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We investigated the profound involvement of chymase, an alternative angiotensin II-generating enzyme, in angiogenesis using a hamster sponge implant model. In vivo transfection of human pro-chymase cDNA or a direct injection of purified chymase into the sponges implanted resulted in marked increment of hemoglobin contents in the sponge granuloma tissues, demonstrating that chymase has an ability to elicit angiogenesis and is a potent angiogenic factor. Daily injection of basic fibroblast growth factor into the sponges implanted also induced angiogenesis, which was suppressed by the treatment with chymostatin, an inhibitor of chymase, or TCV-116, an antagonist of angiotensin II (Ang II) type 1 receptor. Expression of chymase mRNA and production of Ang II in the granuloma tissues were enhanced by the stimulation with basic fibroblast growth factor. Chymase activity in the sponge granulomas increased in parallel with the rise in hemoglobin contents, and mast cells observed in the granuloma tissues were positively stained with anti-chymase antibody. Exogenous administration not only of Ang II but of angiotensin I (Ang I) directly into the sponges could enhance angiogenesis. Chymostatin inhibited the angiogenesis induced by Ang I but not Ang II, suggesting the presence of a chymase-like Ang II-generating activity in the sponge granulomas. Our results may suggest a potential ability of chymase to promote angiogenesis through the local chymase-dependent and angiotensin-converting enzyme-dependent Ang II generating system in pathophysiological angiogenesis.

beta1-null GD25 fibroblasts adherent to vitronectin fail to bind the N-terminal 70-kDa matrix assembly domain of fibronectin or to assemble fibronectin (Sakai, T., Zhang, Q., Fässler, R., and Mosher, D. F. (1998) J. Cell Biol. 141, 527-538). We have made four observations that extend this finding. First, the presence of vitronectin on a substrate that otherwise can support fibronectin assembly has a dominant-negative effect on assembly. Second, the dominant-negative effect is lost when active beta1A is expressed. Third, beta1A containing the extracellular D130A inactivating mutation has a dominant-negative effect on fibronectin assembly. Fourth, beta1-null cells adherent to vitronectin are flat and lack filopodia, whereas beta1-null cells adherent to fibronectin or beta1A-expressing cells adherent to either vitronectin or fibronectin are contracted and exhibit numerous filopodia. These results reveal, therefore, that GD25 cells adherent to vitronectin can only assume a shape suitable for assembly of fibronectin when there is a countervailing signal from functional beta1-integrins.

High-molecular-weight kininogen has been isolated from rat plasma in three steps in a relatively high yield. The purified preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence and presence of 2-mercaptoethanol, and the apparent Mr was estimated as 100,000. On incubation with rat plasma kallikrein, rat high Mr kininogen yielded a kinin-free protein consisting of a heavy chain (Mr = 64,000) and a light chain (Mr = 46,000), liberating bradykinin. The kinin-free protein was S-alkylated, and its heavy and light chains were separated by a zinc-chelating Sepharose 6B column. The amino acid compositions of rat high Mr kininogen and its heavy and light chains were very similar to those of bovine high Mr kininogen and its heavy and fragment 1.2-light chains, respectively. A high histidine content in the light chain of rat high Mr kininogen indicated the presence of a histidine-rich region in this protein as in bovine high Mr kininogen, although this region was not cleaved by rat plasma kallikrein. Rat high Mr kininogen corrected to normal values the prolonged activated partial thromboplastin time of Brown-Norway Katholiek rat plasma known to be deficient in high Mr kininogen and of Fitzgerald trait plasma. The kinin-free protein had the same correcting activity as intact high Mr kininogen. Rat high Mr kininogen also accelerated approximately 10-fold the surface-dependent activation of rat factor XII and prekallikrein, which was mediated with kaolin, amylose sulfate, and sulfatide. These results indicate that rat high Mr kininogen is quite similar to human and bovine high Mr kininogens in terms of biochemical and functional properties.

We report a remarkable Fe K? fluorescence line profile in the Seyfert 1.9 galaxy MCG -5-23-16. The line is complex, consisting of a narrow core located at the galaxy's systemic velocity and wings to the red and blue sides of the core. This represents only the second detection with ASCAof an Fe K? line profile with significant broadening blueward of 6.4 keV, consistent with Doppler boosting. The line core has an EW of ~60 eV and the wings have a combined EW of ~200 eV. The entire profile cannot be easily modeled with an emission line predicted from standard accretion disk theories. Instead, the line is marginally triple-peaked and is best described with three Gaussians having rest energies of 5.37+ 0.23?0.19keV (FWHM = 30,200 km s-1), 6.37 ± 0.04 keV (FWHM < 6,600 km s-1), and 6.58+ 0.35?0.38keV (FWHM = 75,300 km s-1). The line can also be modeled with a narrow Gaussian at ~6.4 keV and an emission line from an accretion disk viewed at an inclination angle of ~30° to 65°, depending on the ionization state of the gas. Within the context of the unified model, the most likely physical description of the complex profile is a superposition of an emission line from an accretion disk and a line that arises far from the disk in either the obscuring torus or the broad line region. This represents the first strong evidence for emission from two distinct X-ray reprocessors within a single Seyfert 1-type galaxy.

Two T-kininogens (TI- and TII-kininogens) found in plasma of Freund's adjuvant-treated rats were purified by several chromatographic procedures. The isolated TI- and TII-kininogens showed different mobilities on polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate, but were indistinguishable in the presence of sodium dodecyl sulfate. They were also indistinguishable in amino acid composition and antigenicity, but differed in sialic acid content. The NH2- and COOH-terminal sequences were determined. In the 30 NH2-terminal residues, 2 were different. The kinin regions in the COOH-terminal portions of the two kininogens have sequences that demonstrate TI-kininogen contains a mixture of two kinin-containing regions, with substitution of 4 amino acid residues, one of which is identical to the COOH-terminal portion of alpha 1-major acute phase protein (Cole, T., Inglis, A. S., Roxburgh, C. M., Howlett, G. J., and Schreiber, G. (1985) FEBS Lett. 182, 57-61) and the other to the COOH-terminal portion of TI-kininogen (Furuto-Kato, S., Matsumoto, A., Kitamura, N., and Nakanishi, S. (1985) J. Biol. Chem. 260, 12054-12059), both predicted from cDNA sequences. The amino acid sequence of the kinin-containing region from TII-kininogen is the same as the COOH-terminal portion of TII-kininogen predicted from the cDNA. These results indicate that T-kininogens from the plasma of adjuvant-treated rats consist of a family of kininogens, that is, TI- and TII-kininogens (separable on DEAE-Sephadex A-50), and that TI-kininogen consists of at least two variants (TI alpha and TI beta) which correspond to the alpha 1-major acute phase protein reported by Cole et al. and TI-kininogen reported by Furuto-Kato et al., respectively. Immunoblotting studies with plasmas from non-inflamed and adjuvant-treated rats also indicate that T-kininogen which was previously isolated from non-inflamed rat plasma corresponds to TI-kininogen and that TII-kininogen is newly generated after treatment of rats with adjuvants.

Two kinds of low molecular weight kininogens (identified as A and B) were isolated from pooled plasma of Sprague-Dawley rats. They show a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of 2-mercaptoethanol, and the molecular weights are 68,000 for low Mr kininogen A and 73,000 for low Mr kininogen B. Although the molecular weights and amino acid compositions of the low Mr kininogens are similar, rat submaxillary and urinary kallikreins released bradykinin from low Mr kininogen B, whereas low Mr kininogen A was resistant to these enzymes. The COOH-terminal portion of low Mr kininogen A was isolated after cyanogen bromide treatment, and the amino acid sequence of the COOH-terminal 55 residues including the T-kinin (Ile-Ser-bradykinin) was determined. The COOH-terminal portion consists of two sequences with substitution of 4 residues. One peptide corresponds to alpha 1-major acute phase protein (Cole, T., Inglis, A. S., Roxburgh, C. M., Howlett, G. J., and Schreiber, G. (1985) FEBS Lett. 182, 57-61) and the other to the TI-kininogen predicted from a cDNA study (Furuto-Kato, S., Matsumoto, A., Kitamura, N., and Nakanishi, S. (1985) J. Biol. Chem. 260, 12054-12059). The results demonstrate that there exist at least two kinds of low Mr kininogens with clearly different function in rat plasma: one of them, low Mr kininogen A, is a precursor of T-kinin and is resistant to kallikreins, and the second, low Mr kininogen B, is sensitive to tissue kallikreins and shares properties with bovine and human low Mr kininogens. The results also demonstrate that T-kininogen is a mixture of two isoproteins which correspond to alpha 1-major acute phase protein or TI-kininogen, respectively. We could not detect the low Mr kininogen corresponding to the TII-kininogen predicted from the cDNA study of Furuto-Kato et al.