Electrostatic interaction and complex formation between gum arabic and bovine serum albumin.

The interaction of gum arabic (GA) and bovine serum albumin (BSA) has been investigated through turbidity and light scattering intensity measurements and by the use of dynamic light scattering, laser Doppler velocimetry, and isothermal titration calorimetry. It has been shown that GA and BSA can form soluble and insoluble complexes depending on the solution pH and the mixing ratio and is a function of the net charge on the complex. Soluble complexes were obtained when the electrophoretic mobility was greater than ±1. 5 µm s(-1) V(-1) cm(-1). Changes in the value of the isoelectric point of the complexes with mixing ratio and isothermal titration calorimetric data indicated that complexes formed at pHs 3 and 4 consisted of ∼60 BSA molecules for every GA molecule, while at pH 5 there were ∼10 BSA molecules per GA molecule. Calorimetric studies also indicated that the interaction occurred in two stages at both pH 3 and pH 4, but that the nature of the interaction at these two pH values was significantly different. This was attributed to differences in the relative magnitude of the positive and negative charges on the BSA and GA, respectively, and possibly due to changes in the BSA conformation. The fact that there is an interaction at pH 5, which is above the isoelectric point of the BSA, is due to the interaction of the carboxylate groups on the GA with positive patches on the BSA or to the charge regulation of the protein-polysaccharide system brought about by changes in dissociation equilibria. Complexation is reduced as the ionic strength of the solvent increases and is prevented at a NaCl concentration of 120 mM.

Training tissue bank operators: the International Atomic Energy Agency (IAEA)/National University of Singapore (NUS) 10 years of experience.

National University of Singapore (NUS) was appointed by IAEA to become IAEA/NUS Regional Training Centre (RTC) for Asia and the Pacific region in September 1996. The Government of Singapore (represented by the Ministry of Environment) with the National Science and Technology Board as the funding agency awarded a grant of S$225,500 to build a new purpose-built tissue bank to be the Regional Training Centre. National University Hospital provided a space of 2,000 square feet for this purpose. The first Diploma Course was launched on 3 November 1997 with 17 candidates with the first NUS Diploma Examination being held in October 1998. Between November 1997 and April 2007, a total of nine courses were conducted by RTC with a total of 180 tissue bank operators, 133 from Asia and the Pacific region (13 countries including 2 from Iran), 14 from Africa (Algeria, Egypt, Libya, Egypt, South Africa and Zambia), 6 from Latin America (Brazil, Chile, Cuba, Peru and Uruguay), 9 from Europe (Greece, Slovakia, Poland, Ukraine) and 2 from Australia. The last batch (ninth batch) involved twenty students registered in April 2007 and will be due to sit for the terminal examination only in April 2008.

New insights into the structural characteristics of the arabinogalactan-protein (AGP) fraction of gum arabic.

The structural characteristics of the gum exudate of Acacia senegal (gum arabic) have been investigated by monitoring the composition and physicochemical properties before and after treatment with proteolytic enzyme and various alkaline systems. Molecular mass ( M w) and radius of gyration ( R g) measurements were performed using gel permeation chromatography (GPC) coupled to refractive index, UV absorbance, and multiangle light scattering detectors and indicated that the macromolecules present have a compact structure. It was found that treatment with proteolytic enzyme caused the arabinogalactan-protein component (AGP) with average molecular mass approximately 2 x 10 (6) Da to degrade, yielding material of molecular mass approximately 4 x 10 (5) Da, whereas the bulk of the material corresponding to the protein-deficient arabinogalactan component (AG) with molecular mass 4 x 10 (5) remained unaffected. Barium hydroxide was found to hydrolyze the polysaccharide component (AG) itself in addition to the proteinaceous component as demonstrated in control experiments using dextran. However, sodium borohydride/sodium hydroxide treatments were unable to hydrolyze dextran and were assumed to hydrolyze only the proteinaceous component of gum arabic. The AGP component was completely degraded, yielding material of molecular mass approximately 4.5 x 10 (4) Da. It has been concluded, therefore, that the enzyme did not fully hydrolyze all of the protein present and that the AGP component of gum arabic consists of carbohydrate blocks of approximately 4.5 x 10 (4) Da linked to a polypeptide chain consistent with the wattle blossom structure. Because the AGP was degraded to differing extents using a mild and more severe sodium borohydride/sodium hydroxide treatment, it was concluded that the polysaccharide moieties were linked through both O-serine and O-hydroxyproline residues. The gum arabic sample was deglycosylated by treatment with anhydrous hydrogen fluoride and revealed the presence of two putative core proteins of approximately 3 x 10 (4) and approximately 5 x 10 (3) Da, respectively, which correspond to proteins of approximately 250 and 45 amino acids in length. A new model for the structure of the AGP component has been proposed.

Chain scission of hyaluronan by carbonate and dichloride radical anions: potential reactive oxidative species in inflammation?

The reactions of the carbonate and dichloride radical anions, CO3- and Cl2-, with the extracellular matrix glycosaminoglycan hyaluronan (HA) have been studied using the kinetic technique of pulse radiolysis and also by steady-state irradiation combined with gel permeation chromatography/multiangle laser light scattering(gpc/MALLS) to measure the rates of reaction with HA and the yield of HA chain scission, respectively. For comparison, the same measurements were made for the reactions of the free radicals *OH, Br2*-, and N3*. The carbonate and dichloride radical anions were found to react relatively quickly with HA (7.0 x 10(5) and 6.9 x 10(6) dm3 mol(-1) s(-1), respectively) although they are much less reactive than the hydroxyl radical, *OH. Significant yields (20 and 38%, respectively) of chain scission of HA by these radical anions were also determined from the gpc/MALLS experiments, providing some support for their potential participation in the depolymerization of HA in vivo. These results are compared with data obtained for the other free radicals (hydroxyl, azide radicals, and dibromide radical anions) investigated in this study in order to gain an insight into their mechanism of reaction with HA. Earlier chain scission yields of HA by hydroxyl radicals determined by the authors have also been revised using the gpc/MALLS technique employed in the current study. The yields of 52% (absence of air) and 44% (in air) are much lower than the previous values. In the current study, the effect of oxygen on the yields of HA chain breaks is discussed in terms of the reactivity of HA peroxyl radicals in the presence of superoxide radical anions. The relevance of the results of this study to mechanisms of inflammation is discussed.

Butyrate modulates TGF-beta1 generation and function: potential renal benefit for Acacia(sen) SUPERGUM (gum arabic)?

Anecdotal evidence suggests that high fibre supplementation of dietary intake may have health benefits in renal disease related to alterations in circulating levels of short-chain fatty acids. The aim of the study was to examine the hypothesis that dietary manipulation may increase serum butyrate and thus have potential beneficial effects in renal disease. We examined the effect of dietary supplementation with a gum arabic sample of standardized molecular characteristics, Acacia(sen) SUPERGUM EM2 (SUPERGUM), on systemic levels of butyrate in normal human subjects. In an in vitro study, we also examined the potential role of butyrate in modifying the generation of the profibrotic cytokine transforming growth factor-beta (TGF-beta1) by renal epithelial cells. Following 8 weeks of dietary supplementation with 25 g/day of SUPERGUM, there was a two-fold increase in serum butyrate (n=7, P=0.03). In vitro work demonstrated that exposure of renal epithelial cells to elevated concentrations of butyrate suppressed both basal and stimulated TGF-beta1 synthesis. The action of butyrate was mediated by suppression of the extracellular signal-regulated kinase/mitogen-activated protein kinase signalling pathway. In addition, butyrate exposures reduced the response of renal epithelial cells to TGF-beta1 as assessed by luciferase activity of a TGF-beta-responsive reporter construct. Attenuation of TGF-beta1 signalling was associated with reduced phosphorylation of Smad 3 and decreased trafficking of TGF-beta1 receptors into signalling, non-lipid raft-associated membrane fractions. In conclusion, the data demonstrate that dietary supplementation with SUPERGU increased serum butyrate, which at least in vitro has beneficial effects on renal pro-fibrotic cytokine generation.

Chain scission of hyaluronan by peroxynitrite.

The reaction of peroxynitrite with the biopolymer hyaluronan has been studied using stopped-flow techniques combined with detection of molecular weight changes using the combination of gel permeation chromatography and multiangle laser light scattering. From the effect of peroxynitrite on the yield of hyaluronan chain breaks, it was concluded that the chain breaks were caused by hydroxyl radicals which escape a cage containing the *OH NO*(2) radical pair. The yield of free hydroxyl radicals was determined as 5+/-1% (as a proportion of the total peroxynitrite concentration). At high peroxynitrite concentrations, it was observed that the yield of chain breaks leveled out, an effect largely attributable to the scavenging of hydroxyl radicals by nitrite ions present in the peroxynitrite preparation. These experiments also provided some support for a previous proposal that the adduct formed between ONOOH and ONOO(-) might itself produce hydroxyl radicals. The rate of this reaction would have to be of the order of 0.05 s(-1) to produce hydroxyl radical yields that would account quantitatively for chain break yields at high peroxynitrite concentrations. By carrying out experiments at higher hyaluronan concentrations, it was also concluded that an additional yield of chain breaks was produced by the bimolecular reaction of the polymer with ONOOH at a rate constant of about 10 dm(3)mol(-1)s(-1). At 5.3 x 10(-3)mol dm(-3) hyaluronan, this amounted to 3.5% chain breaks (per peroxynitrite concentration). These conclusions support the proposal that the yield of hydroxyl radicals arising from the isomerization of ONOOH to nitrate ions is relatively low.

Comparative rheological behavior of hyaluronan from bacterial and animal sources with cross-linked hyaluronan (hylan) in aqueous solution.

Using a variety of rheological techniques, the behavior of hyaluronan (M(w) 0.8-2.2 x 10(6)), cross-linked hyaluronan (hylan) (M(w) 1.8-12.5 x 10(6)), and Healon (M(w) approximately 5 x 10(6)) (a proprietary hyaluronan) was studied over a large range of molecular weights. The object was to study the effect of the cross-links in hylan on the various rheological parameters, in comparison with linear hyaluronan. There are significant differences. The Huggins constant and the critical overlap parameter C*[eta] are considerably lower for hylan and an increase in moduli at low frequencies was observed for hylan compared with the hyaluronan samples at all molecular weights studied. The results point to a difference in structure in dilute solution for hylan due to the ability to form networks, which can be removed by pressure filtration. In contrast, we do not find an increase of the steady shear viscosity and elastic modulus at higher concentrations when a homogeneous entangled network is reached. We attribute this behavior to the semirigid character of the hyaluronan chain and to the predominance of entanglements over the cross-link points present in hylan in the semidilute domain. Due to the higher apparent molecular weights that are possible with hylan structures but not with the hyaluronans currently available, a wider range of applications can be achieved with hylans when viscoelasticity is required, particularly for the viscosupplementation of synovial fluid damaged by osteoarthritis.

The effect of hydroxyl radicals on the rheological performance of hylan and hyaluronan.

Shear flow, dynamic oscillation and extensional viscosity measurements were used to compare the rheological performance of several hylan samples (M(v) 1.6, 3.2, 3.7, 4.7 and 5.6x10(6)) and hyaluronan (M(v) 1.4 and 1.8x10(6)) before and after hydroxyl radicals (*OH) induced degradation. It was found that the higher molecular weight cross-linked structure of hylan was more resistant to degradation than hyaluronan and that this superior stability was reflected in various rheological parameters. The *OH degradation of the initial hylan and hyaluronan samples produced a range of polysaccharides based on hylan and hyaluronan with molecular weight covering a range from 0.5-5.6x10(6). The rheological parameters associated with the polysaccharides could then also be studied. Zero shear values of the complex viscosity (eta*), dynamic viscosity (eta') and shear viscosity (eta) were calculated using the method of Morris(1) and shown to approach the same value at zero shear or frequency. An adaptation of the method of Gibbs et al. gave a 'master curve' for the storage and loss modulus of hyaluronan and hylan, which encompasses a 10-fold molecular weight and a 5-fold concentration variation. In all instances for hylan, the storage modulus predominates over the loss modulus, whereas for hyaluronan, the reverse is true, demonstrating the greater elasticity of hylan throughout the whole experimental range of molecular weights and concentrations.

Acacia gum (Gum Arabic): a nutritional fibre; metabolism and calorific value.

Gum Arabic (Acacia gum, INS 414: E414) is extensively used as a food additive, but there is no regulatory or scientific consensus about its calorific value. It is a complex polysaccharide, primarily indigestible to both humans and animals, not degraded in the intestine, but fermented in the colon under the influence of microorganisms. Despite a range of animal studies, there are no usable data for humans which can quantify the utilizable energy of Gum Arabic. Estimates in the literature from animal experiments vary from 0 to 4 kcal/g. After certain allowances are made for the energy losses from volatile and gaseous fermentation products, an upper level of 2 kcal/g for rats has been set. The situation in man is demonstrably different, with greatly reduced amounts of such products, and the need to adapt for varying periods before Gum Arabic is attacked by colonic bacteria. In the absence of an agreed scientific assignment, the FDA in the USA insist upon 4 kcal/g in nutritional labelling, whereas in Europe, no value has been assigned to soluble dietary fibre, such as Gum Arabic. This review argues that based on present scientific knowledge only an arbitrary value can be used for regulatory purposes.

The application of shear and extensional viscosity measurements to assess the potential of hylan in viscosupplementation.

The shear and extensional viscosity characteristics have been compared for hyaluronan and two samples of a cross-linked derivative, hylan, of different molecular weights. While shear thinning behavior was observed for all systems in shear flow, strain thickening was observed in extensional flow for the relatively dilute systems. However, there was a progressive transition to shear thinning behavior as the polymer concentration was increased. It is evident from the results that the shear flow techniques alone provide an incomplete picture of the rheological properties of these materials and that extensional flow characteristics are potentially dominant. For example, at relatively high deformation rates of 500 s-1 and above, our results show that the extensional viscosities of aqueous solutions of the various polymers are at least two orders of magnitude greater than their corresponding shear flow viscosities. The incremental differences in viscosity with concentration increased with increasing molecular mass of the polymers and were greater in extensional flow than shear flow. These results demonstrate that the dynamic network structure formed by the higher molecular mass hylans offer potentially better physical and mechanical properties for viscosupplementation of diseased osteoarthritis joints compared with the parent hyaluronan.

E.s.r. study of the conformational transition of spin-labelled xanthan gum in aqueous solution.

The order to disorder transition of xanthan molecules in aqueous solutions has been studied using e.s.r. spectroscopy. Nitroxide spin-label was covalently attached to carboxyl groups on the xanthan side chains. The e.s.r. spectra obtained for aqueous spin-labelled xanthan solutions at varying ionic strengths contained both isotropic and anisotropic components at room temperature. The anisotropic component was attributed to the association of the side chains with the xanthan cellulosic backbone and was found to be present in greater proportions at increasing ionic strength. The spectra gradually changed with rising temperature and the proportion of anisotropic component decreased. This spectral change reflected the disruption of the side chain association with the backbone during the conformational change. Hysteresis effects were observed following sequential heating and cooling cycles suggesting that chain aggregation occurred.

Effect of sugars and polyols on water in agarose gels.

Effects of ribose, glucose, sucrose, ethylene glycol, glycerin, propylene glycol, and sorbitol on water in concentrated agarose gels were studied by differential scanning calorimetry at low temperatures. Changes in the phase transition temperatures of 40% agarose gels, induced by the addition of these chemical reagents, are discussed, together with rheological and thermal data for the same systems at ambient and higher temperatures. Both sugars and polyols are believed to reduce the amount of freezable water and to promote plasticization and molecular mobility of agarose chains in gels, thus shifting the glass transition temperatures to lower temperatures. However, the effects of decreasing freezable water, and the direct effect on the junction zones, produced by sugars seem to be different from the effects produced by polyols.

The kinetics of hydroxyl-radical-induced strand breakage of hyaluronic acid. A pulse radiolysis study using conductometry and laser-light-scattering.

Hydroxyl radicals were generated radiolytically in N2O- and N2O/O2(4:1)-saturated aqueous solutions of hyaluronic acid. The hydroxyl radicals react rapidly with hyaluronic acid mainly by abstracting carbon-bound H atoms. As a consequence of subsequent free-radical reactions, chain breakage occurs the kinetics of which has been followed using the pulse radiolysis technique. In the absence of oxygen, strand breakage was followed by the change in conductivity induced by the release of cationic counterions condensed at the surface of hyaluronic acid which is a polyanion consisting of subunits of glucuronic acid alternating with N-acetyl-glucosamine. It appears that strand breakage is not due to one single first-order process, however, the contributions of the different components cannot be adequately resolved. At pH 7 the overall half-life is 1.4 ms, in both acid and basic solutions the rate of free-radical induced strand breakage is accelerated (at pH 4.8, t1/2 = 0.6 ms; at pH 10, t1/2 = 0.18 ms). In the absence of oxygen there is no effect of dose rate on the kinetics of strand breakage. In the presence of oxygen in addition to conductometric detection, strand breakage was also followed by changes in low-angle laser light-scattering. These two techniques are complementary in that in this system the conductometry requires high doses per pulse while the light-scattering technique is best operated in the low-dose range. In the presence of oxygen a pronounced dose-rate effect is observed, e.g. at pH 9.7 after a dose of 9.4 Gy the overall half-time is approx. 0.5 s, while after a dose of 6.6 Gy the half-time is approx. 0.23 s. Both the yield and the rate of strand breakage increase with increasing pH, e.g. at pH 7 G(strand breaks) = 0.7 x 10(-7) mol J-1 and at pH 10.4, 4.8 x 10(-7) mol J-7. The radiolytic yields of CO2, H2O2, organic hydroperoxides, O2.- and oxygen consumption have been determined in gamma-irradiated N2O/O2(4:1)-saturated solutions of both hyaluronic acid and beta-cyclodextrin.

One-electron reduction of 5-deazalumiflavin in aqueous solution: a pulse radiolysis study.

The one-electron reduction of 5-deazalumiflavin has been studied in aqueous solution in the acidity range H0 = -1 to pH 13 using the reducing species CO2-, e-aq and (CH3)2COH radicals. The spectral and other properties of the deazaflavin radicals formed were found to be independent of the reductant used. Four protolytic forms of the radical were distinguished with associated pKa values of 1.3 +/- 0.3, 6.0 +/- 0.3 and 10.7 +/- 0.3.

Superoxide radical reactions in aqueous solutions of pyrogallol and n-propyl gallate: the involvement of phenoxyl radicals. A pulse radiolysis study.

The reactions of O2-. in aqueous solutions of pyrogallol 1 and the antioxidant n-propyl gallate 2 have been studied. In both cases the initial reaction gives hydrogen peroxide and the corresponding phenoxyl radical (k(1 + O2-.) = 3.4 x 10(5), k(2 + O2-.) = 2.6 x 10(5) dm3 mol-1S-1). These phenoxyl radicals have been produced independently by reacting 1 and 2 with Br2-. and their spectra and first pKa values measured (pKa(phenoxyl radical from 1) = 5.1, pKa(phenoxyl radical from 2) = 4.1). It is necessary to correct the observed spectra for the contribution of the H-adducts, formed by the reaction of radiolytically produced H atoms with the substrates (k(1 + H) = 2.5 x 10(9), k(2 + H) = 3.8 x 10(9) dm3 mol-1 S-1). The H-adduct spectra are given. In the reactions of O2-. with the substrates the initial transient absorbances are characteristic of the phenoxyl radicals; however at longer times a new transient absorbing around 500 nm (epsilon congruent to 10(4) dm3 mol-1 cm-1) appears. This is believed to be the deprotonated hydroxy-orthoquinone, formed by the reaction of phenoxyl radicals with O2-. (k congruent to 1.5 x 10(8) dm3 mol-1 S-1, from kinetic curve-fitting). The absorbance due to the hydroxy-orthoquinones decays by first-order kinetics (1.6 x 10(2) in the case of 1 and 1.1 x 10(2) s-1 in the case of 2). This is thought to be mainly the result of the conversion of the hydroxy-orthoquinone into its hydrate. Similar experiments were carried out with catechol and ethyl protocatechuate. The chemistry appears to be similar to that of the pyrogallol derivatives. The rate constant for reaction of these compounds with O2-. is, however, only less than or equal to x 10(4) dm3 mol-1 s-1.

The reactivity of various free radicals with hyaluronic acid: steady-state and pulse radiolysis studies.

The reactions of the primary water radicals with the biopolymer hyaluronic acid have been studied by pulse radiolysis. Bimolecular rate constants, expressed in terms of the disaccharide repeating sub-unit of hyaluronic acid, for OH., H. and eaq- were found to be 7 X 10(8) M-1 X s-1, 5 X 10(7) M-1 X s-1 and less than 5 X 10(6) M-1 X s-1, respectively. By comparing the viscosities of samples, gamma-irradiated in the steady state under a variety of conditions, with unirradiated controls, the efficiencies with which selected radicals cause chain breakage have been determined. Efficiencies of 30%, 15%, 0%, 0.2% and 5% were estimated for OH., H., eaq-, methanol radicals and tert-butanol radicals, respectively. The presence of oxygen during irradiation increased the extent of chain breakage by a factor of 1.75.

Evidence for the addition of the superoxide anion to the anti-oxidant n-propyl gallate in aqueous solution.

n-Propyl gallate reacts with the superoxide radical anion in aqueous solution (k = 5.1 x 10(5) mol-1 dm3 s-1). The spectrum of the transient species so formed has been measured (absorbance maximum at 550 nm, epsilon = 1360 mol-1 dm3 cm-1). Electron or H atom transfer processes as well as proton abstraction have been excluded as possible mechanisms, and it is proposed that an addition reaction takes place.