An Arginine Specific Protease from Spirulina platensis

An arginine specific protease, Sp-protease, was purified by column chromatography from freeze-dried Spirulina platensis using a five-step process. Purified Sp-protease has a molecular weight of 80 kDa. It hydrolyzed the synthetic substrates containing arginine residue in the P1 position but did not hydrolyze synthetic substrates containing other amino acid residues, including lysine residue in the P1 position. Among the synthetic substrates tested, a substrate of plasminogen activator (Pyr-Gly-Arg-MCA) was hydrolyzed most effectively with the enzyme (K_m = 5.5 × 10⁻⁶ M), and fibrin gel was solubilized via activation of intrinsic plasminogen to plasmin with the enzyme. Activity was inhibited completely with camostat mesilate (K_i = 1.1 × 10⁻⁸ M) and leupeptin (K_i = 3.9 × 10⁻⁸ M) but was not inhibited with N^α-tosyl-L-lysine chloromethyl ketone (TLCK). The optimum pH of the enzyme has a range of pH 9.0 to pH 11.0. The optimum temperature was 50°C; the enzyme was stable at 0–50°C.     

Statins suppress oxidized low density lipoprotein-induced macrophage proliferation by inactivation of the small G protein-p38 MAPK pathway

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) ameliorate atherosclerotic diseases. Macrophages play an important role in the development and subsequent stability of atherosclerotic plaques. We reported previously that oxidized low density lipoprotein (Ox-LDL) induced macrophage proliferation through the secretion of granulocyte/macrophage colony-stimulating factor (GM-CSF) and the consequent activation of p38 MAPK. The present study was designed to elucidate the mechanism of the inhibitory effect of statins on macrophage proliferation. Mouse peritoneal macrophages were used in our study. Cerivastatin and simvastatin each inhibited Ox-LDL-induced [(3)H]thymidine incorporation into macrophages. Statins did not inhibit Ox-LDL-induced GM-CSF production, but inhibited GM-CSF-induced p38 MAPK activation. Farnesyl transferase inhibitor and geranylgeranyl transferase inhibitor inhibited GM-CSF-induced macrophage proliferation, and farnesyl pyrophosphate and geranylgeranyl pyrophosphate prevented the effect of statins. GM-CSF-induced p38 MAPK phosphorylation was also inhibited by farnesyl transferase inhibitor or geranylgeranyl transferase inhibitor, and farnesyl pyrophosphate and geranylgeranyl pyrophosphate prevented the suppression of GM-CSF-induced p38 MAPK phosphorylation by statins. Furthermore, we found that statin significantly inhibited the membrane translocation of the small G protein family members Ras and Rho. GM-CSF-induced p38 MAPK activation and macrophage proliferation was partially inhibited by overexpression of dominant negative Ras and completely by that of RhoA. In conclusion, statins inhibited GM-CSF-induced Ras- or RhoA-p38 MAPK signal cascades, thereby suppressing Ox-LDL-induced macrophage proliferation. The significant inhibition of macrophage proliferation by statins may also explain, at least in part, their anti-atherogenic action.     

Forest gap dynamics and the Ising model

The vegetation height in forest ecosystems is spatially heterogeneous. Canopy gaps (sites with low vegetation) are formed by treefalls, and they recover to canopy sites (with high vegetation) either by growth of small trees or by branch extension of surrounding trees. The dynamics of canopy gaps have been studied using a spatial Markov chain with nearest neighbor interaction. (1) If the canopy recovery rate is constant and if the gap formation rate for a site increases exponentially with the number of neighboring gap sites, the equilibrium distribution is the same as the one generated by the Ising model in statistical mechanics. Here, we extend the equivalence to the situation in which both the gap formation and canopy recovery depend on the neighborhood, as shown in recent forest data. (2) We develop a statistical test of whether a given spatial pattern is a random sample from the Ising model. The test is based on the conditional probability of configurations on a partial lattice. We apply the method to vegetation height data from the Ogawa forest reserve, Japan, measured on a 5x5 m grid in 1976, 1981, 1986, and 1991. The spatial pattern of the original forest data deviates significantly from the Ising model. We examine whether a larger sampling distance or the removal of the effects of the topography can reduce this deviation.     

How should we define goodness?--reputation dynamics in indirect reciprocity

Theory of indirect reciprocity is important in explaining cooperation between humans. Since a partner of a social interaction often changes, an individual should assess his partner by using social information such as reputation and make decisions whether to help him or not. To those who have 'good' social reputation does a player give aid as reciprocation, whereas he has to refuse to help those who have 'bad' reputation. Otherwise benefits of altruism is easily exploited by them. Little has been known, however, about the definition of 'goodness' in reputation. What kind of actions are and should be regarded as good and what kind of actions bad? And what sort of goodness enables sustaining exchange of altruism? We herein challenge this question with an evolutionary perspective. We generalize social reputation as 'Honor-score' (H-score) and examine the conditions under which individuals in a group stably maintain cooperative relationships based on indirect reciprocity. We examine the condition for evolutionarily stable strategies (ESSs) over 4096 possible cases exhaustively. Mathematical analysis reveals that only eight cases called 'leading eight' are crucial to the evolution of indirect reciprocity. Each in the leading eight shares two common characteristics: (i) cooperation with good persons is regarded as good while defection against them is regarded as bad, and (ii) defection against bad persons should be regarded as a good behavior because it works as sanction. Our results give one solution to the definition of goodness from an evolutionary viewpoint. In addition, we believe that the formalism of reputation dynamics gives general insights into the way social information is generated, handled, and transmitted in animal societies.     

Global mutations and local mutations have very different effects on evolution, illustrated by mixed strategies of asymmetric binary games

We study the evolutionary effect of rare mutations causing global changes in traits. We consider asymmetric binary games between two players. The first player takes two alternative options with probability x and 1−x; and the second player takes options with probability y and 1−y. Due to natural selection and recurrent mutation, the population evolves to have broad distributions of x and y. We analyze three cases showing qualitatively different dynamics, exemplified by (1) vigilance-intrusion game, (2) asymmetric hawk-dove game and (3) cleaner-client game. We found that the evolutionary outcome is strongly dependent upon the distribution of mutants’ traits, more than the mutation rates. For example in the vigilance-intrusion game, the evolutionary dynamics show a perpetual stable oscillation if mutants are always close to the parent (local-mutation mode), whilst the population converges to a stable equilibrium distribution if mutants can be quite different from the parent (global-mutation mode), even for extremely low mutation rate. When common local mutations and rare global mutations occur simultaneously, the evolutionary outcome is controlled by the latter.     

Functional characterization of D-galacturonic acid reductase, a key enzyme of the ascorbate biosynthesis pathway, from Euglena gracilis

D-Galacturonic acid reductase, a key enzyme in ascorbate biosynthesis, was purified to homogeneity from Euglena gracilis. The enzyme was a monomer with a molecular mass of 38-39 kDa, as judged by SDS-PAGE and gel filtration. Apparently it utilized NADPH with a Km value of 62.5+/-4.5 microM and uronic acids, such as D-galacturonic acid (Km=3.79+/-0.5 mM) and D-glucuronic acid (Km=4.67+/-0.6 mM). It failed to catalyze the reverse reaction with L-galactonic acid and NADP(+). The optimal pH for the reduction of D-galacturonic acid was 7.2. The enzyme was activated 45.6% by 0.1 mM H(2)O(2), suggesting that enzyme activity is regulated by cellular redox status. No feedback regulation of the enzyme activity by L-galactono-1,4-lactone or ascorbate was observed. N-terminal amino acid sequence analysis revealed that the enzyme is closely related to the malate dehydrogenase families.     

Evolution of resistance during clonal expansion

Acquired drug resistance is a major limitation for cancer therapy. Often, one genetic alteration suffices to confer resistance to an otherwise successful therapy. However, little is known about the dynamics of the emergence of resistant tumor cells. In this article, we consider an exponentially growing population starting from one cancer cell that is sensitive to therapy. Sensitive cancer cells can mutate into resistant ones, which have relative fitness alpha prior to therapy. In the special case of no cell death, our model converges to the one investigated by Luria and Delbrück. We calculate the probability of resistance and the mean number of resistant cells once the cancer has reached detection size M. The probability of resistance is an increasing function of the detection size M times the mutation rate u. If Mu < 1, then the expected number of resistant cells in cancers with resistance is independent of the mutation rate u and increases with M in proportion to M(1-1/alpha) for advantageous mutants with relative fitness alpha>1, to l nM for neutral mutants (alpha = 1), but converges to an upper limit for deleterious mutants (alpha<1). Further, the probability of resistance and the average number of resistant cells increase with the number of cell divisions in the history of the tumor. Hence a tumor subject to high rates of apoptosis will show a higher incidence of resistance than expected on its detection size only.     

Mathematical Study of the Role of Delta/Notch Lateral Inhibition during Primary Branching of Drosophila Trachea Development

A wide range of cellular developmental processes employ intercellular signaling via the Delta/Notch lateral inhibitory pathway to achieve stable spatial patterning. Recent genetic experiments have shown the importance of Delta/Notch lateral inhibition for regulating the number of tip cells in the tracheal primary branching of Drosophila. To examine the role of Delta/Notch regulation in the tip-cell selection, we analyzed a mathematical model of a simple lateral inhibitory system having input signals. Mathematical and numerical analyses revealed that the lateral inhibition did not amplify the signal difference between neighboring cells over the parameter ranges in which the spatial pattern of tip selection was realized. We also show that the number of tip cells becomes less affected by a fluctuation of the input gradient signal as the lateral inhibition becomes stronger. In addition, we demonstrate that the lateral inhibitory regulation enhances the robustness of the tip-cell selection compared with a system regulated by self-inhibition, an alternative means of inhibitory regulation. These results suggest that the lateral inhibition promotes the robustness of tip-cell selection in the tracheal development of Drosophila.     

Role of the Actin Ala-108–Pro-112 Loop in Actin Polymerization and ATPase Activities

Actin plays fundamental roles in a variety of cell functions in eukaryotic cells. The polymerization-depolymerization cycle, between monomeric G-actin and fibrous F-actin, drives essential cell processes. Recently, we proposed the atomic model for the F-actin structure and found that actin was in the twisted form in the monomer and in the untwisted form in the filament. To understand how the polymerization process is regulated (Caspar, D. L. (1991) Curr. Biol. 1, 30–32), we need to know further details about the transition from the twisted to the untwisted form. For this purpose, we focused our attention on the Ala-108–Pro-112 loop, which must play crucial roles in the transition, and analyzed the consequences of the amino acid replacements on the polymerization process. As compared with the wild type, the polymerization of P109A was accelerated in both the nucleation and the elongation steps, and this was attributed to an increase in the frequency factor of the Arrhenius equation. The multiple conformations allowed by the substitution presumably resulted in the effective formation of the collision complex, thus accelerating polymerization. On the other hand, the A108G mutation reduced the rates of both nucleation and elongation due to an increase in the activation energy. In the cases of polymerization acceleration and deceleration, each functional aberration is attributed to a distinct elementary process. The rigidity of the loop, which mediates neither too strong nor too weak interactions between subdomains 1 and 3, might play crucial roles in actin polymerization.