranes, along with other polymer-modifying enzymes and the polysaccharide substrate. The resulting product showed an apparently random distribution ofN-acetyl andN-sulfate groups, instead of the predominantly block-wise arrangement achieved through membrane-associated biosynthesis.O-Sulfation occurred mainly at C2 of the iduronic acid units in the membrane-bound polysaccharide but at C6 of the glucosamine residues in the presence of detergent.A capsular polysaccharide fromEscherichia coli K5, previously found to have a structure identical to that of the nonsulfated heparin-precursor polysaccharide, was sulfated in the solubilized system in a fashion similar to that of the endogenous substrate, but was not accessible to the membrane-bound enzymes.These findings suggest that the regulation of the polymer-modification process, and hence the structure of the final polysaccharide product, depends heavily on the organization of the enzymes and their proteoglycan substrate in the endoplasmic membranes of the cell.Abbreviations PAPS 3-phosphoadenylylsulfate - Hepes 4-(2-hydroxy-ethyl)piperazineethanesulfonic acid - GlcUA glucuronic acid This is Paper XIV of a series in which the preceeding reports are refs 10 and 12. A preliminary report has appeared [28].     

A phylogenetic analysis of the Euterpeinae (Palmae; Arecoideae; Areceae) based on morphology and anatomy

The Euterpeinae contains six neotropical genera. There has been continual disagreement on generic and subgeneric boundaries in the subtribe.Euterpe andPrestoea, andJessenia andOenocarpus, have been repeatedly united and separated. A phylogenetic analysis based on 54 morphological and anatomical characters gave one tree of 127 steps.Euterpe is separate fromPrestoea, butJessenia andOenocarpus are best treated as one genus. Subgeneric relationships ofEuterpe andOenocarpus are also analyzed and discussed.     

Identification of two Drosophila TGF-β family members in the grasshopper Schistocerca americana

Intercellular signaling molecules of the transforming growth factor- (TGF-) superfamily are required for pattern formation in many multicellular organisms. The decapentaplegic (dpp) gene of Drosophila melanogaster has several developmental roles. To improve our understanding of the evolutionary diversification of this large family we identified dpp in the grasshopper Schistocerca americana. S. americana diverged from D. melanogaster approximately 350 million years ago, utilizes a distinct developmental program, and has a 60-fold-larger genome than D. melanogaster. Our analyses indicate a single dpp locus in D. melanogaster and S. americana, suggesting that dpp copy number does not correlate with increasing genome size. Another TGF- superfamily member, the D. melanogaster gene 60A, is also present in only one copy in each species. Comparison of homologous sequences from D. melanogaster, S. americana, and H. sapiens, representing roughly 900 million years of evolutionary distance, reveals significant constraint on sequence divergence for both dpp and 60A. In the signaling portion of the dpp protein, the amino acid identity between these species exceeds 74%. Our results for the TGF- superfamily are consistent with current hypotheses describing gene duplication and diversification as a frequent response to high levels of selective pressure on individual family members.     

Epicuticular wax alkanes ofEricaceae andEmpetrum from alpine and sub-alpine heaths in Austria

Alkane distribution patterns were determined in the epicuticular wax of the leaves of 13 species and a hybrid fromEricaceae and one species ofEmpetrum (Empetraceae). As chemotaxonomic indicators, the results are of limited use only. The most uniform genus wasRhododendron, the most heterogeneousVaccinium. The dominant effect of genetic over environmental factors was apparent in most cases.