Hyaluronic acid (HA) is a high molecular weight glycosaminoglycan involved in a wide variety of cellular functions. However, its turnover in living cells remains largely unknown. In this study, CD44, a receptor for HA, and hyaluronidase-1, -2, and -3 (Hyal-1, -2 and -3) were stably expressed in HEK 293 cells and the mechanism of HA catabolism was systematically investigated using fluorescein-labeled HA. CD44 was essential for HA degradation by both endogenous and exogenously expressed hyaluronidases. Hyal-1 was not able to cleave HA in living cells in the absence of CD44. Intracellular HA degradation was predominantly mediated by Hyal-1 after incorporation of HA by CD44. Although Hyal-1 was active only in intracellular space in vivo, a certain amount of the enzyme was secreted to extracellular space. This extracellular Hyal-1 was found to be incorporated by cells and such uptake of Hyal-1 was, in part, involved in the intracellular degradation of HA. Hyal-2 was involved in the extracellular degradation of HA. Hyal-2 activity was also dependent on the expression of CD44 in both living cells and enzyme assays. Immunofluorescent microscopy demonstrated that both Hyal-2 and CD44 are present on the cell surface. Without CD44 expression, Hyal-2 existed in a granular pattern, and did not show hyaluronidase activity, suggesting that localization change could contribute to Hyal-2 function. A convenient and quantitative enzyme assay was established for the measurement of Hyal-2 activity. Hyal-2 activity was detected in the membrane fraction of cells co-expressing Hyal-2 and CD44. The pH optimum for Hyal-2 was 6.0 -7.0. The membrane fraction of cells expressing Hyal-2 alone did not show hyaluronidase activity. Hyal-3 did not show any hyaluronidase activity in our experimental conditions. Based on these findings, Hyal-1 and -2 contribute to intracellular and extracellular catabolism of HA, respectively, in a CD44-dependent manner, and their HA degradation occurs independently from one another.Hyaluronic acid (HA) 3 is a negatively charged, high molecular weight glycosaminoglycan found predominantly in the extracellular matrix. It is the simplest of the glycosaminoglycans, the only one not covalently linked to core protein, and is unbranched and composed of repeating alternating units of glucuronic acid and N-acetylglucosamine. Despite the simplicity of its composition, HA has a great number of biological functions. It not only functions as a biological glue that participates in lubricating joints or holding together gel-like connective tissues, but also functions as a microenvironmental cue that co-regulates cell behavior during embryonic development and morphogenesis (1, 2), wound healing (3, 4), repair and regeneration, inflammation (5-7), and tumor progression and invasion (8, 9).There are 15 g of HA in a 70-kg individual, of which 5 g is replaced daily. In the skin, which contains 50% of the total body HA, the half-life of HA is about 1 day, and even in as seemingly inert a tissue as cartilage, HA turns over with a ha...

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In an attempt to investigate the origin of the intracellular symbiont of the aphid (Buchnera), aphid gut aerobic bacteria were isolated, and their phylogenetic relations to other prokaryotes were examined based on nucleotide sequences of 16S rDNA. It turned out that there are seven aerobic bacterial groups which constitute major flora of the aphid's gut. As three of the isolated bacteria were identified as members of the family Enterobacteriaceae, and share the common ancestor with the intracellular symbiont, the nucleotide sequences of 16S rDNA were determined for 15 representative strains of the family Enterobacteriaceae. One of the gut microbes belonging to the family Enterobacteriaceae was identified as Erwinia herbicola that is found mainly on plant surfaces. This fact may suggest that the intracellular symbiont of aphid is derived from a habitant of plant on which host insects feed.Pea aphid, Acyrthosiphon pisum, harbors prokaryotic intracellular symbionts in the mycetocytes, huge cells in the abdomen, which are differentiated specifically to accommodate the symbionts (1). The date of establishment of the symbiotic association is estimated to be 160-280 million years ago based on 16S rDNA molecular clock calibrated by aphid fossils (12). The aphid and its intracellular symbionts, which are often referred to as Buchnera species (14), are in a closely mutualistic relationship, and neither of them is able to propagate without the other (8,15). Buchnera, essential for the normal growth and reproduction of the aphid, are maternally inherited through generations of the host insect, and have not been successfully cultivated outside the host mycetocyte.

Five strains of Gram-negative, oxidase-negative, facultatively anaerobic, fermentative, motile, rodshaped bacterium with the general characteristics of the family Enterobacteriaceae were isolated from the gut of multiple specimens of the pea aphid. All the strains caused aphid mortality when ingested by insects via a synthetic diet. The results of biochemical tests showed that these strains are most related to Erwinia herbicola and Pantoea agglomerans. According to DNA-DNA hybridization, the five strains showed more than 96% relatedness to each other, indicating that these organisms are members of a single species. These strains were most closely related to Erwinia herbicola (22% DNA relatedness).Phenotypic differentiation of these strains from Erwinia herbicola, which was also detected from aphid gut, was based on negative reactions in tests of yellow pigment production, gelatin liquefaction, acid production from inulin, starch and dulcitol, and positive acid production from melibiose, inositol, cellobiose and glycerol. On the basis of these data, the name Erwinia aphidicola is proposed for the new organism. The type strain is strain X 001 (=IAM 14479). Key WordsErwinia aphidicola; insect gut; pea aphidIn a previous study, aerobic bacteria were isolated from aphid gut in order to investigate insect-bacteria interaction, and seven groups were identified as major constituents of the gut flora of the pea aphid, Acyrthosiphon pisum (Harada et al., 1996). Based on the molecular phylogenetic analyses of 16S rDNA, three of the seven bacterial groups were identified as members of the family Enterobacteriaceae, and tentatively named bacterium T, W and X. DNA-DNA hybridization testing and a phylogenetic tree of 16S rDNA indicated that bacterium T was identical to Erwinia herb/cola (Harada et al., 1996). Bacterium X was detected from almost all the healthy specimens, and was the largest in number among the seven bacterial groups which constituted the major flora of the insect gut (Harada and Ishikawa, 1993;Harada et al., 1996). In enterobacterial groups we examined, only bacterium X, and Er. herb/cola successfully infected the gut of the insects that had been kept aseptically (Harada and Ishikawa, 1997). Bacterium X grew very well in aphid gut and caused aphid mortality in the laboratory, whereas Er. herb/cola seemed to have no effect on insect health.In this study, the five strains of predominant bacterial species in aphid gut were examined biochemically and by DNA-DNA hybridization. Our results indicated that these bacteria belong to a new species in the genus Erwinia, for which the name Erwinia aphid/cola is proposed. Materials and MethodsBacterial strains. Strains X 001, X 081, X 151, X 171 and X 181 were isolated from the guts of healthy apterous aphids as previously described (Harada et al., 1996). Whole bodies of the insect, which were surface-sterilized by dipping into 70% ethanol for 5 min, were washed in sterilized Carlson's solution (0.7% NaCI (w/v), 0.02% KCI (w/v), 0.02% CaCl2.2H2O (wlv), 0.01% MgCl2 • 6H2O (...