Identification of Heparan-Sulfate Rich Cells in the Loose Connective Tissues of the Axolotl (Ambystoma mexicanum) with the Potential to Mediate Growth Factor Signaling during Regeneration.

Limb regeneration is the outcome of a complex sequence of events that are mediated by interactions between cells derived from the tissues of the amputated stump. Early in regeneration, these interactions are mediated by growth factor/morphogen signaling associated with nerves and the wound epithelium. One shared property of these proregenerative signaling molecules is that their activity is dependent on interactions with sulfated glycosaminoglycans (GAGs), heparan sulfate proteoglycan (HSPG) in particular, in the extracellular matrix (ECM). We hypothesized that there are cells in the axolotl that synthesize specific HSPGs that control growth factor signaling in time and space. In this study we have identified a subpopulation of cells within the ECM of axolotl skin that express high levels of sulfated GAGs on their cell surface. These cells are dispersed in a grid-like pattern throughout the dermis as well as the loose connective tissues that surround the tissues of the limb. These cells alter their morphology during regeneration, and are candidates for being a subpopulation of connective tissue cells that function as the cells required for pattern-formation during regeneration. Given their high level of HSPG expression, their stellate morphology, and their distribution throughout the loose connective tissues, we refer to these as the positional information GRID (Groups that are Regenerative, Interspersed and Dendritic) cells. In addition, we have identified cells that stain for high levels of expression of sulfated GAGs in mouse limb connective tissue that could have an equivalent function to GRID cells in the axolotl. The identification of GRID cells may have important implications for work in the area of Regenerative Engineering.

Glycoside primers of Psittacanthus cucullaris.

Bioassay-directed chromatographic separation of the ethyl acetate extract of the whole plant of Psittacanthus cucullaris afforded a new phenolic xyloside, ellagic acid-4-O-beta-xyloside-3,3', 4'-trimethyl ether (1) together with four known compounds, ellagic acid-4-O-beta-xyloside-3,3'-dimethyl ether (2), gallic acid, beta-sitosterol, and beta-sitosterol beta-D-glucoside. The structure of the new compound was determined by spectroscopic methods. Like other beta-D-xylosides, compounds 1 and 2 stimulated the formation of glycosaminoglycan chains when fed to the cultured Chinese hamster ovary cells.

Primers of glycosaminoglycan biosynthesis from Peruvian rain forest plants.

We have developed a rapid, high throughput screening assay for compounds that alter the assembly of glycosaminoglycan chains in Chinese hamster ovary cells. The assay uses autoradiography to measure the binding of newly synthesized [35S]proteoglycans and [35S]glycosaminoglycans to a positively charged membrane. Screening over 1000 extracts from a random plant collection obtained from the Amazon rain forest yielded five plants that stimulated glycosaminoglycan assembly in both wild-type cells and a mutant cell line defective in xylosyltransferase (the first committed enzyme involved in glycosaminoglycan biosynthesis). Fractionation of an extract of Maieta guianensis by silica gel and reverse-phase chromatography yielded two pure compounds with stimulatory activity. Spectroscopic analysis by NMR and mass spectrometry revealed that the active principles were xylosides of dimethylated ellagic acid. One of the compounds also contained a galloyl group at C-3 of the xylose moiety. These findings suggest that plants and other natural products may be a source of agents that can potentially alter glycosaminoglycan and proteoglycan formation in animal cells.