Inhibition of rat vascular smooth muscle proliferation in vitro and in vivo by bone morphogenetic protein-2.

Vascular proliferative disorders are characterized by the proliferation of vascular smooth muscle cells (SMCs) and excessive extracellular matrix synthesis. We found that bone morphogenetic protein-2 (BMP-2) inhibited serum-stimulated increases in DNA synthesis and cell number of cultured rat arterial SMCs in a fashion quite different from that in the case of transforming growth factor-beta1 (TGF-beta1). In addition, TGF-beta1 stimulated collagen synthesis in SMCs, whereas BMP-2 did not. In an in vivo rat carotid artery balloon injury model, the adenovirus-mediated transfer of the BMP-2 gene inhibited injury-induced intimal hyperplasia. These results indicate that BMP-2 has the ability to inhibit SMC proliferation without stimulating extracellular matrix synthesis, and suggest the possibility of therapeutic application of BMP-2 for the prevention of vascular proliferative disorders.

Sequence and over-expression of subunits of adenosine triphosphate synthase in thermophilic bacterium PS3.

The primary structures of all the subunits of thermophilic ATP synthase were determined, and its alpha, beta and gamma subunits could be over-expressed in Escherichia coli, because these subunits were stable and reconstitutable. DNA of 7500 base pairs in length was found to contain a cluster of nine genes for subunits of ATP synthase. The order of their reading frames (size in base pairs) was: I(381): a(630): c(216): b(489): delta(537): alpha(1507): gamma(858): beta(1419): epsilon(396), I being a gene for a small hydrophobic, basic protein expressed in vitro. All the termini of TF0F1 subunits were confirmed by peptide sequencing. Large quantities of the overexpressed thermophilic alpha, beta and gamma subunits were prepared from the extract of E. coli, by a few purification steps.

Heterotopic ossification of degenerating rat skeletal muscle induced by adenovirus-mediated transfer of bone morphogenetic protein-2 gene.

In vivo gene transfer is a recently developed device for efficient delivery of a therapeutic recombinant protein. We formulated the hypothesis that a high level of expression of bone morphogenetic protein 2 (BMP-2) could be a future therapeutic modality in terms of inducing substantial bone formation in vivo. First, to test this hypothesis, adenoviruses carrying BMP-2 gene were directly injected into the soleus muscle of adult rat. The BMP-2 gene was successfully overexpressed in the target muscle by adenovirus-mediated transfer, whereas bone formation in and around the muscle failed to occur in this case. Second, to recruit putative osteoprogenitor cells, we then induced ischemic degeneration of the target muscle by orthotopically grafting it simultaneously with the gene transfer. The combination of BMP-2 gene transfer and orthotopic muscle grafting resulted in successful ossification of almost the whole grafted muscle, whereas neither muscle grafting alone nor the combination of muscle grafting and adenovirus-mediated transfer of reporter gene LacZ induced any bone formation in the muscle. The ossification process was evident by positive von Kossa staining of the histological sections and roentgenographical radio-opacity of the region. It was also found that the BMP-2 transgene overexpressed in grafted muscles inhibited muscle regeneration, which should otherwise follow the muscle degeneration. We further demonstrated an up-regulation of BMP receptor type IA in grafted muscles, suggesting its involvement in the bone-formation process. In conclusion, overexpression of BMP-2 gene induced massive heterotopic ossification in skeletal muscles under graft-induced ischemic degeneration, which possibly up-regulates osteoprogenitor cells in situ.

Alpha 3 beta 3 complex of thermophilic ATP synthase. Catalysis without the gamma-subunit.

A complex of the alpha- and beta-subunits of thermophilic ATP synthase showed about 25% of the ATPase activity of the alpha beta gamma complex. The alpha 3 beta 3 hexamer structure was analyzed by sedimentation (11.2 S) and gel filtration (310 kDa). Dilution of the alpha beta complex caused dissociation of the complex and rapid loss of ATPase activity which was restored by addition of the gamma-subunit. A previous method using urea for isolating the subunits resulted in an alpha beta complex with lower activity than that prepared by over-expression of the genes. The alpha beta-ATP complex was formed from the alpha beta complex, ADP and Pi in the presence of dimethyl sulfoxide.

[Biochemistry of bone matrix].

Bone matrix proteins can be divided into several groups according to their function; structural proteins, cell adhesive molecules, growth factors, proteinases and others. 1) Type I collagen is a major structural protein. 2) Osteopontin and bone sialoprotein (BSP) are RGD-containing cell adhesive proteins. Cadherins, OSF-2 and MGP also act as cell adhesive molecules in cell to cell or cell to matrix signal transition systems. 3) BMPs, TGF-b, IGFs, FGFs and PDGF are bone growth factors associated to bone matrix. 4) Collagenases and cathepsins are well known organic bone matrix degrading enzymes.

Gene structure of heat shock proteins 61KDa and 12KDa (thermophilic chaperonins) of thermophilic bacterium PS3.

Heat shock proteins 60 (hsp60) and 10 (hsp10) are essential for the formation and restoration of many supramolecular structures. For reconstitution of these structures, we isolated stable hsps of 61kDa and 12kDa, which are similar to hsp60 and hsp10, respectively, from the supernatant fraction of thermophilic bacterium PS3 by ATP-Agarose chromatography. Using synthetic DNA of the deduced sequence, the 1.6kbp double stranded DNA encoding both proteins was obtained by the polymerase chain reaction (PCR). The complete sequence of the resulting reading frames showed high homology to those of the genes encoding GroEL (hsp60) and GroES (hsp10) of E. coli, and hsp60s and hsp10s of several other species. The genes for the 12K and 61K were present in the same operon. 61K was also partially similar to the F1 alpha subunit of thermophilic ATP synthase, which is highly reconstitutable to form the alpha beta complex.