Quantification of the extracellular matrix of the Listeria monocytogenes biofilms of different phylogenic lineages with optimization of culture conditions.

AIMS: The purpose of this study was to quantify the extracellular matrix of Listeria monocytogenes biofilm. A preliminary study was carried out to establish a relationship between phylogenetic lineage of 27 strains and their ability to form biofilm in various conditions. METHODS AND RESULTS: Biofilm formation on microtitre plates of 27 strains of L. monocytogenes belonging to lineages I or II was evaluated in different conditions [two temperatures (37 and 22°C) and two media (tryptone soy broth yeast extract medium (TSBYE) and MCDB 202 defined medium)] using crystal violet assay. Lineage II strains produced significantly more biofilm than lineage I strains. In microtitre plates assay, biofilm quantities were greater in MCDB 202 vs TSBYE medium [confirmed by scanning electron microscopy (SEM) analysis] and at 37 vs 22°C. Cultivable bacteria from biofilm population on Petri dishes were enumerated in greater quantities in TSBYE than in MCDB 202 medium. The SEM investigation established that L. monocytogenes biofilms produce extracellular matrix in both media at 37°C. The amount of exopolymers in the extracellular matrix and the pH values were significantly higher in TSBYE than in MCDB 202 medium. The exception was the ScottA strain that presented similar pH values and exopolymer contents in both media. Proteins were the most abundant exopolymer components, followed by DNA and polysaccharides. CONCLUSIONS: The interpretation of results of biofilm quantification was depending on the growth conditions, the viability of the bacteria and the analysis method. The quantities of proteins, DNA and polysaccharides were different according to the strains and the medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study screened the potential of a wide panel of L. monocytogenes strains to synthesize exopolymers in biofilm growing condition. The characterization of L. monocytogenes biofilm composition may help to develop new strategies to prevent the formation of biofilms and to remove the biofilms.

O-glycan variability of egg-jelly mucins from Xenopus laevis: characterization of four phenotypes that differ by the terminal glycosylation of their mucins.

Eggs from Xenopus laevis are surrounded by several layers of jelly that are needed for proper fertilization. Jelly coat is composed of high-molecular-mass glycoconjugates to which are bound many globular proteins. O-glycans released from the jelly coat of X. laevis have been partially described in previous studies. In this study, we compared the glycosylation pattern of the egg jelly coat isolated from six specimens of X. laevis. The O-glycans were released from jelly coats by alkali/borohydride treatment. Structural characterization was performed through a combination of one- and two-dimensional (1)H-NMR and methylation analysis. This allowed the description of a new family of sulphated O-glycans present in jelly coats of all X. laevis. However, the jelly O-glycans showed a low extent of polymorphism between specimens. This intra-specific variability was restricted to the terminal substitution of O-linked oligosaccharides. The differential expression of two glycosyltransferase [an alpha-(1-->4) galactosyltransferase and an alpha-(1-->3) fucosyltransferase] activities resulted in the characterization of four phenotypes of X. laevis. Furthermore, electrophoretic analysis suggested that the high-molecular-mass fraction of jelly coat was mostly composed of mucin-type glycoproteins. Blot analysis with lectins confirmed that the glycan variability was borne by these mucin-type components. However, fertilization assays suggested that the glycan polymorphism had no repercussion on egg fertilizability.

Single-step gas chromatography-mass spectrometry analysis of glycolipid constituents as heptafluorobutyrate derivatives with a special reference to the lipid portion.

In a previous work (Zanetta et al. Glycobiology 9, 255-266 (1999)), it was reported that all constituents of gangliosides could be obtained as heptafluorobutyrate derivatives after methanolysis in a single gas chromatography analysis. This report demonstrates that gas chromatography coupled with mass spectrometry in the electron impact mode allows identification and quantification of long-chain bases and fatty acids without interference from monosaccharides. On the basis of ions specific for families and for individual compounds, sphingosines, sphinganines, and phytosphingosines (including ramified, unsaturated, hydroxylated, and etherified compounds) can be identified. Fatty acid methyl esters, including linear, ramified, unsaturated, and hydroxylated species, are identified and quantified in the same way. Possible extensions of this method to the fatty moiety of other lipids (alkylacylglycerol and dimethyl acetal) are discussed.

[A biophysical approach to the investigation of physiological processes]. / Biofizicheskii podkhod k issledovaniiu fiziologicheskikh protsessov.

It was shown that understanding the mechanism of rhythmic excitation in cells and tissues requires the combination of physiological and biophysical approaches. Systemic studies of changes in the physicochemical characteristics of the object were carried out by a protocol that takes into account the mode of rhythmic excitation and the functional sate of the object being studied. The validity of the approach was proved in studies of rhythmic excitation in somatic nonmyelinic and myelinic nerves, and in model systems. The approach can be used in studies of many physiological processes.

Triterpenoid saponins from Herniaria fontanesii.

Two new saponins have been isolated from the aerial parts of Herniaria fontanesii. Their structures were established as 28-O-[alpha-L-rhamnopyranosyl-(1-->2-[(alpha-L- rhamnopyranosyl)-(1-->3)-]-(beta-D-4-acetoxyfucopyranosyl] ester of 3-O-[alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranosyl uronic]-2 beta-3 beta-16-alpha-trihydroxy-olean-12-ene-23, 28-dioic acid and 28-omicron-[alpha-L-rhamnopyranosyl-(1-->2-[alpha-L-rhamnopyranosyl (1-->3)]-(beta-D-fucopyranosyl] ester of 3-omicron-[alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranosyl uronic]-2 beta-3 beta-16-alpha-trihydroxy-olean-12-ene-23, 28 dioic acid.

Structural studies on the Escherichia coli O101 lipopolysaccharide found in association with F41 and K99 fimbriae.

In this study it was shown that the O101 lipopolysaccharide isolated from Escherichia coli B41 did not contain an O-specific polysaccharide and that its sugar moiety is probably restricted to the core oligosaccharide. It is characterized by the presence of galactose, glucose, N-acetylglucosamine, heptose and 3-deoxy-D-manno-2-octulosonic acid and the fatty acid composition is typical of an Enterobacteriaceae lipopolysaccharide. Methylation analysis indicated terminal non-reducing galactose and glucose and also 1,2-linked glucose which is a substitution pattern typical of an E. coli lipopolysaccharide core oligosaccharide. The obtained structural information is sufficient to explain the previously observed interactions between the O101 lipopolysaccharide and the K99 lectin.

Herniaria saponin B. a novel triterpenoid saponin from Herniaria fontanesii.

The aerial parts of Herniaria fontanesii have yielded a new saponin named herniaria saponin B. Its structure was characterized by means of mass spectrometry. NMR techniques, and chemical analysis gave 28-O-[alpha-L-rhamnopyranosyl-(1-->2)-[(alpha-L-rhamnopyranosyl-(1--> 3)]-beta-D-xylopyranosyl (1-->2)-beta-D-fucopyranosyl) ester of 3-O-[beta-D-glucopyranosyl uronic-(1-->4)-alpha-L-rhamnopyranosyl]-2 beta. 3 beta-16 alpha-trihydroxy-olean-12-ene-23, 24-dioic acid.

Structure of the O-specific polysaccharide chain from Klebsiella pneumoniae O1K2 (NCTC 5055) lipopolysaccharide.

The structure of the O-specific polysaccharide of Klebsiella pneumoniae O1K2 lipopolysaccharide was investigated by use of methylation, periodate oxidation, partial hydrolysis, and 1H- and 13C-n.m.r. spectroscopy. It was shown to consist of a linear chain composed of two disaccharide repeating units, [----3)-alpha-D-Galp-(1----3)-beta-D-Galp-(1----] and [----3)-alpha-D-Galp-(1----3)-beta-D-Galf-(1----].

[The role of membrane protein SH-groups in regulating ouabain and bungarotoxin binding in the crab nerve]. / Rol' SH-grupp membrannykh belkov v reguliatsii sviazyvaniia ouabaina i bungarotoksina v nerve kraba.

Structural alterations in plasma membrane proteins promoting transition from inactive forms of Na-pump and acetylcholine receptor to active ones during propagation of rhythmic excitation were examined. The role of SH-groups of the nerve (fibre and glial cell) plasma membrane proteins in providing regulation of the pump and receptor state by generalized alterations in the membrane structure is discussed.

[Levels of free fatty acids and calcium absorption in giant squid axons at rest and upon stimulation]. / Soderzhanie svobodnykh zhirnykh kislot i pogloshchenie Ca v gigantskom aksone kal'mara v pokoe i pri vozbuzhdenii.

Nine free fatty acids (FFA) were discovered in the squid axons at rest: 12:0, 14:0, 15:0, 16:0, 16:1, 18:0, 18:1, 20:3, 20:4. The relative amount of FFA equaled 14.1 micrograms per mg of lipids. Stimulation of nervous conductors decreased the amount of acids--14:0, 15:0, 16:1, 20:3. Simultaneously a considerable increase of the content of 20:4 was observed and three new fatty acids 20:0, 20:1, 22:2 appeared. The content of FFA increased to 27.2 micrograms/mg of lipids. Calcium absorption in the squid axons was equal to 23 nmol/g, but under stimulation this parameter increased to 37 nmol/g. Exogenous fatty acids brought about also an increase of calcium absorption. The accumulation of FFA under excitation was suggested to be the result of the increase of phosphoinositides content and related to the regulation of Ca transport.

Specificity towards oligomannoside and hybrid type glycans of the endo-beta-N-acetylglucosaminidase B from the basidiomycete Sporotrichum dimorphosporum.

We have previously shown that an endo-beta-N-acetylglucosaminidase (EC 3.2.1.96) named "Endo B", isolated from culture filtrates of the basidiomycete Sporotrichum dimorphosporum cleaves asialo-, and to some extent, monosialylated bi-antennary glycans of the N-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 the N-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- and N-acetyllactosamine-type glycans from N-glycopeptides and N-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.

Purification of the lipopolysaccharide fraction from Klebsiella pneumoniae O1 K2 by high-performance liquid chromatography.

An high-performance liquid chromatography technique was applied to purify the lipopolysaccharide fraction from a lysate of Klebsiella pneumoniae O1 K2 (NCTC 5055). The separation of the lipopolysaccharide fraction from the proteins was carried out with a reversed-phase column. By this method the lipopolysaccharide fraction was obtained in a pure state, devoid of proteins but possessing the same biological properties as the lipopolysaccharide fraction prepared by the classical phenol-water technique.

[Distribution of calcium in unmyelinated and myelinated nerve trunks]. / Raspredelenie kal'tsii v nemielinovykh i mielinovykh nervnykh stvolakh.

Exchange of Ca2+ between non-myelinated and myelinated nerves at the surface of axoglial complex of the nerve seems to be one of the extracellular systems controlling the Ca2+ level.

[Effect of changes in temperature on the cholesterol and phospholipid ratio in nerve fibers of the frog and squid]. / Vliianie izmenenii temperatury na sootnoshenie kholesterina i fosfolipidov v nervnykh provodnikakh liagushki i kal'mara.

The character of changes in molar correlation of cholesterol and phospholipids has been studied at different functional states in frog myelinic nerve and squid nonmyelinic nerve trunk. The correlation cholesterol/phospholipid (C/P) found for both nerves in rest increases at temperature 38 degrees C. The electrical stimulation on the background of higher temperature action leads to unequal shift of C/P: the increase compared with the rest in frog and decrease in squid.

[Binding of 3H-bungarotoxin by nerve trunks of the frog during excitatory conduction]. / Sviazyvanie 3H-bungarotoksina nervnymi stvolami liagushki pri provedenii vozbuzhdeniia.

In the frog nerve, 3H-bungarotoxin and 3H-acetylcholine binding increased whereas the acetylcholinesterase activity decreased in rhythmic stimulation. The enhancement of 3H-bungarotoxin binding to nerve during the rhythmic stimulation seems to stem from transformation of "inactive" forms of acetylcholine receptor of glial membrane to "active" ones.

[Changes in the permeability of nerve fiber membranes during the initiation of lipid peroxidation]. / Izmenenie pronitsaemosti membran nervnykh volokon v usloviiakh initsiatsii perekisnogo okisleniia lipidov.

The role of lipid oxidation in the mechanism of ion transport was investigated. During initiation of lipid oxidation by actin "Fe2+--ascorbic acid" in the crab nerve 45Ca and 22Na accumulation and level of lipid oxidation were increased, but electric stimulation of the nerve greatly changed ion accumulation. Increased Na+Ca2+ accumulation during the initiation of lipid oxidation is explained not only by changes in the lipid phase of excitable membranes but by the effects of rhythmic excitation on potential-dependent channels.

[Calcium ion binding in somatic nerves during conduction of rhythmic excitation]. / Sviazyvanie ionov kal'tsiia v somaticheskikh nervakh pri provedenii ritmicheskogo vozbuzhdeniia.

Increase of Ca entry upon excitation depended on stimulation frequency and the types of nerves in crab, frog and squid. The amount of absorbed Ca was high in myelinated nerves whereas the velocity of absorption was higher in nonmyelinated nerves. The maximal level of Ca entry during rhythmic excitation was observed in the frog nerve. The data obtained suggest a mechanism of Ca entry in somatic nerves during rhythmic excitation.