Somite chondrogenesis in vitro: 2. Changes in the hyaluronic acid synthesis

The role of hyaluronic acid in limb morphogenesis (chondrogenesis) has been well defined. In the present study, we found that hyaluronic acid synthesis in somite explants steadily increased until day 6, then decreased, and inclusion of notochord did not accelerate the rate of synthesis. Analysis of hyaluronidase activity in the somite explants indicated an increase in the enzyme level in day-6 cultures. Again, inclusion of notochord did not alter this pattern. The decrease in hyaluronic acid after day 6 and the increase in sulfated proteoglycan synthesis from day 6 resemble the pattern described during limb development. Subsequent studies showed that, with time, the size of the hyaluronic acid synthesized by somites increased and, again, inclusion of notochord did not influence this pattern. The results indicate that unstimulated somites are capable of synthesizing cartilage-specific proteoglycans in a relatively restricted manner, and the inclusion of notochord resulted in accelerated synthesis of stable proteoglycan aggregates typical of differentiated chondrocytes. Metabolic events in somites related to hyaluronic acid are not influenced by the notochord.     

Hyaluronic acid synthesis in articular cartilage: an inhibition by hydrogen peroxide

The synthesis of hyaluronic acid by bovine articular cartilage in culture was inhibited after treatment with xanthine oxidase and hypoxanthine. Through the use of catalase, superoxide dismutase and the specific iron chelator diethylenetriaminepentaacetic acid, the active species responsible for inhibition was shown to be hydrogen peroxide. Hydrogen peroxide generated by glucose oxidase was also inhibitory. Some recovery of hyaluronic synthesis was evident after a further period of culturing. Proteoglycan synthesis was inhibited in parallel with hyaluronic acid synthesis.     

Change of hyaluronic acid synthesis during differentiation of myogenic cells and its relation to transformation of myoblasts by Rous sarcoma virus

Hyaluronic acid synthesis was examined in cultures of differentiating chick embryo muscle cells before, during and after fusion. Prior to fusion, hyaluronic acid was synthesized and secreted into the medium, but once fusion began this synthesis was reduced significantly. Synthesis then increased again after completion of fusion. Thus, production of hyaluronic acid was lowest at the time of or right before cell fusion. When myoblasts were transformed by Rous sarcoma virus (RSV), a higher amount of hyaluronic acid was synthesized, and cells were not able to fuse. The turnover rate of hyaluronic acid might be different between myotubes and RSV-transformed myoblasts. The addition of exogenous hyaluronic acid to myoblast cultures resulted in the partial inhibition of fusion. The effect was reversible because fusion took place after removal of the exogenous hyaluronic acid. These observations suggest that hyaluronic acid plays an important role in the differentiation of myogenic cells, and that elevated hyaluronic acid synthesis may partly be the reason for inhibition of myotube formation upon transformation by Rous sarcoma virus.     

Genetic control of processes of bacterial interactions with plants in associations

This review is devoted to the mechanisms and genetic control of processes underlying the formation and efficiency of associative relationships between bacteria and plants. The role of different polysaccharides and cellular fibrils in the appearance of associative relations and the biosynthetic pathway of these compounds and structures is considered. The molecular mechanisms of bacterial systems responsible for stimulating plant growth and development--nitrogen fixation and synthesis of a plant hormone, indoleacetic acid--are presented. The properties of associative bacteria are discussed in comparison with the relevant characteristics of the most studied free-living or symbiotic model species of bacteria.     

Hyaluronic acid synthesis and gap junction endocytosis are necessary for normal expansion of the cumulus mass

The application of a quantitative videographic technique has provided an opportunity to compare the quantitative volumetric expansion of cultured oocyte complexes (COCs) to quantitative changes in gap junction down-regulation and hyaluronic acid synthesis and to investigate the effects of physiological agents that influence these processes. Results of these experiments support the idea that the down-regulation of cumulus gap junctions is required for the initial phase of cumulus cell disaggregation and confirm earlier reports that hyaluronic acid synthesis plays a major role in additional expansion of the cumulus. These studies also provide evidence that the degree of expansion observed in culture lacking substrates of hyaluronic synthesis is significantly attentuated when compared with expansion occurring in vivo and that the failure of cultured complexes to expand maximally can be overcome by the addition of substrates of hyaluronic acid synthesis to the culture medium.     

The role of cytokines in the immunoreactivity modulation with bacteria of the Lactobacillus genus

Mechanisms of the immunomodulating and anticancerogenic action of bacteria of the genus Lactobacillus are updated on the basis of recent publications andown research. The impact of lactobacteria on the production of cytokines by the host as one of the most important factors mediating the modulating properties of these microorganisms is considered. Information on the capacity of lactobacteria to exhibit stimulating and suppressing effects on the immune system, to maintain the development and support oral tolerance and to ensure antitumor action is presented. The role of different cell components of lactic acid bacteria in inducing the cytokines synthesis is discussed. The mechanisms of the immunomodulating action of different probiotic preparations for the prophylaxis and correction of immunodeficient states associated with the development of dysbacteriosis and acute enteric infections are analyzed. The data on the oncostatic action of bacteria of the genus Lactobacillus are presented.     

Glycosaminoglycan polysulfate-induced stimulation of hyaluronic acid synthesis in rabbit knee synovial membrane: involvement of binding protein and calcium ion

We have previously reported that naturally occurring sulfated glycosaminoglycans having a chondroitin-type structure and glycosaminoglycan polysulfate (GAGPS, a persulfated derivative of chondroitin sulfate) caused a specific stimulation of hyaluronic acid synthesis in rabbit knee synovial membranes [H. Nishikawa et al. (1985) Arch. Biochem. Biophys. 240, 146-153]. In the present study, the interaction of [3H]GAGPS and the surface of the rabbit knee synovial membranes and the relationship between this interaction and the stimulatory effect of GAGPS on the hyaluronic acid synthesis were examined in order to define the stimulatory mechanism of hyaluronic acid synthesis by GAGPS. A part of the [3H]GAGPS taken up by the synovial membranes was released from the membranes by trypsin treatment. The interaction of [3H]GAGPS and the surface of the isolated synovial membranes was diminished by pretreatment of the membranes with proteases or chelating reagents. Pretreatment of synovial membranes with trypsin or ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid had little effect on the basal hyaluronic acid synthesis but caused the loss of GAGPS-induced stimulation of hyaluronic acid synthesis accompanied by significant decrease (20% P less than 0.05-P less than 0.01) in the interaction between GAGPS and the surface of the synovial membranes. Dermatan sulfate having a chondroitin-type structure also stimulated hyaluronic acid synthesis but this effect was not additive to the stimulation of hyaluronic acid synthesis by GAGPS. Heparin had no effect on either the basal hyaluronic acid synthesis or the GAGPS-induced stimulation of hyaluronic acid synthesis. These results indicate that binding of GAGPS to certain distinct protein components on the surface of synovial membranes is involved in the stimulatory mechanism of hyaluronic acid synthesis by GAGPS, and that the binding may be mediated by Ca2+ ion. The binding was also found to be specific for sulfated glycosaminoglycans having a chondroitin-type structure.     

Molecular weight dependence of hyaluronic acid produced during oncogenic transformation

Cultured chicken embryo fibroblasts synthesize two distinct molecular size classes of hyaluronic acid. The high molecular weight material (form I, 2.98 x 10(6) is the predominant species synthesized by transformed cells, whereas form II (1.42 x 10(5)) is the major product of non-transformed cells. A shift to synthesis of predominantly form I hyaluronic acid is an early transformation event in cells infected with LA24 Rous sarcoma virus and maintained at the permissive temperature for transformation (35 degrees C). Form I hyaluronic acid exhibits greater binding to preparations of cellular fibronectin and to both normal and transformed cells than does form II. Both forms bind more to transformed cells than to normal, uninfected cells. Hyaluronic acid (predominantly form I) isolated from transforming cells stimulates proliferation in growth-retarded, non-transformed cells.     

Hormonal control of hyaluronic acid production in fibroblasts and its relation to nucleic acid and protein synthesis.

Whole serum and elevated pH previously had been found to stimulate both cell multiplication and hyaluronic acid production by chick embryo fibroblasts in culture. In a study to determine whether cell multiplication and hyaluronic acid production both respond to a single well-defined substance, insulin was found to stimulate, and cortisol to inhibit both processes coordinately. It appears, therefore, that multiplication and differentiated function in fibroblasts respond to a common underlying regulatory signal. Inhibition of ribosomal RNA synthesis by actinomycin D does not prevent serum stimulation of hyaluronic acid production, but inhibition of total RNA synthesis does. If total RNA synthesis is inhibited only after the hyaluronic acid production has reached a new high level, it continues at that level for the next five hours. The stimulatory treatment causes an increase in the activity of the enzyme hyaluronate synthetase. Inhibition of protein synthesis prevents any increase in hyaluronic acid production, and reduces the basal level of production. Reduction of the availability of Mg2+ in the medium coordinately inhibits DNA synthesis and hyaluronic acid production. The results are discussed in the light of a model for coordinate control growth and metabolism based on the availability of Mg2+.     

Influence of the cells on the pericellular environment. The effect of hyaluronic acid on proteoglycan synthesis and secretion by chondrocytes of adult cartilage.

The chondrocyte is a specialized cell that synthesizes proteoglycans of a type found only in cartilage and nucleus pulposus. These proteoglycans are distinct in forming multiple aggregates of unique structure in which hyaluronic acid provides a central chain to which many proteoglycan molecules are bound at one end only. Chondrocytes were isolated from adult cartilage and used in suspension culture to test the effect of compounds in the medium on the synthesis of proteoglycans. Hyaluronic acid alone, among a number of compounds extracted from or analogous to those in cartilage, reduced the incorporation of [35S] sulphate into macromolecular material.Oligosaccharides of hyaluronic acid of the size of decasaccharides and above also had this effect but hyaluronic acid already bound to proteoglycan did not. The proportion of total labelled material associated with the cells increased at the expense of that in the medium. Treatment of the cells with trypsin abolished the effect of hyaluronic acid but treatment with chondroitinase did not. It is suggested that hyaluronic acid interacts with proteoglycans at the cell surface by a specific mechanism similar to that involved in proteoglycan aggregation, as a result of which the secretion and synthesis of proteoglycans is reduced.     

Methylenebisphosphonic acid alters the pattern of pericellular glycosaminoglycans and binding properties of CD44 in human endothelial cells

The effect of methylenebisphosphonic acid (MBPA) on glycosaminoglycan metabolism, adhesive and proliferative properties of human endothelial cells has been investigated. It was demonstrated that MBPA (100 microM) inhibited the synthesis of all studied groups of glycosaminoglycans, but promoted the accumulation of heparan sulphate in endothelial pericellular matrix. Simultaneously, the redistribution of hyaluronic acid from pericellular matrix to the conditioned medium was observed. The decreased adhesion of endothelial cells to immobilized hyaluronic acid was not mediated by the alterations of CD44 expression. It was also demonstrated that MBPA affected the proliferative properties of endothelial cells. The alterations of glycosaminoglycan metabolism are considered to be involved in antiangiogenic effects of MBPA.     

Hyaluronic acid synthesis and secretion by rat liver fat storing cells (perisinusoidal lipocytes) in culture

The ability of rat liver fat storing cells to synthesize and to secrete hyaluronic acid was examined in monolayer cultures. The cells produce [3H] glucosamine-labeled hyaluronic acid, of which about 80% are secreted into the medium. The synthesis rate per cell (mg DNA) of labeled total glycosaminoglycans and hyaluronic acid in the medium increases significantly with culture time, but hyaluronic acid expressed as fraction of total glycosaminoglycans declines from about 0.70 in early cultures (up to the 4th day) down to 0.20 in advanced cultures. Cycloheximide increases and beta-D-xylopyranoside decreases significantly the fraction of hyaluronic acid in the medium, colchicine up to 5 microM was without effect. The synthesis of hyaluronic acid is a newly recognized function of this special type of sinusoidal liver cells. The results suggest that fat storing cells are likely to be a major source of hyaluronic acid in normal and probably also in injured liver.     

Stimulatory effect of vanadate on hyaluronic acid synthesis in mesothelial cells from rabbit pericardium

Vanadate dose-dependently stimulated the incorporation of [3H]glucosamine into glycosaminoglycan, especially hyaluronic acid, in mesothelial cells from rabbit pericardium. The activity of hyaluronic acid synthase in the mesothelial cells treated with 50 microM vanadate for 0.5-1 h was stimulated to a level about 2 times over that of the control. Neither DNA synthesis nor protein synthesis in the mesothelial cells under the same experimental conditions was affected. The enhancement of the activity of hyaluronic acid synthase in the mesothelial cells treated with vanadate (50 microM) was not inhibited by the addition of cycloheximide (1 microgram/ml). These results suggest that vanadate stimulates the hyaluronic acid synthesis by activation of hyaluronic acid synthase in mesothelial cells from rabbit pericardium.     

Biosynthesis of hyaluronic acid by Streptococcus.

Synthesis of hyaluronic acid was investigated in a cell-free system derived from a strain of Group A streptococci. Preparative procedures were improved so that an enzyme system 70 times more active than that previously reported was obtained. The hyaluronic acid synthesized could be separated into trichloroacetic acid-soluble and -insoluble fractions. On the basis of pulse-chase experiments, it was shown that the trichloroacetic acid-insoluble fraction is a precursor of the soluble fraction. The release of the trichloroacetic acid-insoluble hyaluronic acid is specifically blocked with p-chloromercuribenzoate, without inhibition of chain elongation. The addition of butanol to trichloroacetic acid resulted in solubilization of all of the hyaluronic acid. No detectable difference in molecular size was observed between the two hyaluronic acid fractions, both of which were estimated to be more than one million daltons in size. Testicular hyaluronidase digestion of either one of the two types of hyaluronic acid yielded no high molecular weight fragments, indicating that hyaluronic acid is not bound covalently to protein. However, following incubation of enzyme assay mixtures with UDP-[14C]GlcUA, even in the absence of UDP-GlcNAc, radioactive high molecular weight hyaluronic acid was obtained which suggests that the enzyme system elongates rather than initiates hyaluronic acid chains. Tunicamycin did not inhibit hyaluronic acid synthesis, indicating lack of participation of an intermediate of pyrophosphorylpolyisoprenol type. The results obtained are consistent with the hypothesis that chain elongation of hyaluronic acid proceeds by alternate addition of monosaccharides from UDP-sugars by a membrane-bound synthesizing system followed by release of completed hyaluronic acid chains.     

Mechanism of action of the migration stimulating factor produced by fetal and cancer patient fibroblasts: Effect on hyaluronic acid synthesis

Summary We have previously demonstrated that confluent fetal fibroblasts migrate into three-dimensional collagen gels to a significantly greater extent than their normal adult counterparts. Recent studies have revealed that this behavioral difference results from the secretion by fetal fibroblasts of a soluble migration-stimulating factor (MSF) which acts on these cells in an autocrine fashion. Adult fibroblasts do not produce MSF but remain responsive to it. Skin fibroblasts from cancer patients resemble fetal fibroblasts (rather than normal adult cells) with respect to their migratory behavior on collagen gels and continued production of MSF. This communication is concerned with elucidating the biochemical basis of MSF activity. Data are presented indicating that a) hyaluronic acid is required for the elevated migratory activity displayed by confluent fetal and breast cancer patient skin fibroblast; b) adult fibroblasts exhibit a bell-shaped dose-response to MSF, with maximal stimulation of migration observed at a concentration of 10 ng/ml; c) the migratory activity of adult fibroblasts pre-incubated with MSF remains high in the absence of additional factor: and d) MSF affects both the quantity and size class distribution of hyaluronic acid synthesized by adult fibroblasts. We have previously speculated that the persistent fetal-like fibroblasts of breast cancer patients play a direct role in disease pathogenesis by perturbing normal epithelial-mesenchymal interactions. The observations reported here suggest that MSF-induced alterations in hyaluronic acid synthesis may contribute to the molecular basis of such perturbations. This work was funded by grants from the Cancer Research Campaign (CRC) and Medical Research Council (MRC), London, England.     

Hyaluronic acid derivatives prepared in aqueous media by triazine-activated amidation

A method is presented for the preparation of hyaluronic acid derivatives obtained through triazine-activated amidation. A number of amines were successfully reacted with hyaluronic acid carboxyl groups using 2-chloro-4,6-dimethoxy-1,3,5-triazine as an activating species in a mixture of water and acetonitrile under neutral conditions. By varying the amount of triazine reagent, it was possible to control the degree of modification. Depending on the amine chosen, degrees of modification ranging from 3 to 20% were obtained when using 0.5 equiv of the triazine to hyaluronic acid carboxyl groups. The possibility to perform the reaction in a mixture of water and acetonitrile facilitates the introduction of a wide range of both hydrophilic and hydrophobic amines. Triazine-activated amidation appears to be a highly versatile, controllable, and relatively mild technique for modification of hyaluronic acid, and we predict that it will be useful in the design of novel hyaluronic acid based biomaterials.     

Transforming growth factor-beta 1 in adipose derived stem cells conditioned medium is a dominant paracrine mediator determines hyaluronic acid and collagen expression profile

Conditioned medium from adipose derived stem cells (ADSC-CM) stimulates both collagen synthesis and migration of fibroblasts, and accelerates wound healing in vivo. Recently, the production and secretion of growth factors has been identified as an essential function of adipose-derived stem cells (ADSCs). However, the main soluble factor of ADSC-CM which mediates paracrine effects and its underlying mechanism has not been elucidated yet. In this study, we considered transforming growth factor-beta1 (TGF-β1) as a strong candidate for paracrine effect of ADSC-CM and investigated collagen synthesis and hyaluronic acid synthase (HAS) expression. After ADSC-CM addition, collagen type I, type III, HAS and hyaluronic acid (HA) expressions on human dermal fibroblasts (HDFs) were evaluated. Furthermore, to clarify effects of TGF-β1 as a paracrine mediator, TGF-β1 antibody and external supplementary TGF-β1 were treated to HDFs. Collagens type I, type III, HAS-1 and HAS-2 mRNA expressions of HDFs were greatly increased by ADSC-CM treatment, however there was no change in TGF-β1 antibody treated HDFs compared with non-treated control. These results strongly demonstrate that TGF-β1 plays an important role as a paracrine mediator of ECM synthesis. The fact that TGF-β1 contained in ADSC-CM not only accelerates collagen deposition but also increase hyaluronic acid synthesis of HDFs through HAS-1 and HAS-2 expression was also elucidated in this study. Therefore, ADSC-CM shows promise for the treatment of cutaneous wounds and accelerates granulation formation during healing process.     

Hyaluronic acid bonded to cell culture surfaces inhibits the program of myogenesis

Primary isolates of chick leg muscle myoblasts cultured on hyaluronic acid substrates have been examined by transmission electron microscopy for evidence of myoblast fusion and subsequent differentiation. Even though these cells form close contacts, no evidence of multinucleated myotubes is found in these cultures. Two-dimensional SDS-polyacrylamide gel electrophoresis shows that the muscle macromolecular biosynthetic program is not initiated in these hyaluronic acid fusion-blocked cells. Further, these fusion-blocked myoblasts continue replicating while cultured on hyaluronic acid surfaces. The inhibition of both fusion and the myogenic expressional program is reversed by replating these myoblasts onto a denatured collagen (gelatin) substrate; both the synthesis of muscle-specific proteins and the formation of multinucleated myotubes are observed when these subcultured cells are introduced onto gelatin substrates. These observations indicate that the hyaluronic acid inhibition of fusion is not permanent and is manifested in a way different from other fusion blockers in that hyaluronic acid inhibits both fusion and the myogenic expressional program.     

调节慢性炎症防治衰老相关疾病的研究进展

衰老是应激、损伤、感染、免疫反应衰退以及代谢障碍等综合作用积累的结果。细胞在衰老的过程中会分泌大量的炎性因子。相关研究表明,老年人的皮肤及粘膜对细菌、病毒感染、外伤等等方面的抵抗和防御能力明显低于青年人,炎性因子不断增多,加速细胞衰老。由此可见,控制慢性炎症可能是干预衰老的有效途径。生物活性透明质酸可能通过诱导防卫素分泌和与CD44/TLR4受体结合,抑制细菌生长和炎症进展,从而发挥抗衰老作用。本文对有关慢性炎症和衰老之间的关系的研究进行综述,探讨生物活性透明质酸对慢性炎症的调节作用及其抗衰老机制。     

Influences of sulfated glycosaminoglycans on biosynthesis of hyaluronic acid in rabbit knee synovial membrane

The effect of various sulfated glycosaminoglycans on glycoconjugates syntheses in synovial membranes of rabbit knee joints in culture was investigated by two different approaches. In the first approach, synovial membranes isolated from rabbit knee joints were cultured in the presence of sulfated glycosaminoglycans and [14C]glucosamine. In the second approach, solutions of sulfated glycosaminoglycans were injected into rabbit knee joints and synovial membranes isolated from the joints were cultured in the presence of [14C]glucosamine. The major part of [14C]glucosamine-labeled glycoconjugates associated with the synovial membranes and secreted into culture medium was hyaluronic acid. Of the natural glycosaminoglycans tested, dermatan sulfate gave the maximum stimulation of hyaluronic acid synthesis followed by chondroitin 4- and 6-sulfate. Heparin, heparan sulfate, keratan sulfate, keratan polysulfate, and hyaluronic acid had no significant effect. Of the chemically polysulfated glycosaminoglycans, GAGPS (a persulfated derivative of chondroitin sulfate) gave high stimulation but N-acetylchitosan 3,6-disulfate had no effect. The effect of sulfated glycosaminoglycans on hyaluronic acid synthesis was the same in both experimental approaches. The increase in the amount of secreted hyaluronic acid in culture medium paralleled that in synovial membranes. The results indicate that the galactosamine-containing sulfated glycosaminoglycans have a specific stimulatory effect on hyaluronic acid synthesis. A high degree of sulfation of the molecules appeared to potentiate the stimulatory effect.