Bimodal solubilization of phospholipid-cholesterol vesicles in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) solutions. Formation of filamentous and helical microstructures depends on negatively charged lipids

Phospholipid/cholesterol vesicles were solu-bilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Above 30 mol% cholesterol (Ch) in the lipid vesicles several remarkable changes of the solubilization process were observed. (i) Two modes of solubilization: The effective detergent to lipid ratio R^c(M) for the formation of mixed micelles decreased from R^c(M) = 43 ± 3 at low lipid concentrations, [L]≤ 0.15 mm, to R^c(M) = 2.4 ± 0.3 above [L] = 0.5 mm (40 mol% Ch, T = 20 °C). (ii) At subsolubilizing CHAPS concentrations, filamentous and helical microstructures were formed, similar to those which were observed in native and model bile. (iii) The number of observed fibers was about two orders of magnitude higher in the presence of the negatively charged lipids phosphatidylglycerol (PG) and phosphatidic acid (PA) compared to the zwitterionic phosphatidylcholine (PC). Fiber formation began after 16–18 h using PG and PA compared to 3–4 days in the presence of PC. Screening of the charged lipids by NaCl effectively reduced the formation of fibers. Assuming binding of Na⁺ to the charged lipid aggregates, an intrinsic binding constant K_int = 0.6 M^–1 was determined by applying the Gouy-Chapman theory. After the addition of CHAPS to PG/Ch vesicles, a fast initial solubilization of the vesicles (<1 min) to mixed micelles (r_h = 2.3 ± 0.2 nm) and small vesicles (r_h = 23 ± 1 nm) was observed, followed by an intermediate period of 2 h, after which the formation of fibers occurred (>15 h). The microstructures are visualized by darkfield and electron microscopy. The method of vesicle solubilization is compared to the dilution of concentrated micellar solutions, which is usually applied to model bile systems. Received: 28 May 1996 / Accepted: 26 July 1996     

Conformation of heparin studied with macromolecular hydrodynamic methods and X-ray scattering

The hydrodynamic characteristics of heparin fractions in a 0.2 M NaCl solution have been determined. Experimental values varied over the following ranges: the sedimentation coefficient (at 20.0 °C), 1.3<s₀×10¹³<3.2 s; the Gralen coefficient (sedimentation concentration-dependence parameter), 10<k_s<70 cm³ g^–1; the translational diffusion coefficient, 3.9<D₀×10⁷<15.4 cm² s^–1; the intrinsic viscosity, 7.9<[]<40 cm³ g^–1. Combination of s₀ with D₀ using the Svedberg equation yielded molecular weights in the range 3.9<M×10^–3<37 g mol^–1. The value of the mass per unit length of the heparin molecule, M_L, was determined using the theory of hydrodynamic properties of a weakly bending rod, giving M_L=570±50 g nm^–1 mol^–1. The equilibrium rigidity, Kuhn segment length (A=9±2 nm) and hydrodynamic diameter (d=0.9±0.1 nm) of heparin were evaluated on the basis of the worm-like coil theory without the excluded volume effect, using the combination of hydrodynamic data obtained from fractions of different sizes. Small-angle X-ray scattering for three heparin fractions allowed an estimate for the cross-sectional radius of gyration as 0.43 nm; from the evolution with the macromolecule contour length of the radius of gyration, a value for the Kuhn segment length of 9±1 nm was obtained. A good correlation is thus observed for the conformational parameters of heparin from hydrodynamic and X-ray scattering data. These values describe heparin as a semi-rigid polymer, with an equilibrium rigidity that is essentially determined by a structural component, the electrostatic contribution being negligible in 0.2 M NaCl.Presented at the conference for Advances in Analytical Ultracentrifugation and Hydrodynamics, 8–11 June 2002, Grenoble, France     

Allometric relationship between body mass and aerobic metabolism in zebrafish Danio rerio

The relationship between body mass (M) and metabolic rate was investigated through the assessment of active (R_A) and standard (R_S) metabolic rate at different life stages in zebrafish Danio rerio (5 day‐old larvae, 2 month‐old juveniles and 6 month‐old adults). Scaling exponents and constants were assessed for standard (R_S = 0·273M^0·965 in mgO₂ g^?1 h^?1) and active metabolic rate (R_A = 0·799M^0·926 in mgO₂ g^?1 h^?1). These data provide the basis for further experiments regarding the effects of environmental factors on aerobic metabolism throughout the life cycle of this species.     

Kinetics of Thermal Denaturation and Aggregation of Bovine Serum Albumin

Thermal aggregation of bovine serum albumin (BSA) has been studied using dynamic light scattering, asymmetric flow field-flow fractionation and analytical ultracentrifugation. The studies were carried out at fixed temperatures (60°C, 65°C, 70°C and 80°C) in 0.1 M phosphate buffer, pH 7.0, at BSA concentration of 1 mg/ml. Thermal denaturation of the protein was studied by differential scanning calorimetry. Analysis of the experimental data shows that at 65°C the stage of protein unfolding and individual stages of protein aggregation are markedly separated in time. This circumstance allowed us to propose the following mechanism of thermal aggregation of BSA. Protein unfolding results in the formation of two forms of the non-native protein with different propensity to aggregation. One of the forms (highly reactive unfolded form, U_hr) is characterized by a high rate of aggregation. Aggregation of U_hr leads to the formation of primary aggregates with the hydrodynamic radius (R_h,1) of 10.3 nm. The second form (low reactive unfolded form, U_lr) participates in the aggregation process by its attachment to the primary aggregates produced by the U_hr form and possesses ability for self-aggregation with formation of stable small-sized aggregates (A_st). At complete exhaustion of U_lr, secondary aggregates with the hydrodynamic radius (R_h,2) of 12.8 nm are formed. At 60°C the rates of unfolding and aggregation are commensurate, at 70°C the rates of formation of the primary and secondary aggregates are commensurate, at 80°C the registration of the initial stages of aggregation is complicated by formation of large-sized aggregates.     

Renaturation of triple helical polysaccharide lentinan in water-diluted dimethylsulfoxide solution

Triple helical lentinan, a β-(1→3)-d-glucan from Lentinus edodes, was denatured in dimethylsulfoxide (DMSO) into single random coils. The randomly coiled lentinan/DMSO solutions were diluted with pure water to w_H (the weight fraction of water in the mixed solvent) of 95%, and their intrinsic viscosity [η], weight-average molecular weight M_w, radius of gyration R_g, and hydrodynamic radius R_h were investigated at 25 °C after over 5-day storage. The [η] and M_w values, especially the conformation parameter ρ (≡R_g/R_h), of the renatured lentinan were close to those of the originally extracted one, suggesting that random lentinan chains in DMSO were reassembled into triple helical structures. Moreover, the renatured lentinan in 95% water/5% DMSO solution exhibited a unique behavior of triple helical glucans that shear modulus G′ decreased sharply at temperature from 8.4 °C to 13.3 °C with increasing temperature, which was ascribed to the intramolecular conformation transition from ordered triple helical I to disordered triple helical II. The AFM images gave was suggested intuitively evidence that the renatured lentinan mainly existed as rod-like chains, supporting that formation of triple helical structure. The optimal lentinan concentration for triple helical configuration formation was estimated to be over 0.04%. The time dependence of R_h and UV absorption of the water-diluted lentinan/DMSO solution with an indicator of azo dye of Congo red suggested that renaturation of triple helix was a very rapid process. Moreover, the blue-shift of UV-vis absorption spectra suggested that the dye molecules of Congo red were assembled into supramolecular structure in the hydrophobic cavity of the renatured triple helical lentinan. All the results showed that the triple helical structure formed once the randomly coiled lentinan/DMSO was diluted to the final water content of 95%.     

Dependence of laser light scattering of DNA on NaCl concentration

We show that the persistence length a of DNA, derived from total intensity laser light scattering of linear Col E₁ DNA and corrected for excluded-volume effects, varies from about 68 nm in 0.005M NaCl to about 40 nm in 0.2M NaCl, leveling off to a constant value (about 27 nm) at high NaCl (1–4M) concentration. These observations do not agree with current views on the effect of electrostatic charge and ionic conditions on DNA dimensions. The apparent diffusion constant D_app, determined from laser light scattering autocorrelation as a function of scattering vector q, at NaCl concentrations 0.005–4M, correctly yields the translational diffusion coefficient D_t at low values of q and scales with molecular dimensions rather than segment length at high values of q; thus, D_app/D_t yields a universal curve when plotted against q²R, where R_g is the radius of the gyration. The sedimentation coefficients s at 0.1 and 0.2M NaCl concentration closely agree with the well-tested empirical relations, and a combination of s, D_t, and the appropriate density increments yield correct molar masses over the whole salt concentration range. Approximate constancy of D_tR_g indicates limited draining in translational flow. We present some observations and thoughts on the regimes in which a dependence of the correlation decay times on q³ rather than q² applies. We conclude that quasielastic laser light scattering discloses little information about dynamics of internal motion of DNA chains.     

Light scattering and viscosity study of heat aggregation of insulin

Aggregation behavior and hydrodynamic parameters of insulin have been determined from static and dynamic light scattering experiments and intrinsic viscosity measurements carried out at pH 4.0, 7.5, and 9.0 in the temperature range 20–40°C in aqueous solutions. The protein aggregated extensively at elevated temperatures in the acidic solutions. Intermolecular interactions were found to be attractive and to increase with temperature. The measured intrinsic viscosity [η], diffusion coefficient D₀, molecular weight M, and radius of gyration R_g exhibited the universal behavior: M[η] = (2.4 ± 02) × 10⁻²⁷ (R_e,η/R_e,D)³(D_0η/T)⁻³ and (D₀√n)₋₁ ≃ (√6 πη₀ζβ/k_BT) [1 + 0.201)(v/β³)√n], where n is the number of segments in the polypeptide. The effective hydrodynamic radii deduced from [η], (R_e, η) and the same deduced from D₀, (R_e,D) showed a constant ratio, (R_e,η/R_e,D = 1.1 ± 0.1). R_e,D/R_g = ξ was found to be (0.76 ± 0.07). From the known solvent viscosity η₀, the segment length β was deduced to be (10 ± 1) Å. The excluded volume was deduced to be (5 Å)³ regardless of pH. The Flory-Huggins interaction parameter was found to be χ = 0.45 ± 0.04, independent of pH and temperature. © 1998 John Wiley & Sons, Inc. Biopoly 45: 1–8, 1998     

Interaction of filipin with dimyristoylphosphatidylcholine membranes studied by 2H-NMR,circular dichroism,electronic absorption and fluorescence

Interaction of the pentene antibiotic filipin with dimyristoylphosphatidylcholine (DMPC) membranes has been monitored by ²H-NMR, circular dichroism (CD), electronic absorption and fluorescence in the temperature range 10° to 60°C. Interaction appears to depend on whether filipin is added before or after membrane formation and also upon the temperature of the system.When filipin is added to preformed DMPC large unilamellar vesicles (LUV), the association constants, as determined by electronic absorption are 39×10³ M ^-1, 15×10³ M ^-1 and 0.6×10³ M ^-1 at 15°, 30° and 50°C, respectively. Under identical conditions, CD spectra of bound filipin exhibit features characteristic of an aggregation over the whole temperature range.When filipin is incorporated in membranes during their preparation, the ²H-NMR spectra of deuterated DMPC indicate that the drug has a slight disordering effect on the lipid matrix below the temperature, T _c ,of the gel-to-fluid phase transition and above T _c +11°C. Between these two temperature boundaries the system consists of two lipid regions of very different dynamic properties. One of the regions, which is attributed to a filipin-lipid complex, has the properties of gel-like lipids whereas the other has those of fluid-like lipids. The latter domain is however more ordered than the pure lipid at corresponding temperatures. CD spectra under the same conditions are found to be identical to spectra when the drug is added to preformed membranes, only in the region T _c to T _c +11°C.Filipin induced carboxyfluorescein release from DMPC-LUV is found to be complete when the filipin-to-lipid ratio is near 1, for temperatures below T _c +11°C.Results are compared to previous data on amphotericin B and provide evidence that the gel-like structure of phospholipid and membrane permeation may be induced by filipin even in the absence of cholesterol.Abbreviations NMR nuclear magnetic resonance - CD circular dichroism - DMPC dimyristoylphosphatidylcholine - EPA egg phosphatidic acid - LUV large unilamelar vesicles - SPC soybean phosphatidylcholine - DMSO dimethylsulfoxide - CF carboxyfluorescein     

Plasma free amino acid kinetics in rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) using a bolus injection of <Superscript>15</Superscript>N-labeled amino acids

To gain insight into the metabolic design of the amino acid carrier systems in fish, we injected a bolus of ¹⁵N amino acids into the dorsal aorta in mature rainbow trout (Oncorhynchus mykiss). The plasma kinetic parameters including concentration, pool size, rate of disappearance (R _d), half-life and turnover rate were determined for 15 amino acids. When corrected for metabolic rate, the R _d values obtained for trout for most amino acids were largely comparable to human values, with the exception of glutamine (which was lower) and threonine (which was higher). R _d values ranged from 0.9 μmol 100 g⁻¹ h⁻¹ (lysine) to 22.1 μmol 100 g⁻¹ h⁻¹ (threonine) with most values falling between 2 and 6 μmol 100 g⁻¹ h⁻¹. There was a significant correlation between R _d and the molar proportion of amino acids in rainbow trout whole body protein hydrolysate. Other kinetic parameters did not correlate significantly with whole body amino acid composition. This indicates that an important design feature of the plasma-free amino acids system involves proportional delivery of amino acids to tissues for protein synthesis.     

Nonnative protein polymers: structure, morphology, and relation to nucleation and growth

Thermally induced aggregates of α-chymotrypsinogen A and bovine granulocyte-colony stimulating factor in acidic solutions were characterized by a combination of static and dynamic light scattering, spectroscopy, transmission electron microscopy, and monomer loss kinetics. The resulting soluble, high-molecular weight aggregates (∼10³-10⁵ kDa) are linear, semiflexible polymer chains that do not appreciably associate with one another under the conditions at which they were formed, with classic power-law scaling of the radius of gyration and hydrodynamic radius with weight-average molecular weight (M_w). Aggregates in both systems are composed of nonnative monomers with elevated levels of β-sheet secondary structure, and bind thioflavine T. In general, the aggregate size distributions showed low polydispersity by light scattering. Together with the inverse scaling of M_w with protein concentration, the results clearly indicate that aggregation proceeds via nucleated (chain) polymerization. For α-chymotrypsinogen A, the scaling behavior is combined with the kinetics of aggregation to deduce separate values for the characteristic timescales for nucleation (τ_n) and growth (τ_g), as well as the stoichiometry of the nucleus (x). The analysis illustrates a general procedure to noninvasively and quantitatively determine τ_n, τ_g, and x for soluble (chain polymer) aggregates, as well as the relationship between τ_n/τ_g and aggregate M_w.     

A complete hyaluronan hydrodynamic characterization using a size exclusion chromatography-triple detector array system during in vitro enzymatic degradation

Size exclusion chromatography coupled with triple detection (online laser light scattering, refractometry, and viscosimetry) (SEC-TDA) was applied for the study of hyaluronan (HA) fragments produced during hydrolysis catalyzed by bovine testicular hyaluronidase (BTH). The main advantage this approach provides is the complete hydrodynamic characterization without requiring further experiments. HA was hydrolyzed using several BTH amounts and for increasing incubation times. Fragments were characterized in terms of weight and number average molecular weights (M_w and M_n, respectively), polydispersity index (M_w/M_n), hydrodynamic radius (R_h), and intrinsic viscosity ([η]). The Mark-Houwink-Sakurada (MHS) curves (log [η] versus log M_w) were then derived directly. Fragments covering a whole range of M_w (10-900 kDa) and size (R_h = 4-81 nm) and presenting a rather narrow distribution of molar masses (M_w/M_n = 1.6-1.7) were produced. From the MHS curves, HA conformation resulted in a change from a random coil toward a rigid rod structure while decreasing the M_w. HA enzymatic hydrolysis in the presence of a BTH inhibitor was also monitored, revealing that inhibition profiles are affected by ionic strength. Finally, a comparison of the kinetic data derived from SEC-TDA with the data from rheological measurements suggested different strengths of the two methods in the determination of the depolymerization rate depending on the hydrolysis conditions.     

Evidence for a folded conformation of the cell wall protein fromAcetabularia (Polyphysa) cliftonii

Summary The cell wall protein fromAcetabularia has a non-random structure in aqueous solution at pH 5.3, as determined on the basis of intrinsic viscosity, sedimentation velocity and small angle X-ray scattering experiments. This non-random structure is stable in a pH range of 4.5–6.8, as observed on the basis of circular dichroism and viscosity measurements, supporting that the cell wall protein has a specific folded structure. All hydrodynamic measurements, including small angle X-ray scattering in solution, in this pH range are consistent with a prolate ellipsoid model for the shape of this protein, with overall dimensions ofc=86.0 Å,b=7.0 Å, anda=7.5 Å, and with a radius of gyration ofR=39.5 Å. The possibility of a coiled shape was investigated using a worm-like chain model, but it was inconsistent with the experimental data. Instead, a filled particle with uniform density which is equivalent in the scattering behavior is proposed. By a comparison of the observed radius of gyration, R_g=39.5 Å, and the radius of gyration of the cross section,R _c =7.5 Å, we were able to describe the cell wall protein in terms of a prolate ellipsoid of revolution. Comparisons of the experimental scattering curve, plotted as logl (h) versus logh, with the corresponding plots of normalized intensities, calculated for particles of particular shape and various axial ratios indicate a very asymmetric shape for the cell wall protein fromAcetabularia.This research was supported by a grant of the Deutsche Forschungsgemeinschaft.     

Light-dependent phosphate uptake of a submersed macrophyte Myriophyllum spicatum L.

The uptake kinetics of phosphate (Pi) by Myriophyllum spicatum was determined from adsorption and absorption under light and dark conditions. Pi uptake was light dependent and showed saturation following the Michaelis-Menten relation (in light: V = 16.91 × [Pi](1.335 + [Pi]), R² = 0.90, p < 0.001; in the dark: V = 5.13 × [Pi](0.351 + [Pi]), R² = 0.77, p < 0.001). Around 77% of the loss of Pi in the water column was absorbed into the tissue of M. spicatum, and only 23% was adsorbed on the surface of the plant shoots. Our study shows that M. spicatum shoots have a much higher affinity (in light: 3.9 μmol g⁻¹ dw h⁻¹ μM⁻¹; in the dark: 3.7 μmol g⁻¹ dw h⁻¹ μM⁻¹) and V_max (maximum uptake rate, shoot light) for Pi uptake than many other aquatic macrophytes (in light: 0.002-0.23 μmol g⁻¹ dw h⁻¹ μM⁻¹; in the dark: 0.002-0.19 μmol g⁻¹ dw h⁻¹ μM⁻¹), which may provide a competitive advantage over other macrophytes across a wide range of Pi concentrations.     

Characterization of group C meningococcal polysaccharide by light-scattering spectroscopy. III. Determination of molecular weight, radius of gyration, and translational diffusional coefficient.

Group-specific polysaccharides isolated by means of a cetavlon procedure are immunogenic in man and induce protective immunity against meningococcal meningitis. Minute quantities of the polymers in solution can act as vaccines. We now report the first characterization of a fractionated (C-1) group C polysaccharide in 0.4KM KCl and 0.05M sodium acetate by means of light-scattering spectroscopy. Independent measurements of refractive index increments, absolute scattered intensities, angular scattering intensities and line widths as a function of scattering angles and delay times at different concentrations using incident wavelengths of 632.8 nm from a He–Ne laser and of 488 nm from an argon–ion laser yield information on aggregation properties, molecular weight (M_r), radius of gyration 〈r⁰_g〉^1/2_z, translational diffusion coefficient 〈D〉⁰_z, and second virial coefficients A₂ and B₂ of C-1 polysaccharide. At relatively high ionic strength (0.04M KCl + 0.05M sodium acetate), we obtain for the C-1 polysaccharide in solution M_r = 5.15 × 10⁵, 〈r²_g〉^1/2_z = 345 Å, A₂ = 1.25 × 10^?4 ml/g, 〈D〉 = 1.16 × 10^?7 cm²/sec with a corresponding Stokes radius of 240 Å and B₂ = 4.4 ml/g. A₂ and B₂ are the second virial coefficients from intensity- and diffusion-coefficient measurements. The C-1 polysaccharide aggregates in solution and behaves hydrodynamically like random coils. Viscosity and sedimentation studies further confirm our conclusions that the fractioned C-1 polysaccharide aggregates in solution and EDTA can partially break up those aggregates. However, the system remains polydisperse even after adding an excess amount of EDTA. The weight-average molecular weight of the C-1 polysaccharide in solution depends upon ionic strength and exhibits a minimum at ～0.2M KCl. Finally, viscosity, light-scattering, and sedimentation results all show that the aggregated macromolecular system behaves like random-coiled polymers with no measurable shape factors.     

Facile purification of mono-PEGylated interleukin-1 receptor antagonist and its characterization with multi-angle laser light scattering

Recombinant human interleukin-1 receptor antagonist (rhIL-1ra) was chemically conjugated with succinimidyl carbonate monomethoxyl polyethylene glycols of 5 kDa (SC-PEG_5k) and 10 kDa (SC-PEG_10k) molecular weight. A facile purification of the conjugates was achieved by one-step cationic exchange chromatography. The purity of mono-PEGylated protein was greater than 95%. The purified conjugate was characterized by multi-angle laser light scattering (MALLS) for determining the apparent gyration radius (r_g) and hydrodynamic radius (r_h). MALLS results showed that the conjugation of PEG markedly enhanced r_g and r_h of parent protein (r_g: from 15.7 to 48.2 nm for the PEG_5k and 81.9 nm for the PEG_10k; r_h: from 4.2 to 58.4 nm for the PEG_5k and 102.3 nm for the PEG_10k). The PEGylated rhIL-1ra retained 44.6% of binding activities to the cell receptor for PEG_5k and 40.2% for PEG_10k, compared to the original protein.     

A New Preparation Method of Coenzyme A with Microorganisms

The structure of aggregates formed by heating dilute BSA solution was analyzed with the fractal concept using light scattering methods. BSA was dissolved in HEPES buffer of pH 7.0 and acetate buffer of pH 5.1 to 0.1% and 0.001% solutions, respectively, and heated at 95°C, varying the heating time t_a. The fractal dimension D_f of the aggregate in the solution was evaluated from static light scattering experiments. The polydispersity exponent τ and the average hydrodynamic radius <R_h> of the aggregates were calculated from dynamic light scattering experiments using master curves obtained by Klein et al. The values of D_f and τ of heat-induced aggregates of BSA at pH 7.0 were about 2.1 and 1.5, respectively, the values of which agreed with those predicted by the reaction-limited cluster–cluster aggregation (RLCCA) model. On the other hand, D_f of heat-induced aggregates at pH 5.1 was about 1.8, which agreed with that predicted by the diffusion-limited cluster–cluster aggregation (DLCCA) model. The dependence of <R_h> for the sample of pH 7.0 on t_a was similar to that of the polystyrene colloids reported previously.     

An Emulsion of Sulfoquinovosylacylglycerol with Long-Chain Alkanes Increases Its Permeability to Tumor Cells

The α-anomer form of sulfoquinovosyl-monoacylglycerol with a saturated C18 fatty acid (α-SQMG-C_18:0) is a natural sulfolipid that is a clinically promising antitumor agent. It forms vesicles, micelles or an emulsion in water, depending on several physicochemical conditions. The type of aggregate formed appears to strongly influence the bioactivity level. Thus, we investigated the nature of the aggregates in relation to their bioactivities. The structure of the α-SQMG-C_18:0 assembly was greatly affected by the type of additive used in the preparation. Emulsification with ethanol and n-decane might be more effective at inhibiting tumor cell growth than the micelle or vesicle preparations. α-SQMG-C_18:0 formed an “emulsion-like-aggregate” in ethanol containing an n-decane concentration in the range of 1.03–103 mM. These ethanol/n-alkane/α-SQMG-C_18:0 aggregates inhibited cell growth in a dose-dependent manner, under optimum conditions (i.e., ethanol containing 103 mM of n-decane or n-dodecane dispersed in phosphate-buffered saline or culture medium). Based on these data, we discuss the relationship between the molecular action of and antitumor activity by α-SQMG-C_18:0.     

Hyaluronic acid depolymerization by ascorbate-redox effects on solid state cultivation of <Emphasis Type="Italic">Streptococcus zooepidemicus</Emphasis> in cashew apple fruit bagasse

The cashew fruit (Anacardium occidentale L.) has been used as a promising agricultural resource for the production of low-molecular weight (M_W) hyaluronic acid (HA) (10⁴–10⁵ Da). The cashew juice is a rich source of vitamin C containing, 1.2–2.0 g L⁻¹. This work explores the effects of the initial concentration of the ascorbate on the solid fermentation of the juice-moisturized bagasse from the cashew apple fruit. The results show that the M_W reduction of HA is proportional to the initial ascorbate concentration. The presence of ascorbate did not influence the Streptococcus zooepidemicus metabolism. However, the HA productivity was increased from 0.18 to 0.28 mg g⁻¹ h⁻¹ when the ascorbate concentration ranged from 1.7 to 10 mg mL⁻¹. These findings contribute to the controlled production of HA in a low M_W range, which is important in cell signalization, angiogenesis and nanoparticles production.     

Structure of native proteoglycan aggregates from chick limb-bud chondrocytes

Laser light-scattering has been used to investigate the size of native proteoglycan aggregates (PGA-aA1) from day-8 chick limb-bud chondrocyte cultures isolated under associative extraction and purification conditions in 0.4M guanidinium chloride (GdnHCl) solution. Dynamic light-scattering measurements yielded a hydrodynamic radius, R_s, of 244 ± 10 nm for PGA-aA1 in 0.4M GdnHCl, and a weight-average molecular weight (M _w) of 150 ± 50 × 10⁶ was obtained from a Zimm plot. Disaggregation in 4.0M GdnHCl aqueous solution yielded proteoglycan subunits (PGS) with R_s = 39 ± 2 nm, M _w = 1.6 ± 0.3 × 10⁶, which reassembled in 0.4M GdnHCl to form “reconstituted native” aggregates (PGA-raA1) with R_s = 121 ± 6 nm, M _w = 17 ± 3 × 10⁶. A second specimen of PGA-aA1 had R_s = 192 ± 10 nm, M _w = 100 ± 10 × 10⁶. The latter value was estimated from an empirical relationship between M _w and R_s. After dissociation, this specimen reassembled to form PGA-raA1 with R_s = 85 ± 5 nm, M _w = 12 ± 1 × 10⁶. These data are compared with those for a specimen of reconstituted aggregate (PGA-A1) that had been extracted under dissociative conditions and then reaggregated by dialysis to 0.4M GdnHCl aqueous solution, for which R_s = 138 ± 9 nm, M _w = 45 ± 8 × 10⁶. From these values, we have calculated the weight-average number of subunits per aggregate N_w: 111 for PGA-aA1 and 12 for raA1 (70 and 7 for the second PGA-aA1 and PGA-raA1 specimen, respectively) as compared to 32 for PGA-A1. The numbers of subunits per aggregate were also determined from electron micrographs of spread specimens. The latter results show the same trends as those obtained by light scattering, but lead in each case to lower numbers of subunits per aggregate. These data demonstrate conclusively that PGA samples exhibit a higher degree of aggregation in solution than visualized in typical electron microscopy (EM) preparations, probably due to disaggregation during EM specimen preparation. Since N_w determined both by light scattering (LS) and by EM are larger for native versus reconstituted aggregate samples, our data point to a more compact aggregation of subunits along the hyaluronic acid (HA) chains in the former.