Regulation of fructosyltransferase activity by carbohydrates, in solution and immobilized on hydroxyapatite surfaces

We tested the effect of several carbohydrates on the activity of cell-free fructosyltransferases (FTF) in solution and immobilized onto hydroxyapatite (HA) and found an inhibitory dose-dependent effect of glucose on FTF activity, both on the surface and in solution. Glucose at 160 mM inhibits FTF activity by 75% both on HA and in solution. Fructose at 160 mM inhibited FTF activity by 25% in solution and by 15% on HA. Levan inhibited FTF activity by 30% in solution, while dextrans and inulin had a limited effect on FTF activity. Circular dichroism and infrared analysis demonstrated no major changes in the chemical structure of fructans synthesized by cell-free FTF on HA and in solution, in the presence or absence of glucose. However, as verified by size-exclusion chromatography, glucose inhibited the synthesis of high molecular-weight fructans. The results indicate that glucose, a byproduct of the FTF enzymatic reaction, is the main carbohydrate affecting FTF activity. Selective inhibition of high molecular-weight fructan production by glucose, may indicate that two mechanisms are involved in the synthesis of fructans, both in solution and on the surface.     

Effect of oxazaborolidines on immobilized fructosyltransferase analyzed by surface plasmon resonance

Dental diseases are among the most prevalent afflictions of humankind. These diseases are associated with the formation of biofilms harboring pathogenic bacteria. Fructosyltransferases (FTF) are extra cellular enzymes of several oral bacteria. FTF are associated with the formation of extracellular polysaccharide matrix (fructans) which play a role in biofilm formation and oral bacteria physiology. Oxazaborolidines have been shown to inhibit biofilm formation. The purpose of this study was to examine if the anti-biofilm effect is, in part, an effect on the immobilized enzymes synthesizing the extra cellular polysaccharide participating in biofilm formation. Eight different oxazaborolidines (BNO1-BNO8) were synthesized and evaluated for their affect on the synthesis of fructans by FTF using the biomolecular interaction analysis (BIAcore) system which involves the use of real-time surface plasmon resonance (SPR) technique. The tested oxazaborolidines demonstrated a significant and immediate inhibitory effect on immobilized FTF activity. This effect was reversible. Our results show that oxazaborolidines can act as enzymatic inhibitors of FTF immobilized on the surface, also at levels lower than their MIC. Part of the anti-biofilm effect of BNOs may be accounted for this enzymatic inhibition.     

Effect of carbohydrates on fructosyltransferase expression and distribution in Streptococcus mutans GS-5 biofilms

Streptococcus mutans produces a fructosyltransferase (FTF) enzyme, which synthesizes fructan polymers from sucrose. Fructans contribute to the virulence of the biofilm by acting as binding sites for S. mutans adhesion and as extracellular nutrition reservoir for the oral bacteria. Antibodies raised against a recombinant S. mutans FTF were used to test the effect of glucose, fructose, and sucrose on FTF expression in S. mutans GS-5 biofilms. Biofilms formed in the presence of fructose and glucose showed a higher ratio of FTF compared to biofilms formed in the presence of sucrose. Confocal laser scanning microscopy images of S. mutans biofilms indicated a carbohydrate-dependent FTF distribution. The layer adjacent to the surface and those at the liquid interface displayed high amounts cell-free FTF with limited amount of bacteria while the in-between layers demonstrated both cell-free FTF and cells expressing cell-surface FTF. Biofilm of S. mutans grown on hydroxyapatite surfaces expressed several FTF bands with molecular masses of 160, 125, 120, 100, and 50 kDa, as detected by using FTF specific antibodies. The results show that FTF expression and distribution in S. mutans GS-5 biofilms is carbohydrate regulated.     

Immobilization of fructosyltransferase from Streptococcus mutans on hydroxyapatite surfaces induces the formation of multimeric complexes

AIMS: To investigate the formation of fructosyltransferase (FTF) complexes on hydroxyapatite (HA) surfaces. METHODS AND RESULTS: Cell-free extracellular FTF from Streptococcus mutans, purified from hyperproducing strain V-1995, was adsorbed onto HA and then eluted from the surface by means of a concentration gradient of potassium phosphate buffer. The FTF monomers loaded onto HA formed, upon adsorption, various complexes ranging from 200 to 700 kDa as demonstrated using native PAGE. All these complexes exhibited enzymatic activity. CONCLUSIONS: Adsorption of FTF onto HA induced the formation of stable and enzymatically-active complexes. SIGNIFICANCE AND IMPACT OF THE STUDY: The formation of these complexes may explain the change of FTF catalytic properties after adsorption onto HA. This study is another step in determining the properties of a-cellular constituents of the oral biofilm.     

Effect of hyaluronic acid molecular weight on the morphology of quantum dot-hyaluronic acid conjugates

The morphological analysis of novel quantum dot-hyaluronic acid (QDot-HA) conjugates was carried out with a transmission electron microscope (TEM). Adipic acid dihydrazide-modified HA (HA-ADH) was synthesized and conjugated to quantum dots (QDots) having carboxyl terminal ligands which were activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS). HA molecules with a molecular weight (MW) of 20K, 234 K and 3000 K were used to investigate the effect of MW on the morphology of QDot-HA conjugates. The TEM micrographs of QDot-HA conjugates showed branched and multi-layered chain type morphology formed by inter- and intra-molecular conjugation of QDots to HA molecules. The size of QDot-HA conjugate increased with the MW of HA. QDot-HA conjugate could be successfully used for real-time bio-imaging of HA derivatives in nude mice. The novel QDot-HA conjugate will be further used to investigate the biological roles of HA with a different MW in the body.     

N-Deglycosylation of fructosyltransferase and invertase from Fusarium oxysporum decreases stability but has little effect on kinetics and synthetic specificity

Most of the carbohydrate moiety of invertase and fructosyltransferase (FTF) from F. oxysporum IMI 172464 was removed by peptide-N-glycosidase F. The molecular weights of native invertase and FTF were 260 kDa and 210 kDa respectively. Deglycosylation lowered the molecular sizes by 42% and 23%, respectively. The K values for sucrose remained unchanged by deglycosylation. However the stability of both enzymes at their optimum pH (4.0 for invertase, 5.0 for FTF) and optimum temperature (45°C for invertase, 35°C for FTF) was decreased: their sensitivity to protease digestion was increased by 36% and 41%, respectively. The synthetic specificity of deglycosylated FTF remained unchanged. The carbohydrate moiety of invertase and FTF contributes to the stability of the enzymes but is not essential in their catalytic function and plays no part in determining their specificity.     

The influence of sulfated polysaccharides on the circulating levels of hyaluronan

When chondroitin sulfate (CS) or dextran sulfate (DxS) was administeredintravenously in rats the levels of circulating hyaluronan (HA)rapidly increased, 70 min after injection the levels were foundto be about 10–20 times the initial values. Saline injectionswere without effect on HA levels. CS given intraperitoneallywas found to give prolonged blocking of liver uptake of labeledHA and increased endogenous serum HA to about 10 times the initiallevel at 180 min. HA excretion in urine was dramatically increasedby CS given intravenously, intraperitoneally as well as subcutaneously.Size-exclusion chromatography showed a mean MW of the circulatingHA of around 50 kDa while urinary HA had a mean MW of about10 kDa. Circulating HA has previously been shown to be veryeffectively cleared via receptor mediated endocytosis by re-ticuloendothelialcells, primarily liver endothelial cells. As CS and DxS bindto the same receptors and inhibits HA clearance, the effectsof sulfated polysaccharides on inflammatory conditions and angiogenesismight be via HA, previously shown to affect these processes.Such a mechanism could also explain increased HA levels as asecondary event to increased CS and other sulfated biologicalpolysaccharides in some physiological and pathological conditions. hyaluronic acid turnover metabolism glycosaminoglycans angiogenesis     

Diene polymerizations with lanthanide coordination catalysts. II. The effects of catalytic system component types and polymerization conditions on molecular characteristics of 1,4-cis-Polybutadienes

Molecular inhomogeneity has been investigated for 1,4-cis-polybutadienes obtained with a lanthanide- containing catalytic system; some kinetic parameters have been estimated for polymerizations. Gel-permeation chromatography was chosen as the main method to find out the average molecular weights (MW) and molecular weight distributions (MWD). The catalyst composition has been shown to effect MW and MWD. The molecular characteristics are determined by both the catalyst composition and the polymerization conditions (reagent concentration, temperature). The broad MWD is considered a peculiarity of butadienes prepared with lanthanide- containing catalysts. The data obtained lead to the assumption that the distribution of the polymerization centres by reactivity is responsible for the broad MWD of the polybutadienes formed.     

pH-Amplified multilayer films based on hyaluronan: influence of HA molecular weight and concentration on film growth and stability

In this study, we investigate the growth and internal properties of polyelectrolyte multilayer films made of poly(l-lysine) and hyaluronan (PLL/HA) under pH-amplified conditions, that is, by alternate deposition of PLL at high pH and HA at low pH. We focus especially on the influence of the molecular weight of HA in this process as well as on its concentration in solution. Film growth was followed by quartz crystal microbalance and by infrared spectroscopy to quantify the deposited mass and to characterize the internal properties of the films, including the presence of hydrogen bonds and the ionization degree of HA in the films. Film growth was significantly faster for HA of high molecular weight (1300 kDa) as compared with 400 and 200 kDa. PLL was found to exhibit a random structure once deposited in the films. Furthermore, we found that PLL-ending films are more stable when they are placed in PBS than their HA counterparts. This was explained on the basis of more cohesive interactions in the films for PLL-ending films. Finally, we quantified PLL(FITC) diffusion into the films and observed that PLL diffusion is enhanced when PLL is paired with the HA of high MW. All together, these results suggest that besides purely physicochemical parameters such as variation in pH, the molecular weight of HA, its concentration in solution, and the possibility to form intermolecular HA association play important roles in film growth, internal cohesion, and stability.     

The role of fructans on dental biofilm formation by Streptococcus sobrinus, Streptococcus mutans, Streptococcus gordonii and Actinomyces viscosus

Dental plaque biofilm plays a pivotal role in the progression of dental diseases. Polysaccharides are of great importance in the ecology of the dental biofilm. We studied the effect of fructans, glucans and a mixture of both fructans and glucans, synthesized in situ by immobilized fructosyltransferase or glucosyltransferase, on the adhesion of Streptococcus sobrinus, Streptococcus mutans, Streptococcus gordonii and Actinomyces viscosus to hydroxyapatite beads coated with human saliva (sHA). The adhesion of A. viscosus to sHA was found to be fructan-dependent. Adhesion of both S. sobrinus and S. mutans was found to be mediated mainly by glucans, while the adhesion of S. gordonii was found to be both glucan- and fructan-dependent. Treatment with fructanase prior to A. viscosus adhesion resulted in a significant reduction in adhesion to sHA, while adhesion of S. sobrinus, S. mutans and S. gordonii was slightly influenced by fructanase treatment. Treatment with fructanase after adhesion of S. gordonii to sHA resulted in a significant reduction in their adhesion to sHA. Our results show that fructans may play a role in the adhesion and colonization of several cariogenic bacteria to sHA, thus contributing to the formation of dental plaque biofilm.     

A Systematic Study of the Effect of Different Molecular Weights of Hyaluronic Acid on Mesenchymal Stromal Cell-Mediated Immunomodulation

Introduction

Osteoarthritis (OA) is associated with chronic inflammation, and mesenchymal stromal cells (MSCs) have been shown to provide pain relief and reparative effects in clinical investigations. MSCs are often delivered with hyaluronic acid (HA), although the combined mechanism of action is not fully understood; we thus investigated the immunomodulatory effects of combining MSCs with different molecular weights (MW) of HA.

Methods

HAs with MWs of 1.6 MDa (hHA), 150 kDa or 7.5 kDa, were added to MSCs alone or MSC-immune cell co-cultures. Gene expression analyses, flow cytometry and cytokine measurements were assessed to determine the effect of HAs on the MSC interactions with immune cells.

Results

MSCs in the presence of HAs, in both normal and lymphocyte-conditioned medium, showed negligible changes in gene expression. While addition of hHA resulted in increased proliferation of activated lymphocytes, both in the presence and absence of MSCs, the overall combined effect was a more regulated, homeostatic one; this was supported by higher ratios of secreted IL10/IFNγ and IL10/IL2, in lymphocyte cultures, than with lower MW HAs or no HA, both in the presence and absence of MSCs. In addition, examination of monocyte-derived macrophages showed an increased M2 macrophage frequency (CD14+CD163+CD206+) in the presence of hHA, both with and without MSCs.

Conclusions

hHA produces a less pro-inflammatory environment than lower MW HAs. Moreover, combining hHA with MSCs has an additive effect on the MSC-mediated immunomodulation, suggestive of a more potent combination treatment modality for OA.     

Identification of a 175 kDa protein as the ligand-binding subunit of the rat liver sinusoidal endothelial cell hyaluronan receptor

The rat liver sinusoidal endothelial cell (LEC) hyaluronan (HA)receptor was previously identified using a photoaffinity HAderivative (J. BioL Chem., 267, 20451–20456, 1992). Twopolypeptides with M_r = 175,000 and 166,000, were consistentlycrosslinked, suggesting that the LEC HA receptor is an oligomer.Whether one or both subunits participate in HA binding, wasnot determined. Here we investigate the HA-subunit interactionsand the potential oligomeric nature of the LEC HA receptor.When Sephacryl-400 gel filtration chromatography was used toenrich the HA receptor, the 175 kDa polypeptide was the majorband seen by SDS-PAGE analysis. Little staining was seen at166 kDa, suggesting that the 175 kDa protein could be separatedfrom the 166 kDa protein and still retain HA-binding activity.A ligand blot assay was used to determine if each individualsubunit could bind HA. LEC proteins were separated by nonreducingSDS-PAGE, and then immobilized onto nitrocellulose. ¹²⁵I-HAbound to a 175 kDa polypeptide but not to the 166 kDa protein.A high molecular weight band of     

Isolation, structural elucidation and immunomodulatory activity of fructans from aged garlic extract

Traditionally, garlic (Allium sativum) is known to be a significant immune booster. Aged garlic extract (AGE) possesses superior immunomodulatory effects than raw garlic; these effects are attributed to the transformed organosulfur compounds. AGE is also known to contain fructans; the amount of fructans in AGE represents a small fraction (0.22%) of the total fructans in raw garlic. In order to evaluate the biological activity of fructans present in AGE, both high molecular weight (>3.5 kDa; HF) and low molecular weight (<3 kDa; LF) fructans were isolated. The structures of purified HF and LF from AGE determined by (1)H NMR and (13)C NMR spectroscopy revealed that both have (2→1) β-D-fructofuranosyl bonds linked to a terminal glucose at the non-reducing end and β-D-fructofuranosyl branching on its backbone. Biological activity of fructans was assessed by immunostimulatory activity using murine lymphocytes and peritoneal exudate cells (source of macrophages). Both HF and LF displayed mitogenic activity and activation of macrophages including phagocytosis. These activities were comparable to that of known polysaccharide immunomodulators such as zymosan and mannan. This study clearly demonstrates that garlic fructans also contribute to the immunomodulatory properties of AGE, and is the first such study on the biological effects of garlic fructans.     

Effect of hydroxamic acids on growth and urease activity in Corynebacterium renale.

Studies were conducted on the effect of four different hydroxamic acids (HA), hydroxyurea, acetohydroxamic acid, p-flurobenzoylhydroxamic acid and sorbylhydroxamic acid, on the growth and urease activity of Corynebacterium renale. The addition of each of these HA, at concentrations ranging form 10(-3) to 10(-5) M, to medium containing urea as the sole nitrogen source resulted in a lengthened lag period of growth the extent of which depended upon the concentration of each HA tested as well as the structure of the compound; that is, the size and (or) complexity of the side chain attached to the common terminal group of the molecule. However, the maximal growth levels achieved following conclusion of the exponential phase were not affected by the HA. Investigations on the effect of these HA on the urease activity of intact cells as well as cell-free extracts revealed that in each case the enzymatic activity was inhibited by each of the HA tested. The extent of inhibition with the intact cells was aobut one-half of that observed with cell-free extracts. Direct incubation of cell-free extracts as well as intact cells with each of the HA tested was required for maximal inhibition.     

Isolation and characterization of a thermolabile beta-2 macroglycoprotein ('thermolabile substance' or 'Hakata antigen') detected by precipitating (auto) antibody in sera of patients with systemic lupus erythematosus.

A novel thermolabile beta-2 macroglycoprotein ('thermolabile substance' (TLS) or 'Hakata antigen' (HA], which was detected by the precipitating (auto) antibodies of patients with systemic lupus erythematosus, was isolated and characterized. The purification procedure entailed the following steps: isoelectric precipitation in the range between pH 5.2-6.1, hydroxyapatite absorption chromatography, 35% saturated ammonium sulfate precipitation, Sephadex G-200 gel filtration, Pevikon block electrophoresis, lentil lectin affinity chromatography and immobilized rabbit anti-human whole serum IgG column chromatography. Utilizing these procedures, 0.1 mg of HA was purified from 3 1 of pooled human serum. The molecular mass of HA was determined as 650 kDa by Sepharose 4B gel filtration. On SDS-PAGE analysis, HA showed a single band at 35 kDa under reduced conditions and numerous ladder bands between 35 kDa to more than 300 kDa under nonreduced conditions. On analytical ultracentrifugation, HA gave a molecular mass of 520 kDa with a single meniscus and a sedimentation constant of 12.0. The amino acid and carbohydrate analysis of reduced and S-pyridylethylated HA revealed that it contained five residues of hydroxyproline and an N-linked type sugar chain.     

Genetic and functional characterization of <Emphasis Type="Italic">Lactobacillus panis</Emphasis> levansucrase

Exopolysaccharides (EPS) can affect the rheological properties of foods, act as stabilizers or stimulate preferential growth of bifidobacteria in the gut and therefore function as prebiotics. The latter is referred to fructans, which are synthesized from sucrose by fructosyl transferases (FTFs). In this work, the FTF enzyme of Lactobacillus panis TMW1.648 isolated from sourdough was characterized. The coding gene was identified, sequenced and expressed heterologously in E. coli. Enzyme activity was maximal at pH 4.0–4.6, 45°C and a substrate concentration of 300 mmol l⁻¹. It produced free fructose, a high molecular fructan and the oligosaccharide kestose from sucrose. Calcium ions proved to be essential for the enzymatic activity. In comparison to published data of other FTF enzymes of lactobacilli the described enzyme showed significant differences.     

Intracervical application of hyaluronan improves cervical relaxation in the ewe

Artificial insemination (AI) using frozen semen is a key method to enable rapid genetic improvement but its use in the sheep industry is currently limited by poor fertility. Laparoscropic AI is most effective but has not gained popularity due to cost and welfare considerations. Transcervical intrauterine AI (TCAI) may offer a practical alternative but the complex anatomy of the ovine cervix limits adequate penetration of the inseminating pipette. Hyaluronan (HA) is a glycoaminoglycan whose content in the cervix increases at oestrus and which may contribute to the degree of natural relaxation observed at this time. This study investigated the effect of intracervical application of HA on the depth of cervical penetration in sheep. Oestrus was synchronised on three occasions in 48 Welsh mountain ewes with progesterone sponges and PMSG. Each animal initially served as its own untreated control. Ewes were subsequently treated intracervically with 25 mg of: (2i) low molecular weight (MW) HA; (2ii) high MW HA or (2iii) both low and high MW HA (n = 16/group) at 52 h after sponge removal or with low MW HA at: (3i) 50 h; (3ii) 52 h or (3iii) both 50 h and 52 h. Depth of cervical penetration measured at 54 h was increased from 1.22 cm to 3.66 cm by treatment with low MW HA (P ≤ 0.001), with no differences between the number of treatments (1 or 2) or the time at which the HA was administered (50 or 52 h). High MW HA alone or together with low MW HA had no effect. In conclusion, intracervical application of low MW HA 52 h after sponge removal increases cervical penetration up to 3.4cm to allow TCAI in sheep.     

Characterization of soluble microbial products (SMP) derived from glucose and phenol in dual substrate activated sludge bioreactors

Soluble microbial products (SMP) generated by activated sludge cultures receiving a mixed feed of phenol and glucose were characterized with respect to molecular weight (MW) distribution, octanol-water partition coefficient (K(ow)), and Microtox toxicity. Short-term batch reactor tests using 14C-labeled substrates were performed to collect SMP derived from each substrate, while long-term tests were performed with SMP accumulated over multiple feed cycles using fed-batch reactors receiving non-labeled substrates. Yield of SMP in the batch tests, 10%-20% for phenol and 2%-5% for glucose, differed for each substrate and was independent of initial concentration. The MW distribution (MWD) of SMP was independent of feed composition, and was bimodal in the < 1 kDa and 10-100 kDa MW ranges for phenol-derived SMP and predominantly < 1 kDa for glucose-derived SMP. In the non-labeled tests, the fraction of SMP of MW > 100 kDa increased with the proportion of glucose in the feed. The K(ow) of phenol-derived SMP was higher compared to glucose-derived SMP, indicating that the phenol-derived SMP were more hydrophobic. This was particularly true at an acidic pH, where the K(ow) was 4.2 +/- 1.0 for phenol-derived SMP versus 0.13 +/- 0.13 for glucose-derived SMP. Toxicity testing indicated that phenol-derived SMP, exerting a mean Microtox inhibition of 1%, were less toxic than phenol itself, and showed little correlation between toxicity and concentration. However, glucose-derived SMP were generally more toxic than glucose itself (a non-toxic substrate), and the toxicity increased linearly with the concentration of SMP.     

Degradation of hyaluronan by ultrasonication in comparison to microwave and conventional heating

Hyaluronan (hyaluronic acid, HA) was depolymerised by ultrasonication (US), microwave irradiation (MW) and conventional heating (CH), and the effect of pH and oxidants was investigated. The degradation was followed by viscometry and size exclusion chromatography coupled with low-angle light scattering. The results demonstrated that depolymerisation of HA by US leveled off to a limiting molecular mass, and the degradation was significantly enhanced by acidic and alkaline pH only in the presence of oxidants. In contrast to US, the course of depolymerisation by MW was strongly pH-dependent, and the degradation rate increased with decreasing pH. The expected enhancement of depolymerisation by MW in comparison to CH was marked only at very short heating time at pH <4. The NMR and FTIR spectral analyses indicated that HA in the whole M_w-range studied retained almost the backbone of the parent polysaccharide independently on the degradation method used. At harsh degradation conditions (long-term treatments, particularly at acidic pH or alkaline pH and in presence of oxidants) the depolymerisation was accompanied by destruction of both constituent sugar residues and formation of unsaturated structures detectable by UV-absorption at 230–240 and 260–270 nm. US-assisted oxidative degradation under mild reaction conditions was shown to be the most appropriate procedure to reduce the molecular mass of HA to 100 kDa without significant chemical modification of the polysaccharide.     

Intracervical application of hyaluronan improves cervical relaxation in the ewe

Artificial insemination (AI) using frozen semen is a key method to enable rapid genetic improvement but its use in the sheep industry is currently limited by poor fertility. Laparoscropic AI is most effective but has not gained popularity due to cost and welfare considerations. Transcervical intrauterine AI (TCAI) may offer a practical alternative but the complex anatomy of the ovine cervix limits adequate penetration of the inseminating pipette. Hyaluronan (HA) is a glycoaminoglycan whose content in the cervix increases at oestrus and which may contribute to the degree of natural relaxation observed at this time. This study investigated the effect of intracervical application of HA on the depth of cervical penetration in sheep. Oestrus was synchronised on three occasions in 48 Welsh mountain ewes with progesterone sponges and PMSG. Each animal initially served as its own untreated control. Ewes were subsequently treated intracervically with 25 mg of: (2i) low molecular weight (MW) HA; (2ii) high MW HA or (2iii) both low and high MW HA (n = 16/group) at 52 h after sponge removal or with low MW HA at: (3i) 50 h; (3ii) 52 h or (3iii) both 50 h and 52 h. Depth of cervical penetration measured at 54 h was increased from 1.22 cm to 3.66 cm by treatment with low MW HA (P ≤ 0.001), with no differences between the number of treatments (1 or 2) or the time at which the HA was administered (50 or 52 h). High MW HA alone or together with low MW HA had no effect. In conclusion, intracervical application of low MW HA 52 h after sponge removal increases cervical penetration up to 3.4cm to allow TCAI in sheep.