Intracervical application of hyaluronan improves cervical relaxation in the ewe

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

Intra-cervical application of Misoprostol at estrus alters the content of cervical hyaluronan and the mRNA expression of follicle stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR) and cyclooxygenase-2 in the ewe

The complex anatomy the of ovine cervix limits the success of transcervical artificial insemination in sheep, but Misoprostol (a PGE₁ analogue) relaxes the cervix and facilitates transcervical artificial insemination. However, the mechanism by which Misoprostol causes cervical relaxation is not known. This study examined if intra-cervical Misoprostol altered the hyaluronan content and the mRNA expression of COX-2, LHR, or FSHR in the cervix of the estrus ewe. Estrus was synchronized in cyclic ewes with progestagen pessaries and 48 h after sponge removal ewes were treated intra-cervically with 0 (controls), 200, or 400 μg Misoprostol. Hyaluronan content was determined by ELISA and mRNA expression of LHR, FSHR, and COX-2 was analyzed by in situ hybridization using digoxigenin-11-uridine-5′-triphosphate labeled riboprobes. The hyaluronan content of the cervix was significantly higher in sheep that received 200 (P < 0.05) or 400 (P < 0.05) μg Misoprostol compared to controls. Moreover, it was significantly (P < 0.05) higher in the vaginal region compared to mid and uterine regions. Misoprostol increased (P < 0.05) the mRNA expression of LHR and COX-2 but not FSHR. The expression for all three genes was highest in the vaginal region and lowest in uterine region. The luminal epithelium and circular smooth muscle layers had higher (P < 0.05) expression for LHR, FSHR, and COX-2 mRNAs, and the sub-epithelial stroma had the lowest (P < 0.05). We propose that the intra-cervical application of Misoprostol induces the mRNA expression of LHR, FSHR, and COX-2 through a positive feedback loop. The data suggest that softening of the cervix by Misoprostol is caused by an increase in the hyaluronan content of the cervix.     

Antisense inhibition of hyaluronan synthase-2 in human osteosarcoma cells inhibits hyaluronan retention and tumorigenicity

Osteosarcoma is a common malignant bone tumor associated with childhood and adolescence. The results of numerous studies have suggested that hyaluronan plays an important role in regulating the aggressive behavior of various types of cancer cells. However, no studies have addressed hyaluronan with respect to osteosarcomas. In this investigation, the mRNA expression copy number of three mammalian hyaluronan synthases (HAS) was determined using competitive RT-PCR in the osteoblastic osteosarcoma cell line, MG-63. MG-63 are highly malignant osteosarcoma cells with an abundant hyaluronan-rich matrix. The results demonstrated that HAS-2 is the predominant HAS in MG-63. Accumulation of intracellular hyaluronan increased in association with the proliferative phase of these cells. The selective inhibition of HAS-2 mRNA in MG-63 cells by antisense phosphorothioate oligonucleotides resulted in reduced hyaluronan accumulation by these cells. As expected, the reduction in hyaluronan disrupted the assembly of cell-associated matrices. However, of most interest, coincident with the reduction in hyaluronan, there was a substantial decrease in cell proliferation, a decrease in cell motility and a decrease in cell invasiveness. These data suggest that hyaluronan synthesized by HAS-2 in MG-63 plays a crucial role in osteosarcoma cell proliferation, motility, and invasion.     

The distribution of renal hyaluronan and the expression of hyaluronan synthases during water deprivation in the Spinifex hopping mouse, Notomys alexis

Hyaluronan (HA) is a glycosaminoglycan that is synthesized by a family of enzymes called hyaluronan synthases (HASs), of which there are three isoforms (HAS1, 2 and 3) in mammals. The HASs have different tissue expression patterns and function, indicating that synthesis of HA and formation of the HA matrix may be regulated by various factors. The HA matrix has an important role in renal water handling and the production of a concentrated urine. We investigated the distribution of HA and the expression of HAS1, HAS2 and HAS3 mRNAs in the kidney of the Spinifex hopping mouse, Notomys alexis, a native Australian desert rodent that is reported to produce the most concentrated urine of any mammal. After periods of three, seven and fourteen days of water deprivation, the distribution of renal HA changed considerably, and there was a general down-regulation of HAS mRNA expression. It is proposed that the regulation of HA synthesis by the different HAS isoforms during water deprivation in N. alexis, could be influenced by the molecular mass of the HA chains produced by each isoform, followed by the rate at which the individual HAS produces HA.     

Biology of hyaluronan: Insights from genetic disorders of hyaluronan metabolism

Hyaluronan is a rapidly turned over component of the vertebrate extracellular matrix. Its levels are determined, in part, by the hyaluronan synthases, HAS1, HAS2, and HAS3, and three hyaluronidases, HYAL1, HYAL2 and HYAL3. Hyaluronan binding proteins also regulate hyaluronan levels although their involvement is less well understood. To date, two genetic disorders of hyaluronan metabolism have been reported in humans: HYAL1 deficiency(Mucopolysaccharidosis IX) in four individuals with joint pathology as the predominant phenotypic finding and HAS2 deficiency in a single person having cardiac pathology. However, inherited disorders and induced mutations affecting hyaluronan metabolism have been characterized in other species. Overproduction of hyaluronan by HAS2 results in skin folding and thickening in shar-pei dogs and the naked mole rat, whereas a complete deficiency of HAS2 causes embryonic lethality in mice due to cardiac defects. Deficiencies of murine HAS1 and HAS3 result in a predisposition to seizures. Like humans, mice with HYAL1 deficiency exhibit joint pathology. Mice lacking HYAL2 have variably penetrant developmental defects, including skeletal and cardiac anomalies. Thus, based on mutant animal models, a partial deficiency of HAS2 or HYAL2 might be compatible with survival in humans, while complete deficiencies of HAS1, HAS3, and HYAL3 may yet be recognized.     

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.     

High-performance capillary electrophoretic analysis of hyaluronan in effusions from human malignant mesothelioma

A procedure to quantify hyaluronan in effusions from human malignant mesothelioma using a highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method is presented. Following ethanol precipitation, hyaluronan and galactosaminoglycans were degraded to Δ^4.5-dissacharides with a mixture of chondroitinases ABC and AC. Heparan sulphate and proteins/glycoproteins were separated by ultrafiltration on a Centricon 3 membrane, and hyaluronan-derived disaccharides were analysed by direct injection of the filtrate into a HPCE system. Determination of hyaluronan in effusions from five healthy individuals and three patients with mesothelioma gave values comparable to those found using the HPLC method. One of the advantages of the HPCE method as compared to HPLC is the low solvent consumption. The much lower detection limit (attomole level) of the HPCE method may also allow the analysis of hyaluronan content in serum. The contribution of HPCE in diagnosis of a neoplasm, such as human malignant mesothelioma, illustrates the great potential of this technique in the field of life sciences.     

Measurement of high-molecular-weight hyaluronan in solid tissue using agarose gel electrophoresis

The size of hyaluronan in solid tissue was measured using a combination of agarose gel electrophoresis and a radiometric assay. Radiolabeled hyaluronan binding proteins, used in the radiometric assay, were also used to detect hyaluronan after transfer to a nylon membrane following gel electrophoresis. Lane intensity on the autoradiograph was linearly related to the amount applied to the gel between 10 and 100ng. The intensity was independent of the hyaluronan molecular weight for standards with molecular weights equal to or greater than 790,000. The radiometric assay was used to measure hyaluronan irrespective of size, while gel electrophoresis was used to measure hyaluronan with molecular weights greater than 0.79x10(6) or 4x10(6). Deferoxamine was used to inhibit depolymerization during the digestion of tissue samples with protease. The molecular weight pattern was similar for skin, skeletal muscle, heart, lung, small intestine, and large intestine despite large differences in hyaluronan content. For all tissues, 58% of the hyaluronan had a molecular weight greater than 4million. All tissues showed an absence of hyaluronan with a molecular weight below 790,000. The procedure can be used to study changes in hyaluronan size in tissue during inflammation and other pathological states.     

Profiling of the eye aqueous humor in exfoliation syndrome by high-performance liquid chromatographic analysis of hyaluronan and galactosaminoglycans

The concentrations of hyaluronan and galactosaminoglycans - i.e., chondroitin sulfate and dermatan sulfate - were measured in the aqueous humor of the eye from patients with exfoliation syndrome and from healthy persons. The glycosaminoglycans/proteoglycans were almost completely precipitated (>97%) with ethanol in the presence of dextran as carrier and, following enzymic digestion, hyaluronan and galactosaminoglycans, were quantitatively converted to Δ^4,5-disaccharides. Non-degraded heparan sulfate and proteins/glycoproteins were removed by ultrafiltration using a Centricon 3 membrane. Separation and determination of hyaluronan- and galactosaminoglycan-derived Δ-disaccharides were performed by ion-suppression HPLC. For an accurate analysis in triplicate, as little as 50 μl of aqueous humor is required. Application of this method to the analysis of samples from six patients with exfoliation syndrome and three healthy persons showed that hyaluronan levels in patients (6.65–16.15 μg ml⁻¹) were significantly higher (3–8 times) than in healthy persons (2.0–2.24 μg ml⁻¹). There was no significant alteration in the galactosaminoglycan concentration. The obtained data open a new area in the deeper understanding of the exfoliation syndrome pathophysiology and in establishing a highly sensitivity and accurate HPLC method for its diagnosis and patient's follow-up.     

The decreased secretion of hyaluronan by older human fibroblasts under physiological conditions is mainly associated with the down-regulated expression of hyaluronan synthases but not with the expression levels of hyaluronidases

Although it has been reported that levels of hyaluronan are decreased in the dermis of aged skin, little is known about the cellular mechanism(s) underlying that hyaluronan deficiency. Since hyaluronan is produced by dermal fibroblasts and is secreted into the surrounding dermal tissues, we examined the secretion of hyaluronan by dermal fibroblasts and characterized its cellular mechanism using real-time RT-PCR and western blotting for its synthesizing and degrading enzymes, hyaluronan synthase and hyaluronidase, respectively. The secretion of hyaluronan by dermal fibroblasts derived from differently aged human donors, was higher in the younger human fibroblasts tested (0 and 19 years old) compared to the older human fibroblasts tested (39, 56 and 77 years old). The relative secretion levels of hyaluronan by the different human fibroblasts tested were attributable to the relative expression of hyaluronan synthases 1, 2, 3 but not hyaluronidases 1, 2 enzymes at the gene and protein levels among those fibroblasts. These findings indicate that the deficiency of hyaluronan in the aged dermis might result from the down-regulation in the potential of older human fibroblasts to secrete hyaluronan and that decrease in secretory potential is mainly associated with the down-regulated expression of hyaluronan synthases, especially hyaluronan synthase 2, but not with the expression levels of hyaluronidases.     

Imaging and tracking of single hyaluronan molecules diffusing in solution

Hyaluronan is an important soluble component of the extracellular matrix of many tissues with well known space-filling, lubricating and signaling functions. As such, hyaluronan can regulate cell adhesion, migration, differentiation and proliferation. Ultrastructural studies showed the existence of fibers and networks of hyaluronan molecules at surfaces, while bulk studies of hyaluronan in solution indicated that the polymer forms random coils. Here, we show that single hyaluronan molecules can be visualized and tracked in three-dimensional samples at room temperature in aqueous buffer. Using a wide-field fluorescence microscope equipped with laser excitation and an sensitive and fast EMCCD camera for fluorescence detection, single FITC-labeled hyaluronan molecules from rooster comb were detected in aqueous solutions. Freely moving hyaluronan-FITC could be tracked over up to 20 images acquired at a frame rate of 98 Hz. Analysis of the trajectories revealed Brownian motion of hyaluronan in tris-buffered saline with an average diffusion coefficient D = 3.0 ± 0.2 μm²/s. These observations confirm the concept that hyaluronan molecules form random coils in solution. The possibility of following the tracks of single hyaluronan molecules in solution facilitates the analysis of processes that lead to the formation of more organized forms of hyaluronan and its interactions with cells with very high spatial and temporal accuracy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Regulation of hyaluronan synthases in mouse uterine cervix

We examined the expression pattern of hyaluronan synthase (HAS) mRNAs in the uterine cervix of pregnant mice. The expression levels of HAS-1 and -2 mRNAs peaked at delivery, whereas that of HAS-3 mRNA peaked on the 15th day of pregnancy. The regulation of HAS mRNA expression was examined in pregnant mouse uterine cervical fibroblasts. The expression of HAS-1, -2, and -3 mRNAs was significantly augmented by interleukin-1beta (IL-1beta). Progesterone significantly interfered with expression of HAS-1 and -2 mRNAs, but significantly increased the expression of HAS-3 mRNA. Low-molecular-weight hyaluronan significantly enhanced only the expression of HAS-1 mRNA. These results indicate that HAS in the uterine cervix is regulated in a complex manner by IL-1beta, progesterone, and low-molecular-weight hyaluronan, of which changes in the cervical tissue and serum closely participate in uterine cervical ripening and/or inflammation.     

The pattern of cervical penetration and the effect of topical treatment with prostaglandin and/or FSH and oxytocin on the depth of cervical penetration in the ewe during the peri-ovulatory period

Artificial insemination in sheep has two major limiting factors: the poor quality of frozen-thawed ram semen and the convoluted anatomy of the sheep cervix that does not allow transcervical passage of an inseminating catheter. It has been demonstrated that in the ewe during estrus, there is a degree of cervical relaxation mediated by ovarian and possibly gonadotrohic hormones, and we set out to investigate factors that might enhance cervical relaxation. Five experiments were conducted on ewes of different breeds to determine: 1) the pattern of cervical penetration during the periovulatory period in ewes of several breeds (Welsh Mountain, Île-de-France, Vendéenne, Romanov and Sarda); 2) the effect of the “ram effect” a socio-sexual stimulus, on cervical penetration; and 3) the effects of the intracervical administration of follicle-stimulating hormone (FSH), oxytocin and a prostaglandin E agonist (misoprostol) on the depth of cervical penetration during the periovulatory period. The results showed that during the periovulatory period in all breeds examined, there was increased penetration of the cervical canal (P < 0.05) by an inseminating catheter. Cervical penetration increased to a maximum 54 h after the removal of progestagen sponges and then gradually declined. Furthermore, the depth of cervical penetration but not its pattern, was affected (P < 0.05) by the breed of ewe. The maximum depth of cervical penetration was lower (P < 0.05) in the Vendéenne breed compared to the Île-de-France and Romanov breeds, which did not differ from one another. In the presence of rams, the depth of cervical penetration was increased at 48 and 54 h after removal of sponges (P < 0.05) and reduced at 72 h (P < 0.05). The local administration of hormones FSH, misoprostol (a PGE agonist) and oxytocin alone and in various combinations did not have any significant effect on the depth of cervical penetration during the periovulatory period. In conclusion, the natural relaxation of the cervix observed in ewes of several breeds occurs at a time during estrus, 54 h after the removal of progestagen sponges, which is the most suitable for artificial insemination. The effect was enhanced by the presence of a ram but not by the local intracervical administration of FSH, misoprostol and oxytocin even though oxytocin and PGE₂ are involved in cervical function. The time of maximum cervical penetration in the preovulatory period (54 h) coincides with high LH and estradiol concentrations suggesting they might be responsible for the relaxation of the cervix probably through an oxytocin-PGE mediated pathway.     

Chain-length dependence of the kinetics of the hyaluronan hydrolysis catalyzed by bovine testicular hyaluronidase

Hyaluronan (HA) has various biological functions that are strongly dependent on its chain length. In some cases, as in inflammation and angiogenesis, long and short chain-size HA effects are antagonistic. HA hydrolysis catalyzed by hyaluronidase (HAase) is believed to be involved in the control of the balance between longer and shorter HA chains. Our studies of native HA hydrolysis catalyzed by bovine testicular HAase have suggested that the kinetic parameters depend on the chain size. We thus used HA fragments with a molar mass ranging from 8x10(2) g mol(-1) to 2.5x10(5) g mol(-1) and native HA to study the influence of the chain length of HA on the kinetics of its HAase-catalyzed hydrolysis. The initial hydrolysis rate strongly varied with HA chain length. According to the Km and Vm/Km values, the ability of HA chains to form an efficient enzyme-substrate complex is maximum for HA molar masses ranging from 3x10(3) to 2x10(4) g mol(-1). Shorter HA chains seem to be too short to form a stable complex and longer HA chains encounter difficulties in forming a complex, probably because of steric hindrance. The hydrolysis Vm values strongly suggest that as the chain length decreases the HAase increasingly catalyses transglycosylation rather than hydrolysis. Finally, two HA chain populations, corresponding to HA chain molar masses lower and higher than approximately 2x10(4) g mol(-1), are identified and related to the bi-exponential character of the model we have previously proposed to fit the experimental points of the kinetic curves.     

Age-related differences in the accumulation and size of hyaluronan in alginate culture

The alginate bead culture system has unique properties that make it possible to study the accumulation and turnover of macromolecules in two distinct matrix compartments of the cartilage matrix: the cell-associated matrix (CM) and the further removed matrix (FRM). Taking advantage of this culture system, the purpose of this study was to examine age-related changes in the metabolism of hyaluronan (HA) in these two compartments. Bovine chondrocytes, isolated from fetal, young adult, and old adult articular cartilage, were cultured in alginate beads. On Days 7 and 14 of culture, the alginate gel was solubilized, the CM and FRM were separated and macromolecules in both compartments were analyzed. When compared to the cells from fetal and old adult animals, the young adult cells proliferated at the fastest rate. Fetal cells produced a more abundant CM that was richer in proteoglycans (PGs) than the CM of young or old adult cells. With increasing age, there was an increased tendency for PG, collagen, and HA to escape incorporation into the CM and to become immobilized in the FRM. Very striking changes also were observed in the ratio of HA to PG, which increased markedly with age, and in the size of the HA molecules, which decreased markedly with age. The results suggest that the metabolism of HA in cartilage undergoes pronounced age-related changes, some of which are retained during culture in alginate gel. The findings also suggest that the previously documented age-related decrease in the size of HA in native bovine cartilage reflects, at least in part, a biochemical process occurring at the time or at least soon after the glycosaminoglycan chain is synthesized. It does not appear to simply be the result of age-related changes occurring slowly with time after synthesis, as was previously suggested to be the case for human articular cartilage.     

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.     

Hyaluronan and its binding proteins during cervical ripening and parturition: Dynamic changes in size, distribution and temporal sequence

The uterine cervix undergoes changes during pregnancy and labor that transform it from a closed, rigid, collagen dense structure to one that is distensible, has a disorganized collagen matrix, and dilates sufficiently to allow birth. To protect the reproductive tract from exposure to the external environment, the cervix must be rapidly altered to a closed, undistensible structure after birth. Preparturition remodeling is characterized by increased synthesis of hyaluronan, decreased expression of collagen assembly genes and increased distribution of inflammatory cells into the cervical matrix. Postpartum remodeling is characterized by decreased hyaluronan (HA) content, increased expression of genes involved in assembly of mature collagen and inflammation. The focus of this study is to advance our understanding of functions HA plays in this dynamic process through characterization of HA size, structure and binding proteins in the mouse cervix. Changes in size and structure of HA before and after birth were observed as well as cell specific expression of HA binding proteins. CD44 expression is localized to the pericellular matrix surrounding the basal epithelia and on immune cells while inter alpha trypsin inhibitor (IalphaI) and versican are localized to the stromal matrix. Colocalization of HA and IalphaI is most pronounced after birth. Upregulation of the versican degrading protease, ADAMTS1 occurs in the cervix prior to birth. These studies suggest that HA has multiple, cell specific functions in the cervix that may include modulation of tissue structure and integrity, epithelial cell migration and differentiation, and inflammatory responses.     

Conformational properties of the disaccharide building units of hyaluronan

The conformational space of the disaccharide building units of hyaluronan, β-(1→4) and β-(1→3)-linked N-acetyl-β-d-glucosamine (GN) and β-d-glucuronic acid (GA), has been investigated by density functional theory calculations at the B3LYP/6-31G^** level. The study covered the anionic disaccharides, the neutral acids as well as the sodium salts in the isolated state and in aqueous solution using the PCM model approach. We elucidated the intramolecular hydrogen bonding interactions characterizing the most favoured conformers. The protonation and salt formation change these secondary interactions in the vicinity of the carboxyl group, resulting often in a considerable alteration of the conformational preferences. The Na⁺ ion in the salt is involved in multiple bonding in the most stable structures: beyond the primary ionic bond with the carboxylate group it forms slightly weaker interactions with neighbouring oxygens. The main effect of protonation and salt formation on the electron density distribution is restricted to the surroundings of the broken/formed interactions near the carboxylate group.     

A study on the nature of intermolecular links in the cryotropic weak gels of hyaluronan

In this study, the influence of acidification and salting effect on the properties of hyaluronan (HA) aqueous solutions and cryotropic weak gels were investigated by dynamic rheometry, polarizing and optical microscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD), differential scanning calorimetry (DSC) and FTIR spectroscopy. The forming mechanism of HA cryotropic weak gels was also discussed. Experimental results indicated that the HA weak gel showing a thermoreversible property was constructed by entangled bundle-like structures that could be melted at elevated temperature above 70 °C, and that the junction knots of three-dimensional polymeric network were not the ordinary microcrystalline zones that are generally of detectable crystallinity and thermal effect. The intermolecular hydrogen bonding induced from -COOH and -NHCOCH₃ in HA chains played a predominant role in respect to the network formation and stabilization of HA weak gel.     

Reaction of peroxynitrite with hyaluronan and related saccharides

The effects of peroxynitrite on hyaluronan has been studied by using an integrated spectroscopical approach, namely electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), and mass spectrometry (MS). The reaction has been performed with the polymer, the tetrasaccharide oligomer as well as with the monosaccharides N-acetylglucosamine and glucuronic acid. The outcome of the presence of molecular oxygen and carbon dioxide has been also evaluated. Although ₁H-NMR and ESI-MS experiments did not revealed peroxynitrite-mediated modification of hyaluronan as well as of related saccharides, from spin-trapping EPR experiments it was concluded that peroxynitrite induce the formation of C-centered carbon radicals, most probably by the way of its hydroxyl radical-like reactivity. These EPR data support the oxidative pathway involved in the degradation of hyaluronan, a probable event in the development and progression of rheumatoid arthritis.