Linus L. Shen

Effects of calcium ion and covalent crosslinking on formation and elasticity of fibrin gels

Abstract The elastic properties of fibrin gels prepared under a variety of conditions were studied using a Couette elastometer. Fibrin gels formed either by addition of thrombin to fibrinogen or by dilution of previously prepared fibrin monomer were seen to be dependent on calcium ion for the expression of maximum elasticity. By dialyzing a fibrinogen solution it was shown that two calcium ions per mole are tightly bound. Factor XIIIa was shown to have an effect on the elastic modulus by means of its crosslinking the α-chains to form α-polymer. γ-γ crosslinking caused no additional increase over calcium in elasticity of fibrin gels. The relative effects of calcium concentration on the rates of crosslinking of α-chains and γ-chains to form α-polymers and γ-γ dimers were determined by means of SDS gel electrophoresis in the presence of dithiothreitol. At calcium concentrations below 2×10 −4 M α- and γ-chain crosslinking proceed at the same rate which depends strongly on the calcium concentration, while at higher concentrations the rate of α-crosslinking remains constant and the γ-chain crosslinking rate is dependent on calcium concentration. Factor XIIIa was shown also to lower the gel point of dilute solutions of fibrinogen clotted with thrombin in the presence of calcium. This demonstrates that Factor XIIIa promotes gelation in very dilute fibrin solution, probably by irreversibly crosslinking intermediate polymers of fibrin.

In Vitro Antibacterial Potency and Spectrum of ABT-492, a New Fluoroquinolone

ABSTRACT ABT-492 demonstrated potent antibacterial activity against most quinolone-susceptible pathogens. The rank order of potency was ABT-492 > trovafloxacin > levofloxacin > ciprofloxacin against quinolone-susceptible staphylococci, streptococci, and enterococci. ABT-492 had activity comparable to those of trovafloxacin, levofloxacin, and ciprofloxacin against seven species of quinolone-susceptible members of the family Enterobacteriaceae, although it was less active than the comparators against Citrobacter freundii and Serratia marcescens. The activity of ABT-492 was greater than those of the comparators against fastidious gram-negative species, including Haemophilus influenzae, Moraxella catarrhalis, Neisseria gonorrhoeae, and Legionella spp. and against Pseudomonas aeruginosa and Helicobacter pylori. ABT-492 was as active as trovafloxacin against Chlamydia trachomatis, indicating good intracellular penetration and antibacterial activity. In particular, ABT-492 was more active than trovafloxacin and levofloxacin against multidrug-resistant Streptococcus pneumoniae, including strains resistant to penicillin and macrolides, and H. influenzae, including β-lactam-resistant strains. It retained greater in vitro activity than the comparators against S. pneumoniae and H. influenzae strains resistant to other quinolones due to amino acid alterations in the quinolone resistance-determining regions of the target topoisomerases. ABT-492 was a potent inhibitor of bacterial topoisomerases, and unlike the comparators, DNA gyrase and topoisomerase IV from either Staphylococcus aureus or Escherichia coli were almost equally sensitive to ABT-492. The profile of ABT-492 suggested that it may be a useful agent for the treatment of community-acquired respiratory tract infections, as well as infections of the urinary tract, bloodstream, and skin and skin structure and nosocomial lung infections.

Fibrin gel structure: influence of calcium and covalent cross-linking on the elasticity.

Abstract Calcium ion causes the development of a higher elastic modulus in fibrinogen solutions clotted by thrombin. By also measuring the development of covalent crosslinks introduced by activated Factor XIII, we find that (a) calcium ion causes an overall increase of the modulus, (b) covalent crosslinking of α-chains causes an increase of the modulus, while (c) covalent crosslinking of γ-chains does not.

A physical standard of fibrinogen: Measurement of the elastic modulus of dilute fibrin gels with a new elastometer

Abstract We describe the construction and use of an inexpensive and easy to use Couette elastometer with buoyant inner cylinder for measurements with dilute gels. With this instrument we have performed measurements of the static elasticity on fibrin gels at concentrations between 0.002 and 1 mg/ml with elastic moduli between 0.02 and 100 dyne/cm 2 . Measurements with gels with lower elastic moduli are feasible; however, at lower fibrin concentrations no gelation is observed. The effects on the modulus of varying the concentration of calcium ion, of cross-linking of fibrin by Factor XIII a and of digestion of fibrinogen by plasmin have all recently been determined. The modulus is found to be a much more sensitive indication of damage done to fibrinogen than is the “clottability”. Consequently, we propose that the elastic shear modulus of dilute fibrin gels measured with this instrument be used as a standard for the characterization of fibrinogen samples.

Mechanism of Action of a Novel Series of Naphthyridine-Type Ribosome Inhibitors: Enhancement of tRNA Footprinting at the Decoding Site of 16S rRNA

ABSTRACT The novel ribosome inhibitors (NRIs) are a broad-spectrum naphthyridine class that selectively inhibits bacterial protein synthesis (P. J. Dandliker et al., Antimicrob. Agents Chemother. 47:3831-3839, 2003). Footprinting experiments, using a range of NRIs and chemical modification agents on Escherichia coli ribosomes, revealed no evidence for direct protection of rRNA. In the presence of tRNA, however, we found that NRIs enhanced the known ribosomal footprinting pattern of tRNA in a dose-dependent manner. The most prominent increase in protection, at A1492/3 and A1413 in helix-44 of 16S RNA, strictly required the presence of tRNA and poly(U), and the effect was correlated with the potency of the inhibitor. Radioligand binding studies with inhibitor [3H]A-424902 showed that the compound binds to tRNA, either in its charged or uncharged form. The dissociation constant for [3H]A-424902 binding to Phe-tRNAPhe was determined to be 1.8 μM, near its translation inhibition potency of 1.6 μM in a cell-free S. pneumoniae extract assay. The compound did not change the binding of radiolabeled tRNA to the 30S ribosomal subunit. Taken together, these results imply that the NRIs exert their effects on protein synthesis by structurally perturbing the tRNA/30S complex at the decoding site.