Annette Schmidt

Use of minimally modified antisense oligonucleotides for specific inhibition of gene expression

Abstract The design and use of minimally modified oligonucleotides for specific inhibition of gene expression is discussed. The “minimal” protection strategy is a combination of the end-capping technique and the protection of internal pyrimidine positions which are the major sites of endonuclease degradation. By reducing the number of phosphorothioate modifications needed to make the oligonucleotide resistant to nuclease degradation, non-sequence-specific effects, which are frequently observed with uniformly phosphorothioate-modified oligonucleotides, can be reduced.

Compartmentation and characterization of different proteoglycans in bovine arterial wall.

Proteoglycans stained specifically with cuprolinic blue have been visualized in electron micrographs of bovine arterial tissue. Three differently sized proteoglycan-cuprolinic blue precipitates, designated as types I, II, and III, could be detected in the extracellular matrix. The precipitates could be distinguished by their length, width, area, topographical distribution, and their characteristic association with other matrix components. By taking into account the available biochemical data and the individual susceptibilities of the precipitates towards specific glycosaminoglycan-degrading enzymes, each type of proteoglycan-cuprolinic blue precipitate could be attributed to a proteoglycan population containing dermatan sulfate, chondroitin sulfate, or heparan sulfate as its main glycosaminoglycan component.

Undersulfation of Proteoheparan Sulfate Stimulates the Expression of Basic Fibroblast Growth Factor and Protein Synthesis but Suppresses Replication of Coronary Smooth Muscle Cells

Heparan sulfate proteoglycans are obligatory for receptor binding and mitogenic activity of the basic fibroblast growth factor (bFGF). In the present study the influence of undersulfated heparan sulfate on the expression of basic fibroblast growth factor and coronary smooth muscle cell (cSMC) proliferation was investigated. Chlorate, known to be an inhibitor of ATP-sulfurylase, was used as a tool to suppress sulfation of heparan sulfate. When cultured cSMC were treated with 10 mM sodium chlorate in sulfate-depleted medium, the cell number and [3H]thymidine incorporation decreased by 76% and 66% respectively, while the protein content per cell was doubled. At the same time the [35S]sulfate incorporation into cell-associated proteoglycans was reduced by 90%. The remaining minimal amount of available [35S]radioactivity was preferably incorporated into heparan sulfate. Under the same conditions the [6-(3)H]glucosamine incorporation into glycosaminoglycans was not impaired. The chlorate-induced increase of cell protein content includes an overexpression of bFGF, which increased from 6-8 ng to 18-22 ng/mg cell protein. However, no changes in the distribution of bFGF between the intracellular and pericellular compartment could be observed. Cell cycle analysis by FACS revealed a G1 arrest of the cell cycle with increase of the G1/S ratio from 2.9 (control) to 6.1 (chlorate) but the DNA content per cell corresponded to normal diploid cells both in control and chlorate-treated cells. The chlorate effect can be abolished by addition of 5 mM sodium sulfate to the cultures. Our results demonstrate an inverse association between the sulfation of heparan sulfate and the expression of bFGF. They suggest that chlorate blocks the cell cycle in the late G1-phage and that mitogenesis of cSMC requires fully sulfated cell-associated proteoheparan sulfate.