Collagen gel delivery of Tgf-beta3 non-viral plasmid DNA in rat osteoblast and calvarial culture.

Different forms of collagen as a carrier for naked plasmid DNA have shown potential as vehicles for therapeutic gene delivery and tissue engineering. The objective of this study was to determine the suitability of a dense collagen gel as a vehicle for sustained delivery of plasmid DNA in cell and organ culture. Plasmid DNA encoding Tgf-beta(3) was combined with collagen gel. DNA released into the media was measured by Pico-Green spectrophotometry. Results showed that DNA was released from the collagen gel at a gradual rate for up to 14 days. To evaluate collagen-mediated transfection in tissue, calvariae were exposed to collagen containing plasmid encoding GFP or DsRed. Transfection was visualized by fluorescence localized to tissue adjacent to the vehicle. To evaluate protein production, fetal rat calvarial osteoblasts were cultured with a collagen/Tgf-beta(3) plasmid mixture or in media containing plasmid alone. Media was collected at various time points to measure Tgf-beta(3) protein production. ELISA assays showed that collagen-transfected osteoblasts demonstrated an elevated Tgf-beta(3) protein production for up to 14 days. Therefore, collagen delivery of viable plasmid DNA created a sustained transient transfection of calvarial osteoblasts resulting in prolonged and elevated growth factor production. Together, these results suggest that use of collagen gel as a vehicle may provide a strategy to achieve localized and controlled, non-viral gene delivery in vivo.

Effect of harvesting and sorting on beta-1 integrin in canine microvascular cells.

BACKGROUND: The goal of seeding prosthetic conduits with endothelial cells (ECs) has focused attention on the role of EC adhesion molecules. Cell preparation techniques may affect adhesion molecule expression and graft seeding. METHODS: Using fluorescent antibody labeling and flow cytometric analysis, this study examined the effectsof monolayer detachment (scraping vs trypsinization), timing of immunolabeling (pre- vs postdetachment), gene transfection (transfected vs nontransfected), and cell selection (antibiotic vs fluorescence sorting) techniques on beta-1 integrin expression in canine microvascular EC (K9MVEC). RESULTS: Cell scraping resulted in a significantly higher beta-1 integrin mean fluorescence intensity than did cell trypsinization (P < 0.05). No difference was observed with immunolabeling prior to versus following monolayer harvesting. Gene transfection had no significant effect on beta-1 integrin expression. No advantage was observed between cell selection methods (P > 0.05). CONCLUSION: This study suggests that the monolayer harvesting technique employed has a significant impact on beta-1 integrin quantification by flow cytometric analysis. Furthermore, microvascular EC expression of beta-1 integrin was not adversely affected by gene transfection.

Isolation of the major component in white snakeroot that is toxic after microsomal activation: possible explanation of sporadic toxicity of white snakeroot plants and extracts.

Tremetone, the major toxic component in white snakeroot (Eupatorium rugosum Houtt) extracts, was isolated following an in vitro bioactivity assay. Microsomal activation was required to produce a product toxic to murine melanoma (B16F1) cells as well as five other mammalian cell cultures. The metabolic activation product(s) of tremetone is suspected to be responsible for the toxic activity of the plant. Tremetone is also smoothly converted to dehydrotremetone in the plant and cell free homogenates, and readily decomposes to dehydrotremetone in extracts. Dehydrotremetone is not toxic even after microsomal activation. The efficient conversion of tremetone to dehydrotremetone may explain why white snakeroot plant material and extracts have varied activities, and why a previous claim that tremetone was responsible for the toxic activity of white snakeroot was withdrawn. Rayless goldenrod extracts show the same toxic activity as white snakeroot and the toxic activity of rayless goldenrod is most likely due to tremetone.

Some synthetic phytotoxins structurally related to rhynchosporoside.

The (2-O)alpha-d-glucopyranoside of 1,2-propanediol and [U-(14)C]glucose were used as substrates in a reaction with almond beta-glucosidase, which resulted in the production of some (2-O)alpha-d-oligoglucosides of 1,2-propanediol. As its substrate, the beta-glucosidase preferred the glucoside isomer that rotates plane-polarized light to the right. Some of the glucosides produced in the enzymic reaction mixture possessed host selective toxin activity. It appears that the biological activity of the toxin is not dependent on the nature of the glycosidic linkage with the aglycone.