Equilibria in the fibrinogen-fibrin conversion. IX. Effects of calcium ions on the reversible polymerization of fibrin monomer

An investigation was made of the role of calcium ions in the reversible stage of fibrin polymerization, using a direct and relatively simple approach. Purified fibrin monomer in solution (7.5 mg/ml) in 1.0 m NaBr (pH 5.3) was polymerized by raising the pH to 5.7–7.7 by the addition of aliquots of standard NaOH solution and the rate and total extent of proton release during polymerization were measured potentiometrically. In the presence of added CaCl₂ (10⁻⁵-10⁻²m) the rate of proton release was increased and the clotting time was decreased. The profile of equilibrium proton release vs pH of polymerization was also shifted, the maximum being increased and occurring at a lower pH. Sedimentation velocity studies in the intermediate pH range (5.7–6.0) showed that the altered profile of equilibrium proton release was due to a broadening of the pH range of polymerization, and that polymerization remained reversible in the presence of CaCl₂. At pH 5.3, where fibrin is essentially monomeric, addition of CaCl₂ resulted in the release of protons and small increases in sedimentation coefficient and reduced viscosity. Under the same conditions, a similar release of protons was observed from fibrinogen, but there was no effect on its sedimentation coefficient. It was concluded that the proton release at pH 5.3 was due mainly to binding of calcium ions to fibrinogen and fibrin monomer. The effect of CaCl₂ on the sedimentation coefficient of fibrin at pH 5.3 was found to decrease with decreasing protein concentration, indicating that it was the result of a small extent of polymerization, rather than a conformational change. Added MgCl₂ had no effect on fibrin monomer at pH 5.3 and no significant effect on the rate or extent of proton release during polymerization at higher pH, indicating that there are specific binding sites for calcium ions in fibrinogen and fibrin. The observed effects of bound calcium ions on reversible fibrin polymerization are explained most simply in electrostatic terms.     

Evidence for two growth steps in microtubule polymerization

For microtubule assembly, the data reported here support an initial nucleation phase followed by a growth or elongation phase. The nucleation phase was not detected kinetically. Evidence for this step was given by the existence of the critical concentration and the dependence of the number of microtubules on oligomer concentration.Kinetic evidence indicated the existence of two consecutive steps in the growth phase of microtubules. The fast process increased and the slow one decreased with the concentration of microtubule protein. Similar kinetics were found upon recombination of tubulin oligomer and dimer which had been resolved by agarose chromatography. The fast process increased with oligomer and decreased with dimer concentration while the slow one depended positively on dimer concentration. Microtubules were formed when the oligomeric fraction only was employed. In contrast, under identical conditions, no microtubule formation was detected turbidimetrically or by electron microscopy from dimer alone. When dimer caused elongation of seed tubules, there was only one growth step with a rate constant of the same order of magnitude as the slow process for the other experiments.     

Central Equilibria in Multilocus Systems. II. Bisexual Generalized Nonepistatic Selection Models

This paper is a continuation of the paper "Central Equilibria in Multilocus Systems I," concentrating on existence and stability properties accruing to central H-W type equilibria in multilocus bisexual systems acted on by generalized nonepistatic selection forces coupled to recombination events. The stability conditions are discussed and interpreted in three perspectives, and the influence of sexual differences in linkage relationships together with sex-dependent selection is appraised. In this case we deduce that the stability conditions of the H-W polymorphism in the bisexual model coincide exactly with the conditions for the corresponding monoecious model, provided that the recombination distribution imposed is that of the arithmetic mean of the male and female recombination distributions. A second concern has the same recombination distribution for both sexes, but contrasting selection regimes between sexes. It is then established that, with respect to discerning the relevance of the H-W equilibrium, there is an equivalent monoecious selection regime which is an appropriate "weighted combination" of the male and female selection forms. Finally, in the case where the selection and recombination structures are both sex dependent, a hierarchy of comparisons is elaborated, seeking to unravel the nature of selection-recombination interaction for monoecious versus diocecious systems.     

Ureteric bud controls multiple steps in the conversion of mesenchyme to epithelia

Conversion of renal mesenchyme into epithelia depends on the ureteric bud, but its specific actions are not established. From conditioned media of ureteric bud cells, we have identified molecules that mimic the growth and epithelialization of mesenchyme in vivo. LIF targets late epithelial progenitors surrounding the ureteric bud, and in combination with survival factors, converts them into nephrons. In contrast, 24p3/Ngal targets early progenitors at the kidney's periphery through an iron-mediated, but a transferrin-independent mechanism. Hence, the ureteric bud controls many steps of cell conversion. A genome wide search for ureteric bud-specific molecules will identify additional pathways that induce morphogenesis.     

Initial steps of ferulic acid polymerization by lignin peroxidase

The major products of the initial steps of ferulic acid polymerization by lignin peroxidase included three dehydrodimers resulting from beta-5' and beta-beta'coupling and two trimers resulting from the addition of ferulic acid moieties to decarboxylated derivatives of beta-O-4'- and beta-5'-coupled dehydrodimers. This is the first time that trimers have been identified from peroxidase-catalyzed oxidation of ferulic acid, and their formation appears to be favored by decarboxylation of dehydrodimer intermediates. After initial oxidation, the coupling reactions appear to be determined by the chemistry of ferulic acid phenoxy radicals, regardless of the enzyme and of whether the reaction is performed in vitro or in vivo. This claim is supported by our finding that horseradish peroxidase provides a similar product profile. Furthermore, two of the dehydrodimers were the two products obtained from laccase-catalyzed oxidation (Tatsumi, K. S., Freyer, A., Minard, R. D., and Bollag, J.-M. (1994) Environ. Sci. Technol. 28, 210-215), and the most abundant dehydrodimer is the most prominent in grass cell walls (Ralph, J., Quideau, S., Grabber, J. H., and Hatfield, R. D. (1994) J. Chem. Soc. Perkin Trans. 1, 3485-3498). Our results also indicate that the dehydrodimers and trimers are further oxidized by lignin peroxidase, suggesting that they are only intermediates in the polymerization of ferulic acid. The extent of polymerization appears to be dependent on the ionization potential of formed intermediates, H(2)O(2) concentration, and, probably, enzyme stability.     

Reversibility and the age of an allele II. Two-allele models,with selection and mutation

A Markov process with absorbing boundaries may be made recurrent by returning the process to the interior whenever a boundary is reached. The age of such a process may be defined as the length of time since the last return event. Examples drawn from two-allele genetic models are discussed, in which reversibility of the return process means that the age of an allele, whose present frequency in the population is known, has the same probability distribution as its future extinction time. Some discrete models are not reversible, yet if approximated by diffusion processes, the (approximate) age distribution is the same as the future extinction time distribution. Various results in the literature are unified by this viewpoint.     

Primary steps in the energy conversion reaction of the cytochrome bc1 complex Qo site

The primary energy conversion (Qo) site of the cytochrome bc1 complex is flanked by both high- and low-potential redox cofactors, the [2Fe-2S] cluster and cytochrome bL, respectively. From the sensitivity of the reduced [2Fe-2S] cluster electron paramagnetic resonance (EPR) spectral g(x)-band and line shape to the degree and type of Qo site occupants, we have proposed a double-occupancy model for the Qo site by ubiquinone in Rhodobacter capsulatus membrane vesicles containing the cytochrome bc1 complex. Biophysical and biochemical experiments have confirmed the double occupancy model and from a combination of these results and the available cytochrome bc1 crystal structures we suggest that the two ubiquinone molecules in the Qo site serve distinct catalytic roles. We propose that the strongly bound ubiquinone, termed Qos, is close to the [2Fe-2S] cluster, where it remains tightly associated with the Qo site during turnover, serving as a catalytic cofactor; and the weaker bound ubiquinone, Qow, is distal to the [2Fe-2S] cluster and can exchange with the membrane Qpool on a time scale much faster than the turnover, acting as the substrate. The crystallographic data demonstrates that the FeS subunit can adopt different positions. Our own observations show that the equilibrium position of the reduced FeS subunit is proximal to the Qo site. On the basis of this, we also report preliminary results modeling the electron transfer reactions that can occur in the cytochrome bc1 complex and show that because of the strong distance dependence of electron transfer, significant movement of the FeS subunit must occur in order for the complex to be able to turn over at the experimental observed rates.