Expression of synthetic genes encoding bovine and human basic fibroblast growth factors (bFGFs) in Escherichia coli

Synthetic genes encoding bovine and human basic fibroblast growth factors (bFGFs) were assembled and cloned using established Escherichia coli expression plasmids. Transformed E. coli cells were able to synthesize either a fusion protein, comprising the first seven amino acids of beta-galactosidase, a linker fragment and bovine FGF, or genomic human bFGF. The two growth factors were purified from E. coli lysates by cation exchange and heparin-Sepharose affinity chromatography. The purified recombinant proteins were biologically active as monitored by their mitogenic activity for bovine aortic endothelial cells and their angiogenic capacity in the rabbit cornea.     

Aldosterone induction of electrogenic sodium transport in the apical membrane vesicles of rat distal colon

Na-H exchange is present in apical membrane vesicles (AMV) isolated from distal colon of normal rats. Because in intact tissue aldosterone both induces amiloride-sensitive electrogenic sodium transport and inhibits electroneutral sodium absorption, these studies with AMV were designed to establish the effect of aldosterone on sodium transport. An outward-directed proton gradient stimulated 22Na uptake in AMV isolated from distal colon of normal and dietary sodium depleted (with elevated aldosterone levels) experimental rats. Unlike normal AMV, proton gradient-dependent 22Na uptake in experimental AMV was inhibited when uptake was measured under voltage-clamped conditions. 10 microM amiloride inhibited the initial rate of proton gradient-dependent 22Na uptake in AMV of normal and experimental rats by 30 and 75%, respectively. In contrast, 1 mM amiloride produced comparable inhibition (90 and 80%) of 22Na uptake in normal and experimental AMV. Intravesicular-negative potential stimulated 22Na uptake in experimental but not in normal AMV. This increase was inhibited by 90% by 10 microM amiloride. An analogue of amiloride, 5-(N-ethylisopropyl) amiloride (1 microM), a potent inhibitor of electroneutral Na-H exchange in AMV of normal rat distal colon, did not alter potassium diffusion potential-dependent 22Na uptake. Increasing sodium concentration saturated proton gradient-dependent 22Na uptake in normal AMV. However, in experimental AMV, 22Na uptake stimulated by both proton gradient and potassium diffusion potential did not saturate as a function of increasing sodium concentration. We conclude from these results that an electrically sensitive conductive channel, not electroneutral Na-H exchange, mediates 22Na uptake in AMV isolated from the distal colon of aldosterone rats.     

Allosteric regulation of tPA-mediated plasminogen activation by a modifier mechanism: evidence for a binding site for plasminogen on the tPA A-chain.

We studied the mechanism responsible for nonlinear double reciprocal plots for tissue type plasminogen activator (tPA)-mediated plasminogen activation reported previously by several groups. We found nonlinear Eadie-Scatchard plots for Glu-plasminogen activation by recombinant single-chain tPA confirming a non-Michaelis-Menten behavior of tPA. In order to characterize this mechanism, enzyme kinetic studies with truncated substrates (Lys- and miniplasminogen) and modified or truncated enzymes (two-chain tPA and tPA B-chain) were performed. Thereby it could be excluded that product-mediated modifications of the enzyme or the substrate are responsible for the nonlinear plots. Linear plots, i.e., Michaelis-Menten kinetics, were only found when tPA B-chain was used as a plasminogen activator, indicating that the tPA A-chain should be responsible for the non-Michaelis-Menten behavior. Binding studies of plasminogen to immobilized tPA A-chain in fact demonstrated a saturable binding of Glu- and miniplasminogen to the A-chain of tPA with a KD approximately 0.1 microM and one binding site per molecule of tPA A-chain. These data suggested a modifier mechanism responsible for the nonlinear plots whereby the substrate plasminogen itself could function as a modifier. When such a mechanism was included into a model for tPA-mediated plasminogen activation, the experimentally obtained data could be fitted into such a model by nonlinear regression analysis with resulting p-values of less than 0.001.     

Fewer protective cytotoxic T-cell epitopes than T-helper-cell epitopes on vesicular stomatitis virus.

Cytotoxic T-lymphocyte (CTL) and T-helper-cell responses in various mouse strains were monitored. Protective CTL responsiveness against three proteins of vesicular stomatitis virus was H-2 linked and inducible only in half of the 15 combinations tested (each of five H-2 haplotypes combined with each of three viral proteins), whereas biologically relevant T-helper-cell responses were inducible in all. This suggests that vesicular stomatitis virus exhibits more T-helper-cell than CTL epitopes.     

Expression of the Cytoskeletal-Associated Protein Filamin in Adult Rat Organs

Filamin is a well-characterized actin-associated protein first isolated from chicken smooth muscle. Subsequently, this polypeptide and its nonmuscle homolog actin-binding protein have been shown to be expressed in avian muscle tissue, mammalian smooth muscle, mammalian macrophages and other blood cell types, as well as several cultured cell lines. In this report, the occurrence of this polypeptide in adult mammalian organs has been investigated. Immunoblot analysis using three anti-filamin monoclonal antibodies showed that this protein was largely detected in adult rat organs that possess a substantial smooth muscle component. Furthermore, the limited expression of filamin in smooth muscle tissue was corroborated by immunohistochemical analysis. In contrast to avian systems, filamin was never found in detectable quantities in either mammalian cardiac or skeletal muscle. Quantitative immunoblot analysis demonstrated that filamin amounts roughly correlated with the abundance of the smooth muscle component of a given organ, comprising as much as 16.5% of the total SDS-extractable protein in bovine aorta. Work in avian systems and cells in culture has suggested that filamin is a rather ubiquitous cytoskeletal element. By contrast, this work demonstrates that filamin is highly restricted in its expression in mammalian organ systems, in situ.     

Investigation of inheritance of chronic inflammatory bowel diseases by complex segregation analysis.

OBJECTIVE--To investigate the mode of inheritance of ulcerative colitis and Crohn''s disease by complex segregation analysis. DESIGN--Cross sectional population based survey of familial occurrence of chronic inflammatory bowel disease. SETTING--Population of the Copenhagen county in 1987. SUBJECTS--662 patients in whom inflammatory bowel disease had been diagnosed before 1979, of whom 637 (96%) provided adequate information. Of 504 patients with ulcerative colitis, 54 had 77 relatives with ulcerative colitis and of 133 patients with Crohn''s disease, five had seven relatives with Crohn''s disease. MAIN OUTCOME MEASURES--Patterns of segregation of either disease as assessed by complex segregation analysis performed with the computer program POINTER. RESULTS--The analysis suggested that a major dominant gene with a penetrance of 0.20-0.26 is present in 9-13% of adult patients with ulcerative colitis. The analysis did not allow for other components in the familial aggregation. For Crohn''s disease the best fitting model included a major recessive gene with complete penetrance, for which 7% of the patients are homozygous. However, this model was not significantly different from a multifactorial model. CONCLUSIONS--The segregation pattern indicates that a major dominant gene has a role in ulcerative colitis, and suggests that a major recessive gene has a role in Crohn''s disease.     

Isolation and composition of the subunits of spinach chloroplast coupling factor protein.

A more convenient method for preparing large amounts of spinach chloroplast coupling factor is described, in which centrifugation of the EDTA-extracted chloroplasts is replaced by batchwise adsorption on DEAE-cellulose followed by filtration through Miracloth. Methods have been developed to purify the subunits from coupling factor dissociated by sodium dodecyl sulfate, involving hydroxylapatite chromatography followed by gel filtration with the detergent still present. The amino acid composition of the subunits purified by these methods was determined, with some differences noted in values for cysteine, tyrosine, phenylalanine, and methionine compared to previously published values. The stoichiometry of the subunits was estimated as 2:2:1:1:2 from their relative adsorption of dye after gel electrophoresis, compared to dye adsorbed by known amounts of the purified subunits. Estimates of subunit stoichiometry are rounded off to nearest whole numbers; actual preparations of coupling factor usually show less than complete amounts of the two smallest subunits.     

Fibronectin decreases the stimulatory effect of fibrin and fibrinogen fragment FCB-2 on plasmin formation by tissue plasminogen activator.

Fibronectin is a dimeric glycoprotein (Mr 440,000) involved in many adhesive processes. During blood coagulation it is bound and cross-linked to fibrin. Fibrin binding is achieved by structures (type I repeats) which are homologous to the "finger" domain of tissue plasminogen activator. Tissue plasminogen activator also binds to fibrin via the finger domain and additionally via the "kringle 2" domain. Fibrin binding of tissue plasminogen activator results in stimulation of its activity and plays a crucial role in fibrinolysis. Since fibronectin might interfere with this binding, we studied the effect of fibronectin on plasmin formation by tissue plasminogen activator. In the absence of fibrin, fibronectin had no effect on plasminogen activation. In the presence of stimulating fibrinogen fragment FCB-2, fibronectin increased the duration of the initial lag phase (= time period until maximally stimulated plasmin formation occurs) and decreased the rate of maximal plasmin formation which occurs after that lag phase mainly by increasing the Michaelis constant (Km). These effects of fibronectin were dose-dependent and were similar with single- and two-chain tissue plasminogen activator. They were also observed with plasmin-pretreated FCB-2. An apparent Ki of 43 micrograms/ml was calculated for the inhibitory effect of fibronectin when plasminogen activation by recombinant single-chain tissue plasminogen activator was studied in the presence of 91 micrograms/ml FCB-2. When a recombinant tissue plasminogen activator mutant lacking the finger domain was used in a system containing FCB-2, no effect of fibronectin was seen, indicating that the inhibitory effect of fibronectin might in fact be due to competition of fibronectin and tissue plasminogen activator for binding to fibrin(ogen) via the finger domain.