The cDNAs encoding the human bone morphogenetic proteins BMP-2 and BMP-4 in an eukaryotic expression vector were permanently transferred into the murine mesenchymal progenitor cell line C3H10T1/2. Originally, these cells are known to differentiate into myotubes, adipocytes, and chondrocytes upon the addition of azacytidine. Permanent transfection of genes encoding human BMP-2 and BMP-4 induces differentiation into the osteogenic lineage. The osteogenic differentiation potential of C3H10T1/2 cells is substantiated by histochemical and genetic analyses of marker genes typical or specific for osteogenesis, including the parathyroid hormone receptor, alkaline phosphatase, osteopontin, osteonectin, and osteocalcin. In addition to osteoblast formation, development into adipocytes and chondrocytes is also observed, suggesting that BMP-2 and BMP-4 induce differentiation into three mesenchymal lineages.

In vitro 1,25-dihydroxycholecalciferol (1,25(0H)2D3) decreased levels of preproparathyroid(preproPTH) hormone mRNA. We have now pursued these studies in vivo in the rat. Rats were administered vitamin D metabolites i.p. and the levels of preproPTH mRNA were determined in excised parathyroidthyroid glands by blot hybridization. PreproPTH mRNA levels were <4% of basal at 48 h after 100 pmol 1,25(OH)2D3, with no increase in serum calcium. Gel blots showed that 1,25(OH)2D3 decreased preproPTH mRNA levels without any change in its size (833 basepair). Microdissected parathyroids after 1,25(0H)2D3 (100 pmol) showed mRNA levels for preproPTH were 40±8% of controls, but for f-actin were 100% of controls.The relative potencies of vitamin D metabolites were: 1,25(0H)2D3> 24,25(0H1)D3> 25(OH)D3 > vitamin D3. In vitro nuclear transcription showed that 1,25(0H)2D3-treated (100 pmol) rats' PTH transcription was 10% of control, while j5-actin was 100%. These results show that 1,25(0H1)D3 regulates PTH gene transcription. PTH stimulates 1,25(OH)2D3 synthesis, which then inhibits PTH synthesis, thus completing an endocrinological feedback loop.

Angiogenesis is essential in bone fracture healing for restoring blood flow to the fracture site. Vascular endothelial growth factor (VEGF) and its receptor have been implicated in this process. Despite the importance of angiogenesis for the healing processes of damaged bones, the role of VEGF signaling in modulation of osteogenic differentiation in human mesenchymal stem cells has not been investigated in great detail. We examined the expression of VEGF-A and VEGFR-1 in human adult mesenchymal stem cells derived from trabecular bone (hTBCs). VEGF-A was found to be secreted in a differentiation dependent manner during osteogenesis. Transcripts for VEGF-A were also seen to be elevated during osteogenesis. In addition, transcripts for VEGF-A and the corresponding receptor VEGFR-1 were upregulated under hypoxic conditions in undifferentiated hTBCs. To investigate the signaling of VEGF-A on osteogenesis recombinant hTBCs were generated. High expression of VEGF-A stimulated mineralization, whereas high expression of sFLT-1, an antagonist to VEGF-A, reduced mineralization suggesting that VEGF-A acts as autocrine factor for osteoblast differentiation. In addition, VEGF-A secreted by hTBCs promotes sprouting of endothelial cells (HUVE) demonstrating a paracrine role in blood vessel formation. In summary, an in vitro analysis of transgene effects on cellular behavior can be used to predict an effective ex vivo gene therapy.

Enterobacterial common antigen (ECA) is a family‐specific surface antigen shared by all members of the Enterobacteriaceae and is restricted to this family. It is found in freshly isolated wild‐type strains as well as in laboratory strains like Escherichia coli K‐12. The family specificity of ECA can be used for taxonomic and diagnostic purposes. ECA is located in the outer leaflet of the outer membrane. It is a glycophospholipid built up by an aminosugar heteropolymer linked to an l‐glycerophosphatidyl residue. In a few rough mutants, in addition, the sugar chain can be bound to the complete lipopolysaccharide (LPS) core. Recently, for Shigella sonnei a lipid‐free cyclic form of ECA was reported. The genetical determination of ECA is closely related to that of lipopolysaccharide. For biosynthesis of ECA and LPS partly the same sugar precursors and the same carrier lipid is used.

On the basis of ribosomal 16S sequence comparison, Brucella abortus has been found to be a member of the alpha-2 subdivision of the class Proteobacteria (formerly named purple photosynthetic bacteria and their nonphototrophic relatives). Within the alpha-2 subgroup, brucellae are specifically related to rickettsiae, agrobacteria, and rhizobiae, organisms that also have the faculty or the obligation of living in close association to eucaryotic cells. Comparison of the Brucella lipid composition with that of the other Proteobacteria also suggests a close phylogenetical relationship with members of the alpha-2 subdivision. The genealogical grouping of Brucella species with pericellular and intracellular plant and animal pathogens as well as with intracellular plant symbionts suggests a possible evolution of Brucella species from plant-arthropod-associated bacteria.Members of the genus Brucella are gram-negative facultative intracellular pathogens that induce abortion and severe clinical symptoms in mammals (10, 30). The importance of the disease, mainly in developing countries, is recognized by the considerable economic losses due to infection of domestic animals and by the zoonotic problems caused through the ingestion or contact of contaminated secretions and products (30).Six species of Brucella have been described (10, 30): B. melitensis, B. abortus, B. suis, B. neotomae, B. ovis, and B. canis. This classification has been based mainly on the animal host specificity, susceptibility to dyes, metabolic patterns, phage typing, and serological testing (10, 30). Recently Verger et al. (72), using DNA similarity, challenged the separation of Brucella into different species and proposed a single species only: B. melitensis, containing several biovars. In addition, De-Ley et al. (14) established taxonomic affiliations of Brucella species with members of the Centers for Disease Control group Vd in the rRNA superfamily IV and close relationship of these organisms to members of the family Rhizobiaceae.In preliminary reports on the Brucella 16S rRNA sequence (18) and on lipid A analyses (42, 47; J. W. Cherwonogrodzky, G. Dubray, E. Moreno, and H. Mayer, in K. Nielsen and B. Duncan, ed., Animal Brucellosis, in press), we have suggested, that Brucella species are related to the alpha-2 subdivision of the class Proteobacteria (64), formerly named "purple photosynthetic bacteria and their * Corresponding author. nonphototrophic relatives" (77), which includes phototrophic and chemoorganotrophic organisms (77,78). In the present study we propose that species of the genus Brucella are closely related to gram-negative bacteria, such as agrobacteria, rhizobiae, and rickettsiae, which also form intimate pericellular or intracellular associations with eucaryotic cells. MATERIALS AND METHODSExtraction and purification of LPS. The characteristics and culture conditions of smooth B. abortus 1119-3, smooth B. melitensis 16M, rough B. abortus 45/20, rough B. melitensis B115, B. canis (strain obtained from R. Diaz, University of Navarra,...