Laser light scattering measurements on vitreous and rooster comb hyaluronic acids

Molecular weights and translational diffusion coefficients have been measured for rooster comb and vitreous hyaluronic acid (HA) at pH 7.2 and 11. The results indicate that the molecular weight, second virial coefficient and translational diffusion coefficient for vitreous HA can be reversibly decreased by increasing the solution pH from 7.2 to 11, whereas the physical properties of rooster comb HA are independent of pH studied. In addition, it is reported that the second virial coefficient for vitreous HA is negative, suggesting intermolecular interactions exist in solution at both neutral and alkaline pH as opposed to rooster comb HA which exhibits a positive second vitrial coefficient associated with decreasing molecular weights may be related to the accessibility and number of hydrogen bond forming groups. Differences in the dependence of molecular weight on pH between vitreous and rooster comb HA may be due to differences in the number of intramolecular interactions per molecule. These studies indicate that molecules of low molecular weight HA are able to form higher molecular weight complexes and differences in the organization of the polysaccharide chains may contribute to the differences in molecular weight of HAs isolated from various tissues.     

Substrate-bonded hyaluronic acid exhibits a size-dependent stimulation of chondrogenic differentiation of stage 24 limb mesenchymal cells in culture

Extracellular matrix molecules including glycosaminoglycans have been implicated in several differentiative and morphogenetic processes including cell aggregation and migration. Previous reports have shown that plating of stage 24 limb mesenchyme cells onto hyaluronic acid (HA) bonded to the culture substrate causes an increase in the number of cells exhibiting chondrogenesis. This increased chondrogenesis is now shown to be dependent upon the source of the HA. When limb mesenchymal cells are plated onto HA from bovine vitreous humor, human umbilical cord, or large molecular weight HA (Healon), increased chondrogenesis is observed only on the bovine vitreous humor HA. Unsulfated chondroitin, which has a structure and charge density similar to those of HA, is capable of enhancing chondrogenesis, while cells plated onto sulfated glycosaminoglycan substrates are indistinguishable from controls. The evidence in this report suggests that the differentiation response is related to the molecular size of the HA bound to the culture substrate. Healon and human umbilical cord HA are ineffective because their molecular weight is too large, while smaller HA derived from these larger molecules or normally present in bovine vitreous humor preparations stimulates the chondrogenic differentiation of stage 24 limb mesenchymal cells in culture. The most active size class of HA elutes from a Sepharose CL-2B column with a Kav between 0.6 and 0.7 and, thus, has a molecular weight of approximately 200,000-400,000. These observations reinforce the hypothesis that local cues have an informational effect on the differentiation of chick limb mesenchymal cells.     

Monitoring in situ circular dichroism of the intact vitreous: a new approach

For the first time, an attempt has been made to study the vitreous humor in situ by circular dichroism (CD). The vitreous, an avascular and acellular gel-like tissue, is optically transparent and homogeneous, and, thus, light scattering is minimal. The macromolecular components of this tissue, hyaluronate (HA), collagen and noncollagenous protein (NC-P), appear to exist in the matrix in a nonoriented fashion. As a result, no linear dichroism was observed. A typical CD of the vitreous shows a minimum at 206 nm with a shoulder at 220 nm and one small positive peak at approximately 252 nm. Gaussian analysis resolves this spectrum into four component bands. CD analysis of individual components reveals that NC-P makes the major contribution to the dichroic strength of the vitreous; contributions of HA and collagen, on the other hand, are small. The positive peak arises largely from ascorbic acid in the vitreous. CD measurement of the intact vitreous appears to be a useful technique for assessing the structure and changes of the constituent molecules in the normal and diseased vitreous.     

A putative homoeolog of human chromosome 12 in the owl monkey

Analyses of Southern blots of rodent x owl monkey somatic cell hybrids permitted syntenic assignment of gene loci coding for triosephosphate isomerase (TPI), antigen CD4(T4), Kirsten rat sarcoma 2(KRAS2) virus, insulin-like growth factor 1 (IGF1), and alpha 2-macroglobulin (A2M) to chromosome 10 of owl monkey karyotype VI(2n = 49, 50). In addition, regional in situ localization of the T4 and KRAS2 loci on the proximal region of the long arm of this acrocentric chromosome and on the corresponding homologous region on the long arm of metacentric chromosome 1 of karyotype IV (2n = 52) substantiated our hypothesis that a single fusion or fission event is responsible for the polymorphism in chromosome number characteristic of owl monkeys from at least three allopatric populations. The study supports a putative homoeology between owl monkey chromosome 10 (K-VI) and human chromosome 12. The morphological differences between these two primate chromosomes indicate evolutionary rearrangements involving at least one pericentric inversion.     

CD44 stimulation by fragmented hyaluronic acid induces upregulation and tyrosine phosphorylation of c-Met receptor protein in human chondrosarcoma cells

Hepatocyte growth factor/scatter factor (HGF/SF) can induce proliferation and motility and promote invasion of tumor cells. Since HGF/SF receptor, c-Met, is expressed by tumor cells, and since stimulation of CD44, a transmembrane glycoprotein known to bind hyaluronic acid (HA) in its extracellular domain, is involved in activation of c-Met, we have studied the effects of CD44 stimulation by ligation with HA upon the expression and tyrosine phosphorylation of c-Met on human chondrosarcoma cell line HCS-2/8. The current study indicates that (a) CD44 stimulation by fragmented HA upregulates expression of c-Met proteins; (b) fragmented HA also induces tyrosine phosphorylation of c-Met protein within 30 min, an early event in this pathway as shown by the early time course of stimulation; (c) the effects of HA fragments are critically HA size-dependent. High molecular weight HA is inactive, but lower molecular weight fragments (M(r) 3.5 kDa) are active with maximal effect in the microg/ml range; (d) the standard form of CD44 (CD44s) is critical for the response because the effect on c-Met, both in terms of upregulation and phosphorylation, is inhibited by preincubation with an anti-CD44 monoclonal antibody; and (e) phosphorylation of c-Met induced by CD44 stimulation is inhibited by protein tyrosine kinase inhibitor, tyrphostin. Therefore, our study represents the first report that CD44 stimulation induced by fragmented HA enhances c-Met expression and tyrosine phosphorylation in human chondrosarcoma cells. Taken together, these studies establish a signal transduction cascade or cross-talk emanating from CD44 to c-Met.     

Embryotropic effect of glycosaminoglycans and receptors in development of porcine pre-implantation embryos

The present study investigated the expression of receptors for glycosaminoglycans (GAGs) and the effect of GAGs supplementation on development of porcine IVF embryos. Total RNA was prepared from oocytes, 2-, 4- and 8-cell embryos, morulae and blastocysts. The expression of hyaluronic acid receptor (CD44) and heparin (HP) interacting protein (HIP) was determined using RT-PCR and Southern blot analysis. The CD44 and HIP mRNA were detected from in vitro matured oocytes and all stages of pre-implantation embryos. The IVF embryos were cultured in modified NCSU-23 medium supplemented with various concentrations (0, 0.1, 0.5 or 1.0 mg/mL) of hyaluronic acid (HA) or heparin. Supplementing with 0.5 mg/mL HA significantly increased total cell number compared to other experimental groups, due to increase in trophectoderm cells. Supplementing with 1.0 mg/mL, HP significantly increased blastocyst formation rate compared to the control group. Supplementing media, in which IVF embryos were cultured with 0.5 mg/mL HA + 1.0 mg/mL HP, significantly increased blastocyst formation rate compared to the control group. In conclusion, the present study demonstrated the expression of HA and HP receptors and the embryotrophic effect of HA or HP on porcine IVF embryos.     

Histological grade in breast cancer: association with clinical and biological features in a series of 229 patients

In order to study the association of histological grade (HG) with specific clinical and biological parameters which may influence the clinical behavior of infiltrating ductal carcinomas of the breast (IDC), we analyzed in 229 tissue samples the cytosolic concentrations of estrogen receptor (ER), progesterone receptor (PR), pS2, cathepsin D, hyaluronic acid (HA) and tissue-type plasminogen activator (t-PA), as well as those of the erbB2 oncoprotein, epidermal growth factor receptor (EGFR), HA, CD44v5 and CD44v6 in the cell membrane fraction. Likewise, we considered size, ploidy, S-phase fraction and axillary node involvement as variables of the study. The transition from HG1 to HG2 and from HG2 to HG3 was accompanied by a number of common features: global increase in size, greater number of tumors >2.0 cm, decrease in membrane hyaluronic acid concentrations, increased cell proliferation (S-phase >7%) and greater aneuploidy. Other events observed during the transition from HG2 to HG3 were a decrease in ER, PR, t-PA and cytosolic hyaluronic acid. These results led us to consider that HG is associated with certain clinical-biological changes that may help explain its value as a prognostic factor in breast carcinomas.     

Interaction of CD44 with different forms of hyaluronic acid. Its role in adhesion and migration of tumor cells

Interaction between hyaluronic acid (HA) and CD44 has been considered a key event in tumor invasion and metastasis. HA is a linear, high molecular weight glycosaminoglycan in its native state, but fragmented low molecular forms are found at sites of neoplastic or inflammatory infiltrates. Both high and low molecular weights HA are involved in diverse biological functions. In this study, we used two clonal variants of a T cell murine lymphoma designated LBLa and LBLc. These cell lines were found to differ in their in vivo and in vitro growth rates. LBLa grew faster and exhibited an enhanced invasive capacity as compared to LBLc. In contrast, cell lines did not differ in the expression of surface markers (CD8, CD24, CD25, CD44, and CD18), or in their capacity to bind HA. However, LBLa cells exhibited higher capacity to migrate to low molecular weight HA than did LBLc. Migration was mediated by CD44 since it was abrogated by anti-CD44 monoclonal antibody as well as by hyaluronidase. We suggest that interaction between CD44 and low molecular weight HA may trigger migration mechanisms in LBLa cells, thus contributing to enhanced invasive cell capacity.     

Hyaluronic acid (HA) binding to CD44 activates Rac1 and induces lamellipodia outgrowth

Both cell adhesion protein CD44 and its main ligand hyaluronic acid (HA) are thought to be involved in several processes ultimately requiring cytoskeleton rearrangements. Here, we show that the small guanine nucleotide (GTP)-binding protein, Rac1, can be activated upon HA binding to CD44. When applied locally to a passive cell edge, HA promoted the formation of lamellipodial protrusions in the direction of the stimulus. This process was inhibited by the prior injection of cells with dominant-negative N17Rac recombinant protein or by pretreatment of cells with monoclonal anti-CD44 antibodies, interfering with HA binding, implying the direct involvement of CD44 in signaling to Rac1.     

Studies on the affinity of hyaluronic acid binding protein to glycosaminoglycans

The affinity of hyaluronic acid binding protein (HBP) to different glycosaminoglycans (GAGs) was examined. The purified protein was pretreated with hyaluronic acid (HA), heparin, glucuronic acid and N-Acetyl-glucosamine and was loaded onto Hyaluronate-Sepharose affinity column. The binding of HBP to HA immobilized on sepharose column was specifically blocked only by pretreatment of HBP to HA and the elution of HBP was decreased proportionately with the addition of higher quantity of HBP. The specificity of HBP to HA was confirmed as it did not bind to Heparin-Sepharose or Chondroitin-4-Sulphate-Sepharose columns. The complex of HBP in association with HA was further shown on Sephadex G-200 and 7.5% polyacrylamide gel. All the experimental findings indicate that HBP binds specifically to HA only.     

Coelomocyte locomotion in the sipunculan Themiste petricola induced by exogenous and endogenous chemoattractants: role of a CD44-like antigen--HA interaction

Cell migration is a key event in the invertebrate immuno-defense system. Microbial products like lipopolysacharide (LPS) and formyl-methyl-leucyl-phenylalanine (fMLP) promote cell recruitment to sites of infection. In mammals, complement activation by factors such as zymosan induces C5a production, which influences leukocyte migration. The endogenous factor hyaluronic acid (HA), an extracellular matrix component, also promotes cell migration through its receptor CD44. We evaluated whether coelomocytes from the sipunculan worm T. petricola migrated towards LPS, fMLP, or zymosan treated plasma (ZTP) and if HA was involved in coelomocyte migration and adhesion. We also evaluated if antibodies specific for mouse HA receptor CD44 inhibited any of the effects induced by HA. Using microchemotaxis chambers we found that coelomocytes migrated towards exogenously and endogenously derived chemoattractants. We also observed that HA was a potent chemotactic signal and that coelomocytes adhered strongly to plates coated with LMW-HA but not with HMW-HA. In addition we found that these HA mediated effects were blocked by the monoclonal antibody IM7 directed to mouse CD44, suggesting that a CD44-like cross-reactive antigen might play a role in HA mediated coelomocyte locomotion.     

The CD44 receptor of lymphoma cells: structure-function relationships and mechanism of activation

Migration of some tumor cells, and their lodgment in target organs, is dependent on the activation of cell surface CD44 receptor, usually detected by its ability to bind hyaluronic acid (HA) or other ligands. In an attempt to reveal the mechanism of tumor cell CD44 activation, we compared the physical and chemical properties of CD44 in nonactivated LB cell lymphoma with those in phorbol 12-myristate 13-acetate (PMA)-activated LB cells and of an LB cell subline (designated HA9) expressing constitutively-active CD44. In contrast to nonactivated LB cells, PMA-activated LB cells and HA9 cells displayed a CD44-dependent ability to bind HA. The ability of activated cell CD44 to bind HA was not dependent on microfilament or microtubule integrity or on changes in CD44 mobility on the membrane plane, indicating that the CD44 activation status is not associated with cytoskeleton function. Aside from the increased expression of CD44 on the surface of PMA-activated LB cells and HA9 cells, qualitative differences between the CD44 of nonactivated and activated LB cells were also detected: the CD44 of the activated lymphoma was (i) larger in molecular size, (ii) displayed a broader CD44 isoform repertoire, including a CD44 variant that binds HA, and (iii) its glycoprotein contained less sialic acid. Indeed, after removal of sialic acid from their cell surface by neuraminidase, LB cells acquired the ability to bind HA. However, a reduced dose of neuraminidase did not confer HA binding on LB cells, unless they were also activated by a low concentration of PMA, which by itself was ineffective. Similarly, under suboptimal conditions, a synergistic effect was obtained with tunicamycin and PMA: each one alone was ineffective but in combination they induced the acquisition of HA binding by the lymphoma cells, while their CD44 expression was not enhanced. Unveiling of the activation mechanism of CD44, by exposing the cells to PMA stimulation or to deglycosylation, is not only academically important, but it also has practical implications, as activated CD44 may be involved in the support of tumor progression.     

The CD44 Receptor of Lymphoma Cells: Structure-Function Relationships and Mechanism of Activation

Migration of some tumor cells, and their lodgment in target organs, is dependent on the activation of cell surface CD44 receptor, usually detected by its ability to bind hyaluronic acid (HA) or other ligands. In an attempt to reveal the mechanism of tumor cell CD44 activation, we compared the physical and chemical properties of CD44 in nonactivated LB cell lymphoma with those in phorbol 12-myristate 13-acetate (PMA)-activated LB cells and of an LB cell subline (designated HA9) expressing constitutively-active CD44. In contrast to nonactivated LB cells, PMA-activated LB cells and HA9 cells displayed a CD44-dependent ability to bind HA. The ability of activated cell CD44 to bind HA was not dependent on microfilament or microtubule integrity or on changes in CD44 mobility on the membrane plane, indicating that the CD44 activation status is not associated with cytoskeleton function. Aside from the increased expression of CD44 on the surface of PMA-activated LB cells and HA9 cells, qualitative differences between the CD44 of nonactivated and activated LB cells were also detected: the CD44 of the activated lymphoma was (i) larger in molecular size, (ii) displayed a broader CD44 isoform repertoire, including a CD44 variant that binds HA, and (iii) its glycoprotein contained less sialic acid. Indeed, after removal of sialic acid from their cell surface by neuraminidase, LB cells acquired the ability to bind HA. However, a reduced dose of neuraminidase did not confer HA binding on LB cells, unless they were also activated by a low concentration of PMA, which by itself was ineffective. Similarly, under suboptimal conditions, a synergistic effect was obtained with tunicamycin and PMA: each one alone was ineffective but in combination they induced the acquisition of HA binding by the lymphoma cells, while their CD44 expression was not enhanced. Unveiling of the activation mechanism of CD44, by exposing the cells to PMA stimulation or to deglycosylation, is not only academically important, but it also has practical implications, as activated CD44 may be involved in the support of tumor progression.     

Aggravation of Allergic Airway Inflammation by Cigarette Smoke in Mice Is CD44-Dependent

Background

Although epidemiological studies reveal that cigarette smoke (CS) facilitates the development and exacerbation of allergic asthma, these studies offer limited information on the mechanisms involved. The transmembrane glycoprotein CD44 is involved in cell adhesion and acts as a receptor for hyaluronic acid and osteopontin. We aimed to investigate the role of CD44 in a murine model of CS-facilitated allergic airway inflammation.

Methods

Wild type (WT) and CD44 knock-out (KO) mice were exposed simultaneously to house dust mite (HDM) extract and CS. Inflammatory cells, hyaluronic acid (HA) and osteopontin (OPN) levels were measured in bronchoalveolar lavage fluid (BALF). Proinflammatory mediators, goblet cell metaplasia and peribronchial eosinophilia were assessed in lung tissue. T-helper (Th) 1, Th2 and Th17 cytokine production was evaluated in mediastinal lymph node cultures.

Results

In WT mice, combined HDM/CS exposure increased the number of inflammatory cells and the levels of HA and OPN in BALF and Th2 cytokine production in mediastinal lymph nodes compared to control groups exposed to phosphate buffered saline (PBS)/CS, HDM/Air or PBS/Air. Furthermore, HDM/CS exposure significantly increased goblet cell metaplasia, peribronchial eosinophilia and inflammatory mediators in the lung. CD44 KO mice exposed to HDM/CS had significantly fewer inflammatory cells in BALF, an attenuated Th2 cytokine production, as well as decreased goblet cells and peribronchial eosinophils compared to WT mice. In contrast, the levels of inflammatory mediators were similar or higher than in WT mice.

Conclusion

We demonstrate for the first time that the aggravation of pulmonary inflammation upon combined exposure to allergen and an environmental pollutant is CD44-dependent. Data from this murine model of concomitant exposure to CS and HDM might be of importance for smoking allergic asthmatics.     

Chromosome assignment of the gene loci ETS1 and THY1 in the owl monkey

Our previous assignment of the gene loci HBB, HRAS1, INS, PTH, LDHA, and CAT to owl monkey chromosome 19 of karyotype VI (K-VI) indicated a putative homology of this owl monkey chromosome with the short arm of human chromosome 11 (HSA 11p). To investigate further the extent of shared homology, we localized in the owl monkey complement two genes known to be on HSA 11q. Segregation analysis of ETS1 and THY1 homologous DNA in three karyotypically different panels of rodent x owl monkey somatic cell hybrids provided evidence for the syntenic assignment of these loci to homologous chromosomes of three owl monkey karyotypes, namely, chromosomes 4 (K-VI), 3 (K-II), and 5 (K-V). The results indicate a disruption of syntenic gene loci on the distal portion of HSA 11q from 11p during primate evolution.     

PARTICLE INTERACTION IN SOLUTIONS DERIVED FROM OX VITREOUS HUMOR

Solutions made by homogenization of vitreous humor exhibit anomalous viscosity at low shear rates due to interaction between hyaluronic acid and a hydroxyproline-containing material similar to collagen. The latter can be removed by filtration or centrifugation to give a solution whose viscosity is due to hyaluronic acid. On concentration the viscosity of the latter remains independent of shear rate and it is concluded that hyaluronic acid in the vitreous humor is not greatly asymmetric. There is no evidence that the soluble protein of vitreous humor contributes to the viscosity. Hyaluronic acid in vitreous humor is not in chemical combination with protein. The interaction between the hyaluronic acid and the collagen-like material is ascribed to weak, coulombic forces.     

金黄色葡萄球菌透明质酸裂解酶HylS的异源表达与特性研究

透明质酸酶能够将透明质酸聚糖降解成具有抗氧化等生物活性的低分子量寡糖.微生物来源透明质酸酶具有酶学性质多样和易于重组表达等特点,是开发透明质酸酶的研究热点.通过基因组测序获得一个潜在的金黄色葡萄球菌来源透明质酸裂解酶基因hylS,将其进行了大肠杆菌BL21(DE3)异源重组表达,并对重组酶进行了酶学特性和酶解产物抗氧化...     

CD44S-hyaluronan interactions protect cells resulting from EMT against anoikis

The detachment of normal epithelial cells from matrix triggers an apoptotic response known as anoikis, during homeostatic turnover. Metastatic tumor cells evade anoikis, by mechanisms that are only partly characterized. In particular, the epithelial–mesenchymal transition (EMT) in a subset of invasive tumor cells confers anoikis-resistance. In some cases, EMT up-regulates the cancer stem cell marker CD44S and the enzyme hyaluronic acid synthase-2 (HAS2). CD44S is the major receptor for hyaluronan in the extracellular matrix. Herein, we demonstrate that CD44S, unlike the CD44E isoform expressed in normal epithelial cells, contributes to the protection against anoikis. This protection requires the interaction of CD44S with hyaluronan (HA). CD44S–HA interaction is proposed to play an important role in tumor metastasis through enhanced cell survival under detached conditions.     

Inflammatory cytokines can enhance CD44-mediated airway epithelial cell adhesion independently of CD44 expression

In airways, the cell surface molecule CD44 is upregulated on bronchial epithelial cells in areas of damage. We have shown that a blocking standard CD44 (CD44s) antibody caused a 77% (+/- 19%) inhibition of cell migration at 3 h after mechanical damage and decreased epithelial cell repair of cells grown on cell culture filter inserts. With the use of primary human bronchial epithelial cells and the bronchial epithelial cell line 16HBE 14o-, a CD44s antibody inhibited >95% (P < 0.01) of cell binding to hyaluronic acid (HA). The cytokines TNF-alpha, IFN-gamma, IL-1 beta, and IL-4 stimulated a 2- to 3.5-fold increase in CD44-dependent cell binding to HA. IFN-gamma treatment did not increase CD44 expression as assessed by flow cytometry, although phorbol myristate acetate treatment did. This indicates that IFN-gamma-induced cell binding to HA did not require increased CD44 expression. These data indicate that CD44 is important for bronchial epithelial cell binding to HA and that cytokines known to be expressed in inflammation can increase HA binding independently of the level of CD44 expression.     

<Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">Ex Vivo</Emphasis> Evaluation of Novel Curcumin-Loaded Excipient for Buccal Delivery

This study aimed to develop a mucoadhesive polymeric excipient comprising curcumin for buccal delivery. Curcumin encompasses broad range of benefits such as antioxidant, anti-inflammatory, and chemotherapeutic activity. Hyaluronic acid (HA) as polymeric excipient was modified by immobilization of thiol bearing ligands. L-Cysteine (SH) ethyl ester was covalently attached via amide bond formation between cysteine and the carboxylic moiety of hyaluronic acid. Succeeded synthesis was proved by H-NMR and IR spectra. The obtained thiolated polymer hyaluronic acid ethyl ester (HA-SH) was evaluated in terms of stability, safety, mucoadhesiveness, drug release, and permeation-enhancing properties. HA-SH showed 2.75-fold higher swelling capacity over time in comparison to unmodified polymer. Furthermore, mucoadhesion increased 3.4-fold in case of HA-SH and drug release was increased 1.6-fold versus HA control, respectively. Curcumin-loaded HA-SH exhibits a 4.4-fold higher permeation compared with respective HA. Taking these outcomes in consideration, novel curcumin-loaded excipient, namely thiolated hyaluronic acid ethyl ester appears as promising tool for pharyngeal diseases.