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 UDPGlcNAc 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 ؋ 10 5 to 1 ؋ 10 6 Da, shorter than those synthesized by HAS1 and HAS2 which have molecular masses of 2 ؋ 10 5 to ϳ2 ؋ 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 ؋ 10 5 to ϳ2 ؋ 10 6 Da), whereas HAS2 generated hyaluronan with a broad but extremely large size (average molecular mass of >2 ؋ 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. Hyaluronan (HA)1 is a major component of most extracellular matrices, particularly in tissues with rapid cell proliferation and cell migration (1). The interaction of HA with various HA-binding proteins and cell-surface receptors plays important roles in regulating fundamental cell behaviors such as cell adhesion, migration, and differentiation (2, 3). Thus, HA has been greatly implicated in morphogenesis, regeneration, wound healing, tumor invasion, and cancer metastasis (4 -6). In addition, HA is an important structural molecule required for the maintenance of various aspects of tissue architecture and function. The physical and biological properties of HA appear to be affected by many factors including HA concentration and chain length. Indeed, high molecular weight HA at high concentrations suppresses endothelial cell growth, whereas low molecular weight HA stimulated cell growth leading to induction of angiogenesis (7). In addition, viscosity of the HA gel and the ability to hydrate large amounts of water were shown to be dependent on the molecular size of the HA chain.HA is a high molecular weight linear polymer composed of GlcUA ␤-1,3-GlcNAc ␤-1,4 disaccharide units and is synthesized by HA synthase at the inner face of the plasma membrane (8). Although a great deal of effort has been made to elucidate the mechanism of HA biosynthesis in mammalian cells, it has remained unclear due to difficulty in biochemical isolation of the active enzyme (9 -11). Recently, three distinct yet highly conserved genes encoding mammali...

Elevated hyaluronan biosynthesis and matrix deposition correlates with cell proliferation and migration. We ectopically expressed three isoforms of hyaluronan synthase (HAS1, HAS2, or HAS3) in nontransformed rat 3Y1 cells and observed a de novo, massive formation of a hyaluronan matrix that resulted in a partial loss of contact-mediated inhibition of cell growth and migration. All three HAS transfectants showed an enhanced motility in scratch wound assays, and a significant increase in their confluent cell densities. In high-density cultures, the HAS transfectants had a fibroblastic cell shape and markedly formed overlapping cell layers. This phenotype was more pronounced in the HAS2 transfectants than HAS1 or HAS3 transfectants, and occurred with significant alterations in the microfilament organization and N-cadherin distribution at the cell-cell border. Inhibition of a phosphatidylinositol 3-kinase (PI3-kinase) pathway resulted in reacquisition of the normal phenotype of HAS2 transfectants, suggesting that the intracellular PI3-kinase signaling regulates diminution of contact inhibition induced by formation of the massive hyaluronan matrix. Our observations suggest that hyaluronan and its matrix can modulate contact inhibition of cell growth and migration, and provide evidence for functional differences between hyaluronan synthesized by the different HAS proteins.H yaluronan (HA) is a linear polysaccharide composed of a, and is widely present as a major component of extracellular matrix in most vertebrate tissues (1, 2). HA is synthesized by three HA synthase isoforms: HAS1, HAS2, and HAS3 (3, 4). The three HAS genes have distinct expression patterns during mouse development (5), and their products have significantly different enzymatic properties and roles in the formation of the HA matrix (6).HA synthesis and matrix formation may have a role in the development, progression and pathogenesis of cancer. For example, the rate of HA synthesis is enormously increased when oncogenic viruses transform fibroblasts (7,8), and elevated levels of HA are associated with the hyperproliferative and malignant phenotypes in melanomas and various carcinomas (9-11).Studies done with cultured cancer cells showed that overproduction of HA enhances their anchorage-independent growth, tumorigenicity, and metastatic potential (12, 13), suggesting an important role for HA in tumor growth and malignant progression. However, it was unclear whether the overproduction could induce a malignant transformation of nontransformed cells.We overexpressed the three HAS isoforms to test the ability of HA to transform nontransformed cells. We also investigated whether the three HAS isoforms are functionally distinct in nontransformed cells. Materials and MethodsCell Culture and Transfection. 3Y1 1-B6 cells were obtained from Riken Cell Bank (Tsukuba, Japan). The 3Y1 cells were cultured in DMEM containing 10% FCS and 2 mM L-glutamine (growth medium). Stable transfectants were established as previously described (6) and were routinely cultured in ...

Malignant transformation of fibroblasts and epithelial cells is often accompanied by increased hyaluronan production and accumulation. Despite recent progress in the study of hyaluronan biosynthesis, the mechanisms underlying the transformation-induced overproduction of hyaluronan have not been elucidated. Here we report that activity and transcriptional levels of hyaluronan synthase (HAS) significantly increased after oncogenic malignant transformation of a rat 3Y1 fibroblast cell line. Of three HAS isoforms (HAS1, HAS2, and HAS3), only HAS2 gene expression was increased in the v-Ha-ras transformed 3Y1 cells, which show less malignancy. In contrast, both HAS1 and HAS2 expressions were elevated in the highly malignant cells transformed with v-src and/or v-fos. To assess the contribution of HAS expression to the oncogenic malignant transformation, we established stable cell transfectants expressing sense and antisense HAS genes. Antisense suppression of the HAS2 expression significantly decreased hyaluronan production in the cells transformed by the oncogenic v-Ha-ras and eventually led to a reduction in tumorigenicity in the rat peritoneum. The introduction of the HAS1 and HAS2 genes promoted the growth of subcutaneous tumors in a manner dependent on the levels of hyaluronan synthesis. Significant growth promotion was observed within a wide range of HAS1 expression. In contrast, the growth stimulation was only seen within a narrow range of HAS2 expression, and high levels of HAS2 expression even inhibited tumor growth. These results suggest that proper regulation of the expression of each HAS isoform is required for optimal malignant transformation and tumor growth.

Blood eosinophil levels were examined in 200 (100 mild and 100 severe) patients with atopic dermatitis. Eosinophil levels roughly correlated with disease severity. However, the pattern of eosinophilia was not homogeneous. Very high eosinophil counts were common in severe cases of atopic dermatitis who had a personal or family history of respiratory atopy, while normal or moderately elevated eosinophil values were obtained in severe cases of 'pure' atopic dermatitis who had neither personal nor family history of respiratory atopy. It is suggested that disease severity and personal or family history of respiratory atopy are important factors in determining high blood eosinophil levels in atopic dermatitis.

Sensitization trials with dinitrochlorobenzene (DNCB) were performed in 150 patients with atopic dermatitis (AD) (24 severe, 86 moderate, and 40 mild cases). Dinitrochlorobenzene challenge tests were positive in 33% (8/24) of severe cases, in 95% (82/86) of moderate cases, and in 100% (40/40) of mild cases, indicating that DNCB contact sensitivity is diminished only in patients with AD who have extensive skin lesions. Of the 20 patients who were nonreactive on the first DNCB challenge tests, 18 became reactive on the second challenge tests that were performed at the time when the dermatitis was well controlled. It is likely that the suppressed contact sensitivity seen in patients with AD is secondary to the disease.

Hyaluronate plays a unique role in the cancer cell microenvironment. In particular, melanoma is the tumor type in which hyaluronate and hyaluronate recognition have been most closely linked to malignancy. In this study we show that a human melanoma cell line stably transfected with hyaluronate synthase cDNA displays enhanced motility. We used a fixed erythrocyte exclusion assay to isolate subsets of the WM793 human melanoma cell line that expressed either high or low amounts of hyaluronate. A cell line with a high level of hyaluronate on its surface (WM793H) displayed significantly higher cell motility on colloidal-gold-coated coverslips than did a line with a low level (WM793L). Next, in order to directly investigate the effects of hyaluronate on melanoma cell migration, we transfected cDNA encoding mouse hyaluronate synthase HAS1 or HAS2 into the re-cloned human melanoma cell line that produced a low amount of hyaluronate (WM793L) by the lipofection method. Several clonal transfectants differentially producing hyaluronate were obtained. There was a positive correlation between total hyaluronate synthesis and formation of the pericellular hyaluronate-rich matrix. We observed an increase in the migration ability of hyaluronate cDNA (HAS1 or HAS2)-transfected cells compared with control cells on glass plates covered with colloidal gold particles. A migration-inhibition assay with anti-CD44 monoclonal antibody showed blocking of the cell motility. It is speculated that the tumor cells might migrate through a hyaluronate-rich extracellular environment when they invade nearby host tissues and that hyaluronate production by the tumor cells could increase this migration. These results suggest that hyaluronate may play a role in the aggressiveness of human melanoma cells.

In order to minimize the damage from viral epidemics, early detection of the causative agent of a viral epidemic and prevention of its immediate spread are urgent social demands. Therefore, in this study, we evaluated the utility of a Mach-Zehnder-type optical waveguide as a sensing device for influenza virus detection. However, it is impossible to detect a 100-nm-size virus using a sol-gel optical biosensor because sol-gel glass has a pore size of only a few nanometers, which makes it impossible for the virus to diffuse into the silica thin film. In order to construct the influenza-specific Mach-Zehnder optical biosensor for influenza detection, a stable antibody immobilization method with resulting high density on the sol-gel surface is strongly required. In this study, the sol-gel glass surface was modified with amino and carboxyl groups, and an anti-H1N1/HA1 antibody was covalently immobilized using a cross-linking agent. We successfully prepared a carboxyl-modified sol-gel surface, using NHS/EDC as the cross-linker, for antibody immobilization, and confirmed the detection of influenza virus using the antibody-immobilized sol-gel glass. After treatment with a 100 μg/mL influenza virus solution for 15 min, a peak wavelength shift (~24 nm) was observed in the output light spectrum.

To ascertain the prevalence of childhood and adolescent atopic dermatitis in a Japanese population, we clinically observed the total body of 5 to 6-year-old children (994 cases), 7 to 9-year-old children (1,240 cases), 10 to 12-year-old children (1,152 cases), 13 to 15-year-old children (1,670 cases), and 16 to 18-year-old adolescents (2,159 cases). The examination was performed in the spring of 1994-96, when exacerbation of childhood and adolescent atopic dermatitis most frequently occurs in Japan. Atopic dermatitis was observed in 24% of the 5 to 6-year group, in 19% of the 7 to 9-year group, in 15% of the 10 to 12-year group, in 14% of the 13 to 15-year group, and in 11% of the 16 to 18-year group. The prevalence of atopic dermatitis in 9 to 12-year-old children was two times and in 18-year-old adolescents five times as high as in similar age groups examined 20 years ago.