Self-organization of the vascular system in plant leaves: inter-dependent dynamics of auxin flux and carrier proteins

The vegetative hormone Auxin is involved in vascular tissues formation throughout the plant. Trans-membrane carrier proteins transporting auxin from cell to cell and distributed asymmetrically around each cell give to auxin a polarized movement in tissues, creating streams of auxin that presume future vascular bundles. According to the canalization hypothesis, auxin transport ability of cells is thought to increase with auxin flux, resulting in the self-enhancement of this flux along auxin paths. In this study we evaluate a series of models based on canalization hypothesis using carrier proteins, under different assumptions concerning auxin flux formation and carrier protein dynamics. Simulations are run on a hexagonal lattice with uniform auxin production. A single cell located in the margin of the lattice indicates the petiole, and acts as an auxin sink. The main results are: (1) We obtain branching auxin distribution patterns. (2) The type of self-enhancement described by the functional form of the carrier proteins regulation responding to the auxin flux intensity in different parts of a cell, has a strong effect on the possibility of generating the branching patterns. For response functions with acceleration in the increase of carrier protein numbers compared to the auxin flux, branching patterns are likely to be generated. For linear or decelerating response functions, no branching patterns are formed. (3) When branching patterns are formed, auxin distribution greatly differs between the case in which the number of carrier proteins in different parts of a cell are regulated independently, and the case in which different parts of a cell compete for a limited number of carrier proteins. In the former case, the auxin level is lower in veins than in the surrounding tissue, while in the latter, the auxin is present in greater abundance in veins. These results suggest that canalization is a good candidate for describing plant vein pattern formation.     

Electron transfer ability from NADH to menaquinone and from NADPH to oxygen of type II NADH dehydrogenase of Corynebacterium glutamicum

Type II NADH dehydrogenase of Corynebacterium glutamicum (NDH-2) was purified from an ndh overexpressing strain. Purification conferred 6-fold higher specific activity of NADH:ubiquinone-1 oxidoreductase with a 3.5-fold higher recovery than that previously reported (K. Matsushita et al., 2000). UV-visible and fluorescence analyses of the purified enzyme showed that NDH-2 of C. glutamicum contained non-covalently bound FAD but not covalently bound FMN. This enzyme had an ability to catalyze electron transfer from NADH and NADPH to oxygen as well as various artificial quinone analogs at neutral and acidic pHs respectively. The reduction of native quinone of C. glutamicum, menaquinone-2, with this enzyme was observed only with NADH, whereas electron transfer to oxygen was observed more intensively with NADPH. This study provides evidence that C. glutamicum NDH-2 is a source of the reactive oxygen species, superoxide and hydrogen peroxide, concomitant with NADH and NADPH oxidation, but especially with NADPH oxidation. Together with this unique character of NADPH oxidation, phylogenetic analysis of NDH-2 from various organisms suggests that NDH-2 of C. glutamicum is more closely related to yeast or fungal enzymes than to other prokaryotic enzymes.     

Regulation of isthmic Fgf8 signal by sprouty2

Fgf8 functions as an organizer at the mes/metencephalic boundary (isthmus). We showed that a strong Fgf8 signal activates the Ras-ERK signaling pathway to organize cerebellar differentiation. Sprouty2 is expressed in an overlapping manner to Fgf8, and is induced by Fgf8. Its function, however, is indicated to antagonize Ras-ERK signaling. Here, we show the regulation of Fgf8 signaling in relation to Sprouty2. sprouty2 expression was induced very rapidly by Fgf8b, but interfered with ERK activation. sprouty2 misexpression resulted in a fate change of the presumptive metencephalon to the mesencephalon. Misexpression of a dominant negative form of Sprouty2 augmented ERK activation, and resulted in anterior shift of the posterior border of the tectum. The results indicate that Fgf8 activates the Ras-ERK signaling pathway to differentiate the cerebellum, and that the hyper- or hypo-signaling of this pathway affects the fate of the brain vesicles. Sprouty2 may regulate the Fgf8-Ras-ERK signaling pathway for the proper regionalization of the metencephalon and mesencephalon.     

Design, synthesis, and biological activity of non-basic compounds as factor Xa inhibitors: SAR study of S1 and aryl binding sites

Compound 7 was identified as the active metabolite of 6 by HPLC and mass spectral analysis. Modification of lead compound 7 by transformation of its N-oxide 6-6 biaryl ring system and fused aromatics produced a series of non-basic fXa inhibitors with excellent potency in anti-fXa and anticoagulant assays. The optimized compounds 73b and 75b showed sub to one digit micromolar anticoagulant activity (PTCT2). Particularly, anti-fXa activity was detected in plasma of rats orally administered with 1mg/kg of compound 75b.     

Transient expression of laminin alpha1 chain in regenerating murine liver: restricted localization of laminin chains and nidogen-1

Most interstitia between epithelial and endothelial cells contain basal laminae (BLs), as defined by electron microscopy. However, in liver, the sinusoidal interstitium (called space of Disse) between hepatocytes and sinusoidal endothelial cells (SECs) lacks BLs. Because laminins are major components of BLs throughout the body, whether laminins exist in sinusoids has been a controversial issue. Despite recent advances, the distribution and expression of laminin chains have not been well defined in mammalian liver. Here, using a panel of antibodies, we examined laminins in normal and regenerating mouse livers. Of alpha chains, alpha5 was widely observed in all BLs except for sinusoids, while the other alpha chains were variously expressed in Glisson's sheath and central veins. Laminin gamma1 was also distributed to all BLs except for sinusoids. Although the beta2 chain was observed in all BLs and sinusoids, the expression of beta1 chain was restricted to Glisson's sheath. Detailed analysis of regenerating liver revealed that alpha1 and gamma1 chains appeared in sinusoids and were produced by stellate cells. The staining of alpha1 and gamma1 chains reached its maximum intensity at 6 days after two-thirds partial hepatectomy (PHx). Moreover, in vitro studies showed that alpha1-containing laminin promoted spreading of sinusoidal endothelial cells (SECs) isolated from normal liver, but not other hepatic cells. In addition, SECs isolated from regenerating liver elongated pseudopodia on alpha1-containing laminin more so than did cells from normal liver. The transient expression of laminin alpha1 may promote formation of sinusoids after PHx.     

Synthesis, structures, and magnetic properties of dicopper(II) and dinickel(II) complexes with a new bis(macrocycle), 7,7-(2-hydoxypropane-1,3-diyl)bis{3,7,11,17-tetraazabicyclo[11.3.1]- heptadeca-1(17),13,15-triene}

A new bis(macrocycle) ligand, 7,7^′-(2-hydoxypropane-1,3-diyl)-bis{3,7,11,17-tetraazabicyclo[11.3.1]heptadeca-1(17),13,15-triene} (HL), and its dicopper(II) ([Cu₂(HL)Cl₂](NO₃)₂ · 4H₂O (4a), [Cu₂(HL)I₂]I₂ · H₂O (4b)) and dinickel(II) ([Ni₂(L)(OH₂)](ClO₄)₃ (5a), [Ni₂(L)(OH₂)]I₃ · 2H₂O (5b), [Ni₂(L)N₃](N₃)₂ · 7H₂O (5c)) complexes have been synthesized. The alkoxide bridged face-to-face structure of the dinickel(II) complex 5c has been revealed by X-ray crystallography, as well as the “half-opened clamshell” form of the bis(macrocyclic) dicopper(II) complex 4b. Variable temperature magnetic susceptibility studies have indicated that there exists intramolecular antiferromagnetic coupling (J=−33.8 cm⁻¹ (5a), −32.5 cm⁻¹ (5b), and −29.7 cm⁻¹ (5c)) between the two nickel(II) ions in the nickel(II) complexes.     

Transmission of force and displacement within the myosin molecule

Myosin is a repetitive impeller of actin, using its catalysis of ATP hydrolysis to derive repeatedly the required free energy decrements. In each impulsion, changes at the myosin active site are transmitted through a series of structural elements to the myosin propeller (lever arm), almost 5 nm away. While the nature of transmission through most elements is evident, that through the so-called converter is not. To investigate how the converter changes linear displacement into rotation, we tested (one at a time) the effect of two Phe residue mutations (at 721 and 775) in the converter on the overall function of a heavy meromyosin (or subfragment 1) system, after first showing by observing kinetic behaviors that neither mutation affects other elements in the transmission. Using three tests (direct movement of the lever arm, activity in a motility assay with actin filaments, and direct force measurement of lever arm function), we found that these mutations affected only movements of the converter and the lever arm. From interpreting our observations in terms of the structure of the converter, we deduce that the linear-rotational transformation in the converter is mediated by a little machine (two Phe residues linked to a Gly) within a machine.     

Analyzing steady states of dynamics of bio-molecules from the structure of regulatory networks

Regulatory relations between biological molecules constitute complex network systems and realize diverse biological functions through the dynamics of molecular activities. However, we currently have very little understanding of the relationship between the structure of a regulatory network and its dynamical properties. In this paper we introduce a new method, named “linkage logic” to analyze the dynamics of network systems. By this method, we can restrict possible steady states of a given complex network system from the knowledge of regulatory linkages alone. The regulatory linkage simply specifies the list of variables that affect the dynamics of each variable. We formalize two aspects of the linkage logic: the “Principle of Compatibility” determines the upper limit of the diversity of possible steady states of the dynamics realized by a given network; the “Principle of Dependency” determines the possible combinations of states of the system. By combining these two aspects, (i) for a given network, we can identify a cluster of nodes that gives an alternative representation of the steady states of the whole system, (ii) we can reduce a given complex network into a simpler one without loss of the ability to generate the diversity of steady states, (iii) we can examine the consistency between the structure of network and observed set of steady states, and (iv) sometimes we can predict unknown states or unknown regulations from an observed set of steady states alone. We illustrate the method by several applications to an experimentally determined regulatory network for biological functions.