Curdlan and other bacterial (1→3)-β-d-glucans

Three structural classes of (13)--d-glucans are encountered in some important soil-dwelling, plant-associated or human pathogenic bacteria. Linear (13)--glucans and side-chain-branched (13,12)--glucans are major constituents of capsular materials, with roles in bacterial aggregation, virulence and carbohydrate storage. Cyclic (13,16)--glucans are predominantly periplasmic, serving in osmotic adaptation. Curdlan, the linear (13)--glucan from Agrobacterium, has unique rheological and thermal gelling properties, with applications in the food industry and other sectors. This review includes information on the structure, properties and molecular genetics of the bacterial (13)--glucans, together with an overview of the physiology and biotechnology of curdlan production and applications of this biopolymer and its derivatives.     

The role of nonspecific serum factors in the antibody response to the ΦX 174 bacteriophage

The role of the “antibody cofactor” and of other heat-labile serum components (complement) in the neutralization of the ?X 174 bacteriophage by means of specific antibodies was studied. Sera of white mice, guinea-pigs and rabbits obtained mainly early after phage administration were investigated. The character of antibodies was estimated from their sensitivity to 2-mercaptoethanol or else by chromatography on Sephadex G-200. Sera from the first days after the administration of the phage containing mostly type 19S antibodies, and sera from later periods after the administration containing mostly type 7S antibodies, were tested. (Some evidence was also obtained about the formation of slowly sedimenting antibodies sensitive to 2-mercaptoethanol in the rabbit.) With a single exception the tested sera showed no significant decrease of the neutralization activity after 30-mins. heating at 56°C or at 60°C and no increase of the neutralization power could be observed after the application of homologous or normal mouse serum. It is concluded that the heat-labile components of normal sera including the complement and the “antibody cofactor” play no role in specific phage neutralization.     

What is essential remains invisible to the eyes? Blood pressure cuffs colonized by bacterial diversity

International Microbiology - Using sphygmomanometers to measure blood pressure is a common practice in the healthcare context. The disinfection and maintenance of these devices is essential in...     

Does long-term fertilization treatment affect the response of soil ammonia-oxidizing bacterial communities to Zn contamination?

Experiments were conducted to examine the effects of long-term fertilization and acute Zn toxicity on the size, nitrification activity and community structure of autotrophic ammonia-oxidizing populations of the β-subgroup of the class Proteobacteria in arable soils. Plots under different long-term fertilization regimes were sampled, and then different concentrations of ZnCl₂ were spiked into soil samples for 8 weeks. It was found that long-term fertilization significantly increased nitrification rates and population size, and there was a positive correlation between them. A shift in the composition of AOB was also detected in samples fertilized with mineral N fertilizer (NPK) and organic matter (OM) as compared to unfertilized sample. EC50 values suggested that there was no significant difference in Zn toxicity to nitrification rates among the three fertilization treatments. Long-term fertilization did not improve the resilience of AOB activity to Zn toxicity.     

Structural analysis of enzyme-resistant isomaltooligosaccharides reveals the elongation of α-(1→3)-linked branches in Weissella confusa dextran

Weissella confusa VTT E-90392 is an efficient producer of a dextran that is mainly composed of α-(1→6)-linked D-glucosyl units and very few α-(1→3) branch linkages. A mixture of the Chaetomium erraticum endodextranase and the Aspergillus niger α-glucosidase was used to hydrolyze W. confusa dextran to glucose and a set of enzyme-resistant isomaltooligosaccharides. Two of the oligosaccharides (tetra- and hexasaccharide) were isolated in pure form and their structures elucidated. The tetrasaccharide had a nonreducing end terminal α-(1→3)-linked glucosyl unit (α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glc), whereas the hexasaccharide had an α-(1→3)-linked isomaltosyl side group (α-D-Glcp-(1→6)[α-D-Glcp-(1→6)-α-D-Glcp-(1→3)]-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glc). A mixture of two isomeric oligosaccharides was also obtained in the pentasaccharide fraction, which were identified as (α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glc) and (α-D-Glcp-(1→6)[α-D-Glcp-(1→3)]-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glc). The structures of the oligosaccharides indicated that W. confusa dextran contains both terminal and elongated α-(1→3)-branches. This is the first report evidencing the presence of elongated branches in W. confusa dextran. The (1)H and (13)C NMR spectroscopic data on the enzyme-resistant isomaltooligosaccharides with α-(1→3)-linked glucosyl and isomaltosyl groups are published here for the first time.     

The location of (1→3)-β-glucans in the walls of pollen tubes of Nicotiana alata using a (1→3)-β-glucan-specific monoclonal antibody

The location of the (13)--glucan, callose, in the walls of pollen tubes in the style of Nicotiana alata Link et Otto was studied using specific monoclonal antibodies. The antibodies were raised against a laminarinhaemocyanin conjugate. One antibody selected for further characterization was specific for (13)--glucans and showed no binding activity against either a cellopentaose-bovine serum albumin (BSA) conjugate or a (13, 14)--glucan-BSA conjugate. Binding was inhibited by (13)--oligoglucosides (DP, 3–6) with maximum competition being shown by laminaripentaose and laminarihexaose, indicating that the epitope included at least five (13)--linked glucopyranose residues. The monoclonal antibody was determined to have an affinity constant for laminarihexaose of 2.7. 10⁴M^–1. When used with a second-stage gold-labelled, rabbit anti-mouse antibody, the monoclonal antibody probe specifically located the (13)--glucan in the inner wall layer of thin sections of the N. alata pollen tubes.Abbreviations BSA bovine serum albumin - PBS phosphate-buffered saline - ELISA enzyme linked immunosorbent assay - DP degree of polymerization - PVC polyvinyl chloride P.J.M. is an Australian Postdoctoral Research Fellow. We wish to thank Joan Hoogenraad for her technical assistance with the tissue culture, and Althea Wright for her assistance in the preparation of this paper.     

Increased plasma α (1 → 3)-l-fucosyltransferase activities in patients with hepatocellular carcinoma

Alpha(1 3)-l-fucosyltransferase (1,3FT) activity was determined in plasma of patients with chronic liver diseases, namely, chronic hepatitis (CH), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). The plasma 1,3FT activity was significantly higher (p<0.01) in chronic liver diseases than that in normal controls. The enzyme activity in plasma of patients with HCC was also significantly higher than that in LC (p<0.05) or that in CH (p<0.01). However, no significant difference was observed in the enzyme activity between LC and CH. Plasma 1,3FT activity in patients with HCC was not significantly changed before and after transcatheter arterial embolization. In addition, the enzyme activity in the homogenate of the cirrhotic liver tissue was higher than that in the preparation of the hepatoma tissue in the same patient. These results suggest that the increased plasma 1,3FT activity in patients with HCC reflects mainly the enzyme activity of cirrhotic liver tissue, not that of hepatoma tissue. The significance of the elevated levels of plasma 1,3FT and its decreased hepatoma tissue activity in patients with HCC, compared with that in LC, remains to be clarified.     

β-D-Glycan synthases and the CesA gene family: lessons to be learned from the mixed-linkage (1→3),(1→4)β-D-glucan synthase

Cellulose synthase genes (CesAs) encode a broad range of processive glycosyltransferases that synthesize (14)-D-glycosyl units. The proteins predicted to be encoded by these genes contain up to eight membrane-spanning domains and four `U-motifs' with conserved aspartate residues and a QxxRW motif that are essential for substrate binding and catalysis. In higher plants, the domain structure includes two plant-specific regions, one that is relatively conserved and a second, so-called `hypervariable region' (HVR). Analysis of the phylogenetic relationships among members of the CesA multi-gene families from two grass species,Oryza sativa and Zea mays, with Arabidopsis thaliana and other dicotyledonous species reveals that the CesA genes cluster into several distinct sub-classes. Whereas some sub-classes are populated by CesAs from all species, two sub-classes are populated solely by CesAs from grass species. The sub-class identity is primarily defined by the HVR, and the sequence in this region does not vary substantially among members of the same sub-class. Hence, we suggest that the region is more aptly termed a `class-specific region' (CSR). Several motifs containing cysteine, basic, acidic and aromatic residues indicate that the CSR may function in substrate binding specificity and catalysis. Similar motifs are conserved in bacterial cellulose synthases, the Dictyostelium discoideum cellulose synthase, and other processive glycosyltransferases involved in the synthesis of non-cellulosic polymers with (14)-linked backbones, including chitin, heparan, and hyaluronan. These analyses re-open the question whether all the CesA genes encode cellulose synthases or whether some of the sub-class members may encode other non-cellulosic (14)-glycan synthases in plants. For example, the mixed-linkage (13)(14)-D-glucan synthase is found specifically in grasses and possesses many features more similar to those of cellulose synthase than to those of other -linked cross-linking glycans. In this respect, the enzymatic properties of the mixed-linkage -glucan synthases not only provide special insight into the mechanisms of (14)-glycan synthesis but may also uncover the genes that encode the synthases themselves.     

Biosynthesis of the storage polysaccharide from the snail Biomphalaria glabrata,identification and specificity of a branching β1→6 galactosyltransferase

Adult snails synthesize in their albumen glands a storage polysaccharide called galactan which is utilized by the developing embryos. With [6-³H]-uridine 5diphosphogalactose the incorporation of labelled d-galactose into the polysaccharide can be traeed in freshly removed glands maintained in a bathing buffer. After centrifugation of homogenized glands, galactosyltrasferase activity is only found in the insoluble fraction. Chaps extracts of this material retain almost all of their activity and can be used for comparison of the incorporation rates into different native galactans or in various oligosaccharides. A highly efficient -(16) galactosyltransferase was detected when methyl 3-O-(-d-galactopyranosyl)--d-galactopyranoside was offered as acceptor. The substitution at the penultimate residue resulted in a branched trisaccharide as demonstrated by ¹H-NMR-spectroscopy and permethylation analysis of the reaction product. Comparable results were obtained with various oligosaccharides containing an internal galactose unit glycosidically linked 13. Attempts to separate and purify the various enzymes involved resulted in the isolation of a fraction which is able to transfer d-Gal exclusively to native galactan, but not to oligosaccharides. A further fraction was obtained from a different resin with activity for native galactan and 6-O-(-d-galactopyranosyl)-d-galactopyranose. but without any for methyl-3-O-(-d-galactopyranosyl)--d-galactopyranose. It is thus concluded that at least three different enzymes are involved in the biosynthesis of this snail galactan.Abbreviation Gal galactose - glc gas-liquid chromatography - Gro glycerol - tlc thin layer chromatography     

11β-Hydroxysteroid dehydrogenase 1 contributes to the pro-inflammatory response of keratinocytes

The endogenous glucocorticoid, cortisol, is released from the adrenal gland in response to various stress stimuli. Extra-adrenal cortisol production has recently been reported to occur in various tissues. Skin is known to synthesize cortisol through a de novo pathway and through an activating enzyme. The enzyme that catalyzes the intracellular conversion of hormonally-inactive cortisone into active cortisol is 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1). We recently reported that 11β-HSD1 is expressed in normal human epidermal keratinocytes (NHEKs) and negatively regulates proliferation of NHEKs. In this study, we investigated the role of 11β-HSD1 in skin inflammation. Expression of 11β-HSD1 was induced by UV-B irradiation and in response to the pro-inflammatory cytokines, IL-1β and TNFα. Increased cortisol concentrations in culture media also increased in response to these stimuli. To investigate the function of increased 11β-HSD1 in response to pro-inflammatory cytokines, we knocked down 11β-HSD1 by transfecting siRNA. Production of IL-6 and IL-8 in response to IL-1β or TNFα stimulation was attenuated in NHEKs transfected with si11β-HSD1 compared with control cells. In addition, IL-1β-induced IL-6 production was enhanced in cultures containing 1 × 10⁻¹³ M cortisol, whereas 1 × 10⁻⁵ M cortisol attenuated production of IL-6. Thus, cortisol showed immunostimulatory and immunosuppressive activities depending on its concentration. Our results indicate that 11β-HSD1 expression is increased by various stimuli. Thus, regulation of cytosolic cortisol concentrations by 11β-HSD1 appears to modulate expression of inflammatory cytokines in NHEKs.     

Change in the power of EEG activity in the α range in response to tonic nociceptive stimulation of the distal joint of the little finger

Examination of 12 healthy volunteers aged 20–56 years was performed to study the EEG changes caused by a tonic squeeze of the distal joint of the little finger of the left and then the right hand. This stimulation caused painful sensations of different intensity (pain on the right was stronger). Spectral power was measured in the ( ₁ (8–10.5 Hz) and (₂ ranges (10.5–13 Hz) under different conditions. Weak pain led to an increase in the power of the (₁ and ₂ ranges in the occipital regions. With strong pain, the power of ₁ waves increased bilaterally in the posterior regions (O ₁, O ₂, T ₆), as well as in the left frontal region (F ₃, F ₇). The powers of the ₁ and ₂ ranges substantially increased relative to the background level after the strong nociceptive stimulation ceased. This finding testified to a latent and inertial character of its effect on the -wave parameters.Translated from Fiziologiya Cheloveka, Vol. 31, No. 2, 2005, pp. 77–84.Original Russian Text Copyright © 2005 by Garkavenko, Gorkovenko, Mankovskaya, Shevko, Lyskov, Kostyukov.     

Two novel isoneolacto-undecaglycosylceramides carrying Galα1→3Lewisx on the 6-linked antenna and N-acetylneuraminic acidα2→3 or Galactose α1→3 on the 3-linked antenna, expressed in porcine kidney

Three sialosylated and three neutral glycosphingolipids sharing a common iso-neolacto core were isolated from porcine kidney cortex. They were purified by preparative HPTLC, and were characterized by partial exoglycosidase hydrolysis followed by thin layer chromatography and immunostaining with anti-Gal13Gal, anti-type 2 lactosamine and anti-Lewisx antibodies, methylation analysis, MALDI-TOF mass spectrometry and 1H-NMR spectroscopy. Among neutral glycolipids, one was a known structure, VI3VI3(Gal)2-iso-nLc8Cer, and two were novel structures differing by the number of Gal3Lewisx determinants: VI3VI3(Gal)2V3Fuc-iso-nLc8, and VI3VI3(Gal)2 V3V3(Fuc)2-iso-nLc8. The single Gal3Lewis x determinant was found on the 6-linked antenna. Among sialosylated glycolipids, two had been previously found in other species and tissues, VI3VI3(NeuAc)2-iso-nLc8, and VI3NeuAcVI3Gal-iso-nLc8. A novel structure was discovered presenting a Gal3Lewisx determinant on the 6-linked antenna and a N-acetylneuraminic acid on the 3-linked antenna, VI3NeuAcVI3GalV3Fuc-iso-nLc8. These results indicate that, in vivo, the porcine kidney 3fucosyltransferase synthesizes the Gal3Lewisx determinant, acting on the 6-linked before the 3-linked Gal3neolactosamine, and appears unable to synthesize the sialosylated Lewisx determinant on neolactoseries glycolipids.     

Effects of Limiting Extension at the αIIb Genu on Ligand Binding to Integrin αIIbβ3

Structural data of integrin αIIbβ3 have been interpreted as supporting a model in which: 1) the receptor exists primarily in a “bent,” low affinity conformation on unactivated platelets and 2) activation induces an extended, high affinity conformation prior to, or following, ligand binding. Previous studies found that “clasping” the αIIb head domain to the β3 tail decreased fibrinogen binding. To study the role of αIIb extension about the genu, we introduced a disulfide “clamp” between the αIIb thigh and calf-1 domains. Clamped αIIbβ3 had markedly reduced ability to bind the large soluble ligands fibrinogen and PAC-1 when activated with monoclonal antibody (mAb) PT25-2 but not when activated by Mn²⁺ or by coexpressing the clamped αIIb with a β3 subunit containing the activating mutation N339S. The clamp had little effect on the binding of the snake venom kistrin (M_r 7,500) or αIIbβ3-mediated adhesion to immobilized fibrinogen, but it did diminish the enhanced binding of mAb AP5 in the presence of kistrin. Collectively, our studies support a role for αIIb extension about the genu in the binding of ligands of 340,000 and 900,000 M_r with mAb-induced activation but indicate that it is not an absolute requirement. Our data are consistent with αIIb extension resulting in increased access to the ligand-binding site and/or facilitating the conformational change(s) in β3 that affect the intrinsic affinity of the binding pocket for ligand.     

One-pot enzymatic synthesis of the Galα1→3Galβ1→4GlcNAc sequence within situ UDP-Gal regeneration

The trisaccharide Gal13Gal14GlcNAc1O-(CH₂)₈COOCH₃ was enzymatically synthesized, within situ UDP-Gal regeneration. By combination in one pot of only four enzymes, namely, sucrose synthase, UDP-Glc 4-epimerase, UDP-Gal:GlcNAc 4-galactosyltransferase and UDP-Gal:Gal14GlcNAc 3-galactosyltransferase, Gal13Gal14GlcNAc1O-(CH₂)₈COOCH₃ was formed in a 2.2 µmol ml^–1 yield starting from the acceptor GlcNAc1O-(CH₂)₈COOCH₃. This is an efficient and convenient method for the synthesis of the Gal13Gal14GlcNAc epitope which plays an important role in various biological and immunological processes.