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 5.6×10⁶) and hyaluronan (M_v 1.4 and 1.8×10⁶) 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 0.5–5.6×10⁶. The rheological parameters associated with the polysaccharides could then also be studied. Zero shear values of the complex viscosity (η*), dynamic viscosity (η′) and shear viscosity (η) were calculated using the method of Morris¹ and shown to approach the same value at zero shear or frequency. An adaptation of the method of Gibbs et al.² 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.     

Hydrodynamic properties of the fractions of mannan formed by Rhodotorula rubra yeast

Aqueous solutions of fractions of an extracellular linear mannan formed by Rhodotorula rubra yeast have been investigated by hydrodynamic methods (high-speed sedimentation, translation isothermic diffusion and viscometry). The molecular weight was determined according to Svedberg ( ) and the polydispersity parameters of the initial sample were also determined (M_w/M_n = 1·20 and M_z/M_w = 1·21). Relationships between the molecular weight (M) and s_o, D_o and [η] in the range were: [η] = 2·33 × 10⁻² M^0.75, D_o = 1·65 × 10⁻⁴ M^0·58, s_o = 2·24 × 10⁻¹⁵ M^0·43. The equilibrium rigidity and hydrodynamic diameter of chains representing mannan molecules were evaluated.     

Studies on the molecular chain morphology of konjac glucomannan

The chain geometry and parameters of konjac glucomannan were studied by using laser light scatter (LLS), gel permeation chromatography (GPC) and viscosimetry. The weight-average molecular weight (M_w), root-mean-square ratio of gyration (S^21/2), second viral coefficient (A₂) and polydispersity index (M_w/M_n) were 1.036×10⁶, 105±0.9 nm, (−1.587±0.283)×10⁻³  mol ml g⁻² and 1.015±0.003 respectively. Mark-Houwink equation was established as , and the molecular chain parameters were as follows: M_L=982.82 nm⁻¹, q=27.93 nm, d=0.74 nm, h=0.26 nm, L=1054.11 nm. To confirm the above results, konjac glucomannan was observed by using atomic force microscopy (AFM) and transmission electron microscope (TEM). The physical image showed directly that the konjac glucomannan molecule was an extending semi-flexible linear chain without branches, and than the molecular dimension also conformed to the parameters above. Therefore the image of molecular chain geometry confirmed the deduction drawn by Mark-Houwink equation and molecular chain parameters magnificently.     

Hydrodynamic characteristics and equilibrium rigidity of pullulan molecules

The hydrodynamic characteristics of the polysaccharide pullulan (polymaltotriose) in water have been investigated and its molecular characteristics have been determined. Experimental values varied over the following ranges: velocity sedimentation coefficient (S): 0.9 < S < 11.2, translational diffusion coefficient (10⁷ cm² s⁻¹): 1.1 < D < 14.7 and intrinsic viscosity (cm³ g⁻¹): 6.7 < [η] < 164, which corresponds to a change in molecular weight (× 10³) in the range 3.9 < M_SD < 644. On the basis of analysis of the literature and our experimental data, excluded volume effects have been shown to have a prevailing influence on the chain length of these polysaccharides. The equilibrium rigidity and hydrodynamic chain diameter of pullulan were evaluated on the basis of the theory of hydrodynamic properties of a wormlike necklace, taking into account excluded volume effects. At low M (< 30 × 10³) the translation friction data (in contrast to viscometric data) cannot be described in the framework of the theory of linear molecules.     

Isolation and characterization of soluble sulfated polysaccharide from the red seaweed Gracilaria cornea

The composition, structure and rheological properties of a soluble sulfated polysaccharide from Gracilaria cornea (Brazilian red marine alga) were investigated. Agarocolloid yield, intrinsic viscosity, monosaccharide composition, sulfate and cation content as well as molecular weight were determined. The main polysaccharide components were 3,6-anhydrogalactose (24.7%) and galactose (64.6%). In addition, minor components such as 6-O-methyl-galactose (8.5%), glucose (1.5%), xylose (0.7%) and sulfated groups (4.8%) were detected. Comparison between sulfate contents determined by Fourier transform IR (FT-IR) spectroscopy and microelemental analysis was made. Data from ¹³C NMR and FT-IR provided evidence of sulfation in C-4 and C-6 of galactose. Sodium, calcium, magnesium and potassium cations were detected in the agarocolloid. The intrinsic viscosities were lower than typical values for agar in the same experimental conditions. No gelation in 1.5, 2.0 and 3.0% (w/v) aqueous solution was observed, even by cooling up to 4 °C. Gel permeation chromatography indicated two major polysaccharide fractions of M_pk 7.4×10⁴ and 1.8×10⁴ g/mol and a minor fraction of M_pk 2.1×10⁶ g/mol, probably a protein–polysaccharide complex.     

Effect of reaction temperature and reaction time on the preparation of low-molecular-weight chitosan using phosphoric acid

Low-molecular-weight chitosan were prepared using 85% phosphoric acid at different reaction temperatures and reaction time. At room temperature, the viscosity average-molecular weights (M_v) of chitosan decreased to 7.1×10⁴ from 21.4×10⁴ after 35 days treatment. The degradation rate decreased with increasing hydrolysis time. The yields of chitosan also continuously decreased from 68.4 to 40.2% after 35 days. At 40, 60 and 80 °C, the molecular weight decreased to 3.70×10⁴, 3.50×10⁴ and 2.00×10⁴ on 8 h hydrolysis, respectively. The yields of chitosan remain at a high level compared with that at room temperature and were 86.5, 71.4 and 61.3% at 40, 60 and 80 °C treatment, respectively. The different reaction time gave chitosan with different molecular weights. At 60 °C, the molecular weight of products decreased to 7.40×10⁴ from 21.4×10⁴ within 4 h, then decreased slowly to 1.90×10⁴ in 15 h. It was also found that the water-solubility of chitosan increased as the molecular weight decreased. Results show the changes in yields and molecular weight of chitooligomers were strongly dependent on the reaction temperature and reaction time.     

Interaction of sialosyl cholesterol with the cell surface of rat astrocytes and its biological activities

The interaction between sialosyl cholesterol (- or neuraminyl cholesterol, - or β-SC) and the plasma membrane of astrocytes was investigated by the use of ¹⁴C-labeled - or β-SC. Both - and β-SC were dose-dependently and time-dependently bound to rat astrocytes. The Scatchard plot analyses showed that rat astrocytes bound apparently 9.69 × 10⁹ molecules of both -SC/cell (apparent K_d = 2.29 × 10⁻⁵ M) and β-SC/cell (apparent K_d = 5.39 × 10⁻⁵ M) at 37°C. Both the binding of -SC to astrocytes and the subsequent inhibition of DNA synthesis were decreased at the low temperature (4°C), and also suppressed by serum proteins including albumin. One molecule of bovine serum albumin (BSA) bound 2.3 molecules of -SC with the slightly lower K_d-value (8.03 × 10⁻⁶ M) than that for the binding site on astrocytes. BSA not only suppressed the -SC-binding to astrocytes but also increased its release from the cells to the culture media. Gangliosides such as GM1 and GM3 unaffected the -SC-binding, promoted the small release of -SC from the cell surface, and inhibited the morphological changes of astrocytes induced by -SC. The mechanism of -SC-binding to cultured astrocytes with reference to the effects of serum or gangliosides is discussed.     

Interaction of anthracycline antibiotics with biopolymers: 7. Equilibrium binding studies on the interaction of iremycin and DNA

We report spectrophotometric equilibrium studies of both the self-association of the new antibiotic iremycin and of its binding to calf thymus DNA in solution (ionic strength 0.2 M; pH 6.0). Iremycin forms dimers in this solution with a dimerization constant K₄=(1.19 ± 0.10) × 10³ M⁻¹. This equilibrium is taken into account in the evaluation of the interaction of iremycin with DNA. The binding behaviour can be completely described by a single binding mechanism of monomeric iremycin to DNA with allowance both for neighbour exclusion and for cooperativity of interaction. The three intrinsic binding parameters for the homogeneous model were determined simultaneously by a least squares fit of the original titration data: equilibrium constant of cooperative binding K = (2.72 ± 0.66) × 10⁵ M⁻¹ cooperativity parameter σ=0.38±3.27 ± 0.32. The binding parameters of iremycin and adriamycin and their microbial activities are compared.     

Bradykinin dilates rat middle cerebral artery and its large branches via endothelial B2 receptors and release of nitric oxide

Ring segments of rat middle cerebral artery (MCA) were prepared for measurement of isometric force and precontracted with 10⁻⁴ M uridine triphosphate (UTP). Concentration-effect curves (CEC) were constructed for bradykinin (BK, 10⁻⁸–10⁻⁵ M) in segments with functionally intect (E+) or denuded (E−) endothelium. E− segments did not dilate to BK. The BK receptor was characterized by application of specific B₁ or B₂ antagonists [des-Arg⁹-Leu⁸] BK (10⁻⁵ M) and [ -Arg⁰-Hyp³-Thi⁵- -Tic⁷-Oic⁸] BK (HOE140,3 × 10⁻⁷ M), respectively, or B₁ agonist [des-Arg⁹] BK (10⁻⁸–10⁻⁴ M). Involvement of nitric oxide (NO) was tested with N^G-nitro- -arginine (LNNA, 10⁻⁴ M). BK induced concentration-dependent relaxation with a maximal effect (E_max) of 40.86 ± 1.50% at 10⁻⁶ M and a pD₂ (−log₁₀ EC₅₀) of 6.818 ± 0.044. This relaxation could be prevented with HOE140 or LNNA, but was not influenced by [des-Arg⁹-Leu⁸] BK. [des-Arg⁹] BK did not induce any effect. These results demonstrate that BK induced relaxation via endothelial B₂ receptors and release of NO in isolated rat MCA.     

Structure of 7S seed globulin from Phaseolus vulgaris L. in solution

The structure of the 7S globulin from Phaseoulus vulgaris L in dilatue solutions has been studied by small angle X-ray scattering (SAXS), by quasi-elastic light scattering (Q ELS), by circular dichroism spectroscopy (c.d.), and by precise density measurements. The molar mass, the radius of gyration, the volume, the maximum dimension and the diffusion coefficient were determined as M = 1.45 × 10⁵ g mol⁻¹, R_G = 4.05 nm, V = 300- nm³, L = 13.0 nm and D_20,w⁰ = 4.5 × 10⁻⁷ cm² s⁻¹, respectively. The molecule has an asymmetrical shape with the dimensions 12.5 × 12.5 × 3.75 nm. The secondary structure of the 7S globulin is characterized by a small portion of -helical structure (14%) and a marked content of β-structure (18%).     

Molecular interaction studies of the hyaluronan derivative, hylan A using atomic force microscopy

Intermolecular self-association of hylan chains can be observed in hylan of molecular weight ca. 1×10⁷, with an indication of specific cross-linking protein points and inter-chain cross-links of molecular weight of between 10,000 and 80,000. When this high molecular weight hylan is autoclaved to M_w 1.8×10⁶, to yield a molecular size of the same order as a conventional hyaluronan, the structural features of hylan are retained, with regions of network disintegration having single chains to which one or two chains are joined. After degradation by ^√OH radicals, extended linear chains are found with some of the straight chains having branch points. These can be attributed to the unwinding of the hylan coils by the movement of a droplet of water across the mica surface. The effect of filtration by 1 μm filter does not reduce the measured M_w (corresponding to an intrinsic viscosity of 8188 at low shear rate). However, when stressed through a 0.45 μm filter the M_w falls to a quarter of its previous value. The cross-linked structure of the original hylan is shown to be equivalent to a hyaluronan of ca. 10×10⁶, based on rheological measurements. The cross-linked structure confers stability to degradation by ^√OH radicals not observed for hyaluronan. This distinctive behaviour of hylan is maintained for the entire range of molecular weights studied. The results confirm the tendency of hylan chains to readily undergo chain–chain association.     

Screening the physical properties of novel Pseudomonas exopolysaccharides by HPSEC with multi-angle light scattering and viscosity detection

The physical properties of three novel acidic exopolysaccharides obtained from P. marginalis types A, B and C, one from P. ‘gingen’, one from P. andropogenis and one from P. fluorescens have been partially characterized. These EPSs were chromatographed on three serially placed SE Shodex OH pak columns covering a molar mass range for pullulans from about 4 × 10⁷ to 1 × 10³. The mobile phase was 0.05 M NaNO₃. Physical measurements were performed on about 30 mg of sample for each EPS. The weight average molar mass of these EPSs ranged from about 0.71 to 2.85 × 10⁶, the weight average intrinsic viscosity from 7.15 to 35.3 dl/ g and the radius of gyration from 62 to 123nm. The polydispersities of these EPSs ranged from 1.01 to 1.37. The large molar mass, size and viscosities of these EPSs may indicate that they have potential for use as thickeners, stabilizers, emulsifiers, and gelling agents in the food and non-food industries.     

Catalysis of plastocyanin electron self-exchange by redox-inert multivalent cations

Electron self-exchange in solutions of the ‘blue’ copper protein plastocyanin is catalysed by the redox-inert multivalent cations Mg²⁺ or Co(NH₃)³⁺₆. Measurements of specific ¹H-NMR line broadening with 50% reduced solutions in the presence of these cations show that electron exchange proceeds through encounters of cation-protein complexes which dissociate at high ionic strength. In the presence of 8mM (5 equivalents/total protein) Co(NH₃)³⁺₆, with 10 mM cacodylate (pH^*6.0) as background electrolyte, the bimolecular rate constant at 25°C is 7 × 10⁴ M⁻¹·s⁻¹. For comparison, the ‘electrostatically screened’ rate constant measured in 0.1 M KCl in the absence of added multivalent cations is ˜ 4 × 10³ M¹·s⁻¹.

Plastocyanin Electron self-exchange NMR Protein-protein interaction Multivalent cation Blue copper protein     

The hydrodynamic characterization of waxy maize amylopectin in 90% dimethyl sulfoxide–water by analytical ultracentrifugation, dynamic, and static light scattering

Static light scattering of high amylopectin waxy maize starch gently dispersed in 90% dimethyl sulfoxide–water yielded a weight average molecular weight M_w and radius of gyration R_g of 560×10⁶ g/mol and 342 nm, respectively. To obtain an independent hydrodynamic characterization of these solutions, we measured the sedimentation coefficient for the main component in an analytical ultracentrifuge. The value of s⁰, the infinite dilution sedimentation coefficient, was 199 S. The translational diffusion coefficient D⁰ in very dilute solutions was measured by dynamic light scattering at 90° and found to be 2.33×10⁻⁹ cm²/s. An effective hydrodynamic radius R_h was calculated from this diffusion constant using the Stokes–Einstein equation and found to be 348 nm. The structure-related parameter ρ=R_g/R_h was calculated to be 0.98. The weight average molecular weight calculated from the Svedberg equation using the values measured for s⁰ and D⁰ was 593×10⁶ g/mol. This result is in reasonable agreement with the light scattering results. As light scattering results are subject to experimental errors due to the possibility of dust contamination, the presence of microgel or aggregates, and the questionable applicability of light scattering theory to interpret results for macromolecular sizes approaching the wave length of light used as a source for scattering, it is advisable to have corroborating hydrodynamic data when possible to further validate light scattering results in this very high molecular weight range.     

Relationship between cyprid energy reserves and metamorphosis in the barnacle Balanus amphitrite Darwin (Cirripedia; Thoracica)

Nauplii batch cultures of Balanus amphitrite were reared with four different diatoms (Skeletonema costatum, Thalassiosira pseudonana, Chaetoceros gracilis, silicate-limited C. gracilis) at three different cells concentrations: 1×10⁵, 5×10⁵, and 1×10⁶ cells ml⁻¹. The cyprid energy reserves were quantified as the ratio of triacylglycerols (TAG) to DNA. Energy reserves of larvae fed on different diatoms at a concentration of 1×10⁶ cells ml⁻¹ were ranked in the order: silicate-limited C. gracilis>C. gracilis>T. pseudonana>S. costatum. There was a significant linear relationship between the TAG content of the diet and cyprid energy reserves. The effect of cyprid energy reserves on metamorphosis to polystyrene surface in the presence and the absence of conspecific settlement factor (SF) was studied after 12, 24, and 48 h of incubation. A strong positive correlation between energy reserves and percent metamorphosis was observed in the absence of SF (r_{12 h}=0.88, r_{24 h}=0.82, r_{48 h}=0.68, P<0.05). A weak positive correlation was observed in the presence of SF (r_{12 h}=0.43, r_{24 h}=0.48, r_{48 h}=0.50, P<0.05). In both treatments, more than 80% of the cyprids with high energy reserves metamorphosed within 24 h. In contrast, a high proportion of cyprids with low energy reserves metamorphosed in response to SF in 24 h. Our results indicate that discriminatory metamorphic behavior of cyprids is closely linked to their TAG/DNA ratio, a proxy for energy reserve.     

One-electron reduction of selenomethionine oxide

Experimental evidence is provided that selenomethionine oxide (MetSeO) is more readily reducible than its sulfur analogue, methionine sulfoxide (MetSO). Pulse radiolysis experiments reveal an efficient reaction of MetSeO with one-electron reductants, such as e^-_aq (k = 1.2 × 10¹⁰M^-1s^-1), CO^·-₂ (k = 5.9 × 10⁸ M^-1s^-1) and (CH₃)₂) C^·OH (k = 3.5 × 10⁷M^-1s^-1), forming an intermediate selenium-nitrogen coupled zwitterionic radical with the positive charge at an intramolecularly formed Se_∴ N 2σ/1σ* three-electron bond, which is characterized by an optical absorption with λ_max at 375 nm, and a half-life of about 70 μs. The same transient is generated upon HO^· radical-induced one-electron oxidation of selenomethionine (MetSe). This radical thus constitutes the redox intermediate between the two oxidation states, MetSeO and MetSe. Time-resolved optical data further indicate sulfur-selenium interactions between the Se_∴ N transient and GSH. The Se_∴ N transient appears to play a key role in the reduction of selenomethionine oxide by glutathione.     

Effects of environmental factors, age and genotype on sperm production and semen quality in Bos indicus and Bos taurus AI bulls in Brazil

The effects of ambient temperature and humidity, month, age and genotype on sperm production and semen quality in AI bulls in Brazil were evaluated. Data from two consecutive years were analyzed separately. Seven Bos indicus and 11 Bos taurus bulls from one artificial insemination (AI) center were evaluated in Year 1 and 24 B. indicus and 16 B. taurus bulls from three AI centers were evaluated in Year 2. Ambient temperature and humidity did not significantly affect sperm production and semen quality, probably because there was little variation in these variables. Month accounted for less than 2% of the variation in sperm production and semen quality. Increased bull age was associated with decreased sperm motility (P<0.10) and increased minor sperm defects (P<0.001) in Year 1. B. indicus bulls had greater (P<0.005) sperm concentration than B. taurus bulls in both years (1.7×10⁹/ml versus 1.2×10⁹/ml in Year 1 and 1.6×10⁹/ml versus 1.2×10⁹/ml in Year 2, respectively). Ejaculate volume was not significantly affected by genotype in Year 1 (6.6 ml versus 6.9 ml in B. indicus and B. taurus bulls, respectively), but B. indicus bulls had greater (P<0.05) total (11.4×10⁹ versus 8.2×10⁹) and viable (6.7×10⁹ versus 4.9×10⁹) numbers of spermatozoa in the ejaculate than B. taurus bulls. In Year 2, B. taurus bulls had greater (P<0.05) ejaculate volume than B. indicus bulls (8.2 ml versus 6.7 ml, respectively) and total and viable number of spermatozoa in the ejaculate were not significantly different between genotypes (10.3×10⁹ versus 9.1×10⁹ and 6.1×10⁹ versus 5.4×10⁹ in B. indicus and B. taurus bulls, respectively). Sperm motility was not significantly affected by genotype (mean, 59%). In Year 1, B. indicus bulls tended (P<0.10) to have more major sperm defects and had more (P<0.05) total sperm defects than B. taurus bulls (11.8% versus 8.7% and 13.6% versus 10.0%, respectively). In Year 2, B. indicus bulls tended (P<0.10) to have more total sperm defects than B. taurus bulls (16.2% versus 13.3%, respectively). In conclusion, neither ambient temperature and humidity nor month (season) significantly affected sperm production and semen quality. B. indicus bulls had significantly greater sperm concentration and B. taurus bulls had significantly fewer morphologically defective spermatozoa.     

SEC of mono-carboxymethyl cellulose (CMC) in a wide range of pH; Mark–Houwink constants

A method for determination of carboxymethyl cellulose (CMC) molecular weight (M_W) and chemical heterogeneity (degree of oxidation (DO)) using a bi-detector HPSEC (UV-detector online with refractometer) has been developed. It has been found that the use of 0.5 N NaOH or 0.4 M acetate buffer as the eluent ensures CMC separation according to M_W. It has been revealed that the universal calibration for the polyelectrolyte CMC and the neutral polymer dextran is valid under the conditions applied. The Mark–Houwink equations for CMC in 0.5 N NaOH and 0.4 M acetate buffer have been estimated to be [η]=5.37×10⁻⁴ M_W^0.73 and [η] =2.24×10⁻⁴ M_W^0.83 dl g⁻¹, respectively. The equation log K=1.64−4.00 ml g⁻¹ for CMC has been estimated. An approach for determining DO from adsorption at 290 or 313 nm has been developed.     

Structure, physical, chemical and photophysical properties of Pt(bph) (CO)2, where bph is the biphenyl dianion

The complex Pt(bph) (CO)₂ crystallizes in the space group Cmcm with a = 18.647(6), B = 9.566(2) and C = 6.4060(5) Å. The geometry of the molecule is slightly distorted from square planar with a Pt---C(CO) bond distance of 1.98(2) Å and a Pt---C(bph) bond distance of 2.04(2) Å. The Pt(bph)(CO)₂ complex serves as a precursor for the preparation of a wide variety of Pt(bph)X₂ complexes, where X = monodentate ligands such as acetonitrile, pyridine, etc., and X₂ = bidentate ligands such as bypyridine, 1,10-phenanthroline, etc. In the solid state, the complex exhibits a green color, but when ground with an alkali metal salt turns deep blue to purple. In CH₂Cl₂, the color disappears but optical transitions are observed at 271 nm (2.7 × 10⁴ M⁻¹ cm⁻¹), 303 nm (1.1 × 10⁴ M⁻¹ cm⁻¹) and 330 nm (5.5 × 10³ M⁻¹ cm⁻¹). The complex is a weak emitter exhibiting a structured spectrum in CH₂Cl₂ at r.t. with maxima located at 562 and 594 nm and an emission lifetime of 3.1 μs when excited at 337 nm.     

DNA-biding of IQ, Me-IQ and Me-IQx, strong mutagens found in broiled foods

Non-covalent DNA-binding has been studied of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (Me-IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (Me-IQx), strong mutagens found in broiled foods. These mutagens are intercalated into DNA, as found by ultraviolet absorption gel electrophoresis. The binding of IQ is stronger with GC pairs than AT pairs in DNA. The binding constants with calf thymus DNA are 1.6 × 10⁶ (Me-IQ), 0.9 × 10⁶ (IQ) and 0.7 × 10⁶ M⁻¹ (Me-IQx) at pH 6.0. This order of DNA affinity agrees with the order of mutagenicity towards Salmonella typhimurium TA98.