Relationship between lymph and tissue hyaluronan in skin and skeletal muscle

The size of hyaluronan was compared between tissue and lymph using a combination of agarose gel electrophoresis and radiometric assay. Prenodal lymph was collected from heel skin and the gastrocnemius muscle in anesthetized rabbits. The major fraction of hyaluronan in both tissues had a molecular weight >4 million. Lymph contained primarily low-molecular-weight hyaluronan (<0.79 x 10(6)), which was absent from tissue. Volume loading produced a preferential increase in the flux of low-molecular-weight hyaluronan, indicating that tissue contains a small quantity of mobile, low-molecular-weight hyaluronan. The maximum daily removal of hyaluronan by lymph was <1% of the tissue content. The amount of lysosomal hyaluronidase activity in tissue was more than enough to account for a rapid turnover of hyaluronan. The data support the conclusion that lymph drainage is not significant in the normal catabolism of hyaluronan and may represent a small amount that becomes detached from the pericellular and extracellular matrixes.     

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.     

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.     

Quantification and characterization of enzymatically produced hyaluronan with fluorophore-assisted carbohydrate electrophoresis

Hyaluronan (HA) is a polysaccharide with high-potential medical applications, depending on the chain length and the chain length distribution. Special interest goes to homogeneous HA oligosaccharides, which can be enzymatically produced using Pasteurella multocida hyaluronan synthase (PmHAS). We have developed a sensitive, simple, and fast method, based on fluorophore-assisted carbohydrate electrophoresis (FACE), for characterization and quantification of polymerization products. A chromatographic pure fluorescent template was synthesized from HA tetrasaccharide (HA4) and 2-aminobenzoic acid. HA4-fluor and HA4 were used as template for PmHAS-mediated polymerization of nucleotide sugars. All products, fluorescent and nonfluorescent, were analyzed with gel electrophoresis and quantified using lane densitometry. Comparison of HA4- and HA4-fluor-derived polymers showed that the fluorophore did not negatively influence the PmHAS-mediated polymerization. Only even-numbered oligosaccharide products were observed using HA4-fluor or HA4 as template. The fluorophore intensity was linearly related to its concentration, and the limit of detection was determined to be 7.4 pmol per product band. With this assay, we can now differentiate oligosaccharides of size range DP2 (degree of polymerization 2) to approximately DP400, monitor the progress of polymerization reactions, and measure subtle differences in polymerization rate. Quantifying polymerization products enables us to study the influence of experimental conditions on HA synthesis.     

Improved agarose gel electrophoresis method and molecular mass calculation for high molecular mass hyaluronan

The molecular mass of the polysaccharide hyaluronan (HA) is an important determinant of its biological activity and physicochemical properties. One method currently used for the analysis of the molecular mass distribution of an HA sample is gel electrophoresis. In the current work, an improved agarose gel electrophoresis method for analysis of high molecular mass HA is presented and validated. HA mobility in 0.5% agarose minigels was found to be linearly related to the logarithm of molecular mass in the range from approximately 200 to 6000 kDa. A sample load of 2.5 μg for polydisperse HA samples was employed. Densitometric scanning of stained gels allowed analysis of the range of molecular masses present in the sample as well as calculation of weight-average and number-average values. The method was validated for a polydisperse HA sample with a weight-average molecular mass of approximately 2000 kDa. Excellent agreement was found between the weight-average molecular mass determined by electrophoresis and that determined by rheological measurement of the solution viscosity. The revised method was then used to show that heating solutions of HA at 100 °C, followed by various cooling procedures, had no effect on the HA molecular mass distribution.     

The influence of ovarian steroids on ovine endometrial glycosaminoglycans

The ovine endometrium is subjected to cyclic oscillations of estrogen and progesterone in preparation for implantation. One response to fluctuating hormonal levels is the degree of hydration of the tissue, suggesting cyclical alterations in glycosaminoglycan/proteoglycan content. The aim of the present study was to quantitate and characterize glycosaminoglycans in the ovine endometrium during estrogen and progesterone dominant stages. Endogenous endometrial glycosaminoglycan content was determined by chemical analysis and characterized by enzyme specific or chemical degradation. [(35)S]-sulphate and [(3)H]-glucosamine labeled proteoglycans/glycosaminoglycans were extracted by cell lysis or with 4M guanidine-HCl. Extracts were purified by anion exchange and gel chromatography and characterized as above. Estrogen and progesterone dominant endometrium contained 3.2 +/- 0.1 and 2.1 +/- 0.1 mg endogenous glycosaminoglycan/g dehydrated tissue, respectively. Characterization of endogenous glycosaminoglycan showed chondroitin sulphate and hyaluronan contributing over 80%. The major difference between hormonal dominant tissue was a higher estrogenic hyaluronan percentage and a higher progestational keratan sulphate percentage (p < 0.001). Estrogen dominant tissue incorporated 1.6-1.9 fold more radiolabeled proteoglycans/glycosaminoglycans (p < 0.001). Analysis of newly synthesized proteoglycans/glycosaminoglycans revealed a heparan/chondroitin sulphate ratio of 1:2.2-2.5. Keratan sulphate was not detected. Estrogenic hyaluronan was 1.6 fold greater in [(3)H]-labeled tissue. Analysis of labeled proteoglycans/glycosaminoglycans revealed two size classes with apparent molecular weights >2.0 x 10(6) and 0.8-1.1 x 10(5) and a charge class eluting between 0.1-0.5 M NaCl. The greater glycosaminoglycan content (particularly hyaluronan) and synthesis in estrogen dominant tissue supports a role for steroid hormones in endometrial glycosaminoglycan/proteoglycan regulation and consequent tissue hydration. It also suggests a role for these macromolecules in endometrial function and possibly the implantation process.     

Application of fluorophore-assisted carbohydrate electrophoresis to analysis of disaccharides and oligosaccharides derived from glycosaminoglycans

Various combinations of fluorescent dyes, polyacrylamide gels, and electrophoresis buffers were tested by fluorophore-assisted carbohydrate electrophoresis (FACE) for the purpose of analyzing sulfated and nonsulfated glycosaminoglycan (GAG) oligosaccharides in which disaccharides and low-molecular weight oligosaccharides were included. A nonionic fluorescent dye was found to be suitable for analyzing sulfated disaccharides derived from sulfated GAGs (e.g., chondroitin sulfate, dermatan sulfate) because sulfated disaccharides themselves had enough anionic potential for electrophoresis. The migration rates of chondroitin sulfate (CS) disaccharides in polyacrylamide gels were affected by the number of sulfate residues and the conformation of each disaccharide. When an anionic fluorescent dye, 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt (ANTS), was coupled with sulfated GAG oligosaccharides, nearly all of the conjugates migrated at the electrophoretic front due to the added anionic potential. Nonsulfated hyaluronan (HA) oligosaccharides (2-16 saccharides) were subjected to electrophoresis by coupling with a nonionic fluorescent dye, 2-aminoacridone (AMAC), but did not migrate in the order of their molecular size. Especially di-, tetra-, hexa-, and octasaccharides of HA migrated in the reverse order of their molecular size. HA/CS oligosaccharides were able to migrate in the order of their chain lengths by coupling with an anionic fluorescent dye in a nonborate condition.     

Cell-matrix interactions of in vitro human skin fibroblasts upon addition of hyaluronan

Normal human skin fibroblasts were grown in a three-dimensional collagen gel or in monolayer in the presence or absence of high molecular weight hyaluronan (HA) to assess the influence of extracellular HA on cell-matrix interactions. HA incorporated into the collagen gel or added to the culture medium did not modify lattice retraction with time. The effect was independent from HA molecular weight (from 7.5 x 10(5) to 2.7 x 10(6) Da) and concentration (from 0.1 up to 1 mg/ml). HA did not affect shape and distribution of fibroblasts within the gel, whereas it induced the actin filaments to organise into thicker cables running underneath the plasma membrane. The same phenomenon was observed in fibroblasts grown in monolayer. By contrast, vimentin cytoskeleton and cell-substrate focal adhesions were not modified by exogenous HA. The number of fibroblasts attached to HA-coated dishes was always significantly lower compared to plastic and to collagen type I-coated plates. By contrast, adhesion was not affected by soluble HA added to the medium nor by anti-CD44 and anti-RHAMM-IHABP polyclonals. After 24-h seeding on collagen type I or on plastic, cells were large and spread. Conversely, cells adherent to HA-coated surfaces were long, thin and aligned into rows; alcian blue showed that cells were attached to the plastic in between HA bundles. Therefore, normal human skin fibroblasts exhibit very scarce, if any, adhesion to matrix HA, either soluble or immobilised. Moreover, even at high concentration, HA molecules do not exert any visco-mechanical effect on lattice retraction and do not interfere with fibroblast-collagen interactions nor with focal adhesion contacts of fibroblasts with the substrate. This is probably relevant in organogenesis and wound repair. By contrast, HA greatly modifies the organisation of the actin cytoskeleton, suggesting that CD44-mediated signal transduction by HA may affect cell locomotion and orientation, as indicated by the fusiform shape of fibroblasts grown in the presence of immobilised HA. A role of HA in cell orientation could be relevant for the deposition of collagen fibrils in regeneration and tissue remodelling.     

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.     

Quantitative continuous assay for hyaluronan synthase

A rapid, continuous, and convenient three-enzyme coupled UV absorption assay was developed to quantitate the glucuronic acid and N-acetylglucosamine transferase activities of hyaluronan synthase from Pasteurella multocida (PmHAS). Activity was measured by coupling the UDP produced from the PmHAS-catalyzed transfer of UDP-GlcNAc and UDP-GlcUA to a hyaluronic acid tetrasaccharide primer with the oxidation of NADH. Using a fluorescently labeled primer, the products were characterized by gel electrophoresis. Our results show that a truncated soluble form of recombinant PmHAS (residues 1-703) can catalyze the glycosyl transfers in a time- and concentration-dependent manner. The assay can be used to determine kinetic parameters, inhibition constants, and mechanistic aspects of this enzyme. In addition, it can be used to quantify PmHAS during purification of the enzyme from culture media.     

荧光标记电泳对透明质酸寡糖片段鉴定的方法建立

近年来,透明质酸寡糖片段（hyaluronan oligosaccharides, o-HA）的生物学活性引起国外学者的重视,因为o-HA具有一定的生物学活性,如参与免疫调节、刺激新生血管形成等.本研究建立一种经济、简便的ANTS（8-氨基奈-1,3,6-三磺酸）荧光标记电泳对透明质酸寡糖片段大小鉴定的新实验方法.实验原理为,ANTS能与糖分子发生还原反应,在反应时提供3个电子和1个荧光基团,通过高浓度PAGE分离,在特定波长下呈现颜色反应.采用酶消化法得到不同分子量大小的o-HA片段,测得不同片段大小的o-HA聚合度,分别与高效液相色谱（high-performance liquid chromatography, HPLC）和静电喷雾电离质谱（electrospray ionization mass spectrometry, ESI-MS）进行比较,结果吻合.研究提示,用荧光标记电泳法分析寡糖分子量,操作简单、设备低廉、灵敏度较高且检测速度快,是一种检测鉴定寡糖分子的较好方法.     

Agarose and polyacrylamide gel electrophoresis methods for molecular mass analysis of 5- to 500-kDa hyaluronan

Agarose and polyacrylamide gel electrophoresis systems for the molecular mass-dependent separation of hyaluronan (HA) in the size range of approximately 5–500 kDa were investigated. For agarose-based systems, the suitability of different agarose types, agarose concentrations, and buffer systems was determined. Using chemoenzymatically synthesized HA standards of low polydispersity, the molecular mass range was determined for each gel composition over which the relationship between HA mobility and logarithm of the molecular mass was linear. Excellent linear calibration was obtained for HA molecular mass as low as approximately 9 kDa in agarose gels. For higher resolution separation, and for extension to molecular masses as low as approximately 5 kDa, gradient polyacrylamide gels were superior. Densitometric scanning of stained gels allowed analysis of the range of molecular masses present in a sample as well as calculation of weight-average and number-average values. The methods were validated for polydisperse HA samples with viscosity-average molecular masses of 112, 59, 37, and 22 kDa at sample loads of 0.5 μg (for polyacrylamide) to 2.5 μg (for agarose). Use of the methods for electrophoretic mobility shift assays was demonstrated for binding of the HA-binding region of aggrecan (recombinant human aggrecan G1–IGD–G2 domains) to a 150-kDa HA standard.     

Expression of Recombinant Hyaluronan Synthase (HAS) Isoforms in CHO Cells Reduces Cell Migration and Cell Surface CD44

In the present study we investigated the functional properties of the three recombinant hyaluronan synthases (HAS proteins) HAS1, HAS2, and HAS3. HAS3-transfected CHO clones exhibited the highest hyaluronan polymerization rate followed by HAS2 transfectants which were more catalytically active than HAS1 transfectants. In living cells all three HAS proteins synthesized hyaluronan chains of high molecular weight (larger than 3.9 x 10(6)). In vitro, the HAS2 isoform produced hyaluronan chains of a molecular weight larger than 3.9 x 10(6), whereas HAS3 produced polydisperse hyaluronan (molecular weight 0.12-1 x 10(6)), and HAS1 synthesized much shorter chains of an average molecular weight of 0.12 x 10(6). Thus, each HAS protein may interact with different cytoplasmic proteins which may influence their catalytic activity. CHO transfectants with the ability to synthesize about 1 microgram hyaluronan/1 x 10 (5) cells/24 h were surrounded by hyaluronan-containing coats, whereas transfectants generating about 4-fold lower amounts of hyaluronan formed coats only in the presence of chondroitin sulfate proteoglycan. An inverse correlation between hyaluronan production on the one hand and cell migration and cell surface CD44 expression on the other was found; a 4-fold lower migration and a 2-fold decrease of cell surface CD44 receptors was seen when hyaluronan production increased 1000-fold over the level in the untransfected cells. The inverse relationships between hyaluronan production and migration and CD44 expression of cells are of importance for the regulation of cell-extracellular matrix interactions.     

Studies on structural proteins of the rat liver ribosomes. I. Molecular wights of the proteins of large and small subunits.

Structural proteins of active 60-S and 40-S subunits of rat liver ribosomes were analysed by two-dimensional polyacrylamide gel electrophoresis. 35 and 29 spots were shown on two-dimensional gel electrophoresis of proteins from large and small subunits, respectively. It was noted that the migration distances of stained proteins with Amido black 10B remained unchanged in the following sodium dodecyl sulfate-acrylamide gel electrophoresis, although some minor degradation and/or aggregation products were observed in the case of several ribosomal proteins, especially of those with high molecular weights. This finding made it possible to measure the molecular weight of each ribosomal protein in the spot on two-dimensional gel electrophoresis by following sodium dodecyl sulfate-acrylamide gel electrophoresis. The molecular weights of the protein components of two liver ribosomal subunits were determined by this 'three-dimensional' polyacrylamide gel electrophoresis. The molecular weights of proteins of 40-S subunits ranged from 10 000 to 38 000 and the number average molecular weight was 23 000. The molecular weights of proteins of 60-S subunits ranged from 10 000 to 60 000 and the number average molecular weight was 23 900.     

Hyaluronan is not elevated in urine or serum in Hutchinson-Gilford Progeria Syndrome

Elevations in urinary hyaluronan have been used as the principal laboratory indicator for diagnosis of Hutchinson-Gilford Progeria Syndrome (HGPS). Previous reports have provided evidence suggesting that children with HGPS have altered hyaluronan metabolism as indicated by a mean 17-fold increase in urinary hyaluronan over normal values. In addition, adults with Werner's syndrome have elevated urinary hyaluronan and even more prominent elevations in serum hyaluronan over age-matched controls. It is not known whether serum hyaluronan is elevated or whether serum hyaluronan levels correlate with urinary hyaluronan levels in children with HGPS. In a large cohort of 19 HGPS patients, we sought to confirm elevations in urinary hyaluronan concentration, to establish whether serum hyaluronan is elevated, to measure the size of urinary hyaluronan, and to determine whether serum or urine hyaluronidase levels are altered. We have analyzed urinary and serum hyaluronan levels in patients with HGPS and control patients (1) by using an enzyme-linked immunosorbent assay (ELISA)-like method in which sample hyaluronan in solution and hyaluronan in solid phase compete for a solution of biotinylated hyaluronan-binding protein, and (2) by fluorophore-assisted carbohydrate electrophoresis. The size of urinary hyaluronan was measured by using Sepharose CL-6B size exclusion chromatography. Serum and urinary hyaluronidases were evaluated quantitatively, by using ELISA, and qualitatively, by using a gel detection method. HGPS patients did not show a significant elevation in either urinary or serum hyaluronan. We detected no difference in the size of urinary hyaluronan between HGPS children and age-matched controls. Serum and urinary hyaluronidase levels were not significantly different in normal and HGPS patients. These studies indicate that neither serum nor urinary hyaluronan concentration is a reliable diagnostic or prognostic marker for HGPS and underscore a difference between adult and childhood progerias.     

Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties.

Three mammalian hyaluronan synthase genes, HAS1, HAS2, and HAS3, have recently been cloned. In this study, we characterized and compared the enzymatic properties of these three HAS proteins. Expression of any of these genes in COS-1 cells or rat 3Y1 fibroblasts yielded de novo formation of a hyaluronan coat. The pericellular coats formed by HAS1 transfectants were significantly smaller than those formed by HAS2 or HAS3 transfectants. Kinetic studies of these enzymes in the membrane fractions isolated from HAS transfectants demonstrated that HAS proteins are distinct from each other in enzyme stability, elongation rate of HA, and apparent K(m) values for the two substrates UDP-GlcNAc and UDP-GlcUA. Analysis of the size distributions of hyaluronan generated in vitro by the recombinant proteins demonstrated that HAS3 synthesized hyaluronan with a molecular mass of 1 x 10(5) to 1 x 10(6) Da, shorter than those synthesized by HAS1 and HAS2 which have molecular masses of 2 x 10(5) to approximately 2 x 10(6) Da. Furthermore, comparisons of hyaluronan secreted into the culture media by stable HAS transfectants showed that HAS1 and HAS3 generated hyaluronan with broad size distributions (molecular masses of 2 x 10(5) to approximately 2 x 10(6) Da), whereas HAS2 generated hyaluronan with a broad but extremely large size (average molecular mass of >2 x 10(6) Da). The occurrence of three HAS isoforms with such distinct enzymatic characteristics may provide the cells with flexibility in the control of hyaluronan biosynthesis and functions.     

Possibility of shape conformers of the protein inhibitor of the cyclic adenosine monophosphate dependent protein kinase.

The heat-stable, protein inhibitor of the cyclic adenosine monophosphate (cAMP) dependent protein kinase [Walsh, D. A., Ashby, C. D., Gonzalez, C., Calkins, D., Fischer, E., & Krebs, E (1971a) J. Biol. Chem. 246, 1977-1985] has been purified to homogeneity from rabbit skeletal muscle by preparative electrophoresis. Employing a more sensitive assay system, we detected multiple charged forms of the inhibitor on diethylaminoethyl chromatography; the form that has been further characterized is the predominant species in skeletal muscle comprising greater than 70% of the total. The apparent molecular weight of the protein inhibitor, as determined by Sephadex G-75 gel exclusion chromatography, is 22 000 in initial cellular extracts and at all stages during the purification prior to the final purification step of preparative gel electrophoresis, after which the homogeneous protein exhibits a molecular weight of 11 000. These two forms are designated I and I', respectively. The I form migrates with an apparent molecular weight of 10 000 on nondenaturing gel electrophoresis and of 10 500-11 500 on sodium dodecyl sulfate (NaDodSO4) gel electrophoresis; the I' form migrates with an apparent molecular weight of 6500-8300 on NaDodSO4 electrophoresis and has a minimum molecular weight of 10 400 by amino acid analysis. Taking into account the anomalous behavior displayed by low molecular weight proteins with the various techniques employed, we suggest that the I and I' forms of the protein inhibitor may represent shape conformers.     

Asymmetrical-flow field-flow fractionation with on-line multiangle light scattering detection. 1. Application to wormlike chain analysis of weakly stiff polymer chains

Four samples of hyaluronan in the sodium form, ranging in weight-average molecular weight, M(w), from 6.67 x 10(5) to 4.23 x 10(6) were investigated by asymmetrical-flow field-flow fractionation coupled to multiangle light scattering (FlFFF-MALS) in 0.2 M aqueous NaCl at 25 degrees C. M(w) and z-average radii of gyration, R(G)(z)(), obtained via FlFFF-MALS showed a good agreement with the results obtained by conventional static light scattering. Furthermore, the molecular weight dependence of the radius of gyration for sodium hyaluronan obtained via FlFFF-MALS was analyzed on the basis of the Kratky-Porod model for unperturbed wormlike chains combined with the Yamakawa theory for radius expansion factor, and a sufficiently good agreement was observed between the theoretical prediction and experimental data. These results show the potential usage of FlFFF-MALS regarding size separation and molecular characterization even for weakly stiff chains.     

Synthesis of hyaluronan of distinctly different chain length is regulated by differential expression of Xhas1 and 2 during early development of Xenopus laevis

The localization of hyaluronan has been determined in tailbud stage embryos of Xenopus laevis using a neurocan-alkaline phosphatase fusion protein. This polysaccharide was located between the germ layers and enriched in mesenchyme, the lumen of the neural tube, the embryonic gut, the hepatic cavity and the heart. A full-length cDNA for a hyaluronan synthase, Xhas2 has been cloned. The expression pattern of Xhas1 and 2 is closely similar to the distribution of hyaluronan in the embryo. Xhas1 produces hyaluronan with a molecular mass of around 40–200 kDa, while the product formed by Xhas2 has a molecular mass above 1 million Da.