During murine embryogenesis, the Ets factor Erg is highly expressed in endothelial cells of the developing vasculature and in articular chondrocytes of developing bone. We identified seven isoforms for the mouse Erg gene. Four share a common translational start site encoded by exon 3 (Ex3) and are enriched in chondrocytes. The other three have a separate translational start site encoded by Ex4 and are enriched in endothelial cells. Homozygous ErgΔEx3/ΔEx3 knockout mice are viable, fertile and do not display any overt phenotype. By contrast, homozygous ErgΔEx4/ΔEx4 knockout mice are embryonic lethal, which is associated with a marked reduction in endocardial-mesenchymal transformation (EnMT) during cardiac valve morphogenesis. We show that Erg is required for the maintenance of the core EnMT regulatory factors that include Snail1 and Snail2 by binding to their promoter and intronic regions.

The use of fresh allograft tissue to treat osteochondral defects eliminates morbidity associated with harvesting autograft tissue without compromising the results of the surgical procedure.

Background/Aims: Vascular smooth muscle cells contribute both to the structure and function of arteries, but are also involved in pathologic changes that accompany inflammatory diseases such as atherosclerosis. Since inflammation is associated with oxidant stress, we examined the uptake and cellular effects of the antioxidant vitamin ascorbic acid in cultured A10 vascular smooth muscle cells. Methods/Results: A10 cells concentrated ascorbate against a gradient in a sodium-dependent manner, most likely on the sodium-dependent vitamin C transporter type 2 (SVCT2) ascorbate transporter, which was present in immunoblots of cell extracts. Although ascorbate did not affect A10 cell proliferation, it stimulated radiolabeled proline incorporation and type I collagen synthesis. The latter was evident in the cells as increases in proα1(I) collagen and conversion of proα1(I) and proα2(I) collagen to mature forms that were released from the cells and deposited as extracellular matrix. Intracellular type I procollagen maturation was optimal at intracellular ascorbate concentrations of 200 μM and below, which were readily achieved by culture of the cells at plasma physiologic ascorbate concentrations. Conclusion: These results show that the SVCT2 facilitates ascorbate uptake by vascular smooth muscle cells, which in turn increases both the synthesis and maturation of type I collagen.

We have synthesized and characterized bis(sulfo-N-succinimidyl) doxyl-2-spiro-5'-azelate (BSSDA), a membrane-impermeant bifunctional spin-labeling reagent. BSSDA is a nine carbon backbone homologue of bis(sulfo-N-succinimidyl) doxyl-2-spiro-4'-pimelate [BSSDP; Beth et al. (1986) Biochemistry 25, 3824-3832]. Due to its longer backbone, BSSDA can span longer distances between reactive groups on a protein than can BSSDP. However, the purpose of the bifunctional design of these reagents is to provide a tight motional coupling of the spin labels to the surface of a target protein. To test whether the longer backbone of BSSDA results in a greater local flexibility and thereby undermines the effects of bidentate attachment, we have labeled with BSSDA anion-exchange channels of intact human erythrocytes at the same site as we have previously labeled them with BSSDP. Linear and saturation-transfer EPR spectra of BSSDA-labeled anion-exchange channels in intact cells closely approximate the corresponding spectra from BSSDP-labeled channels. Thus, the longer backbone of BSSDA relative to BSSDP does not give rise to significant local flexibility, even when BSSDA is bound to a site that can be spanned by the shorter reagent.

Background. Benign giant cell tumor of bone (GCT) is a primary skeletal neoplasm with an unpredictable pattern of biologic aggressiveness and cytogenetic findings characterized by telomeric associations and telomeric reduction. The role of maintaining telomeric integrity is performed by telomerase. To determine if telomerase activity is present, cell extracts from fibroblasts and tumor cells from five patients with GCT were analyzed and compared with HeLa (a positive control cell line). Methods. Telomerase activity was detected by visualizing the extension of radioactive telomeric repeats on DNA sequencing gels. Telomere reduction was assessed using southern blot analyses of the restriction enzyme Hinf I digested DNA with a radio‐labeled telomere probe. Results. Telomerase or telomerase‐like activity was detected in the cell extracts from HeLa and tumor cells. However, GCT telomerase activity varied and was less than that observed in HeLa, but no activity was detected from fibroblasts. In addition, telomere reduction was seen in DNA isolated from both HeLa and GCT but not in fibroblasts or age‐matched controls. Conclusion. Telomere reduction and telomerase activity may be oncogenic sustaining events required to maintain the transformed phenotype seen in GCT. Cancer 1995;75:1094–9.