Developmental commitment in Dictyostelium discoideum

Upon starvation, Dictyostelium discoideum cells halt cell proliferation, aggregate into multicellular organisms, form migrating slugs, and undergo morphogenesis into fruiting bodies while differentiating into dormant spores and dead stalk cells. At almost any developmental stage cells can be forced to dedifferentiate when they are dispersed and diluted into nutrient broth. However, migrating slugs can traverse lawns of bacteria for days without dedifferentiating, ignoring abundant nutrients and continuing development. We now show that developing Dictyostelium cells revert to the growth phase only when bacteria are supplied during the first 4 to 6 h of development but that after this time, cells continue to develop regardless of the presence of food. We postulate that the cells' inability to revert to the growth phase after 6 h represents a commitment to development. We show that the onset of commitment correlates with the cells' loss of phagocytic function. By examining mutant strains, we also show that commitment requires extracellular cyclic AMP (cAMP) signaling. Moreover, cAMP pulses are sufficient to induce both commitment and the loss of phagocytosis in starving cells, whereas starvation alone is insufficient. Finally, we show that the inhibition of development by food prior to commitment is independent of contact between the cells and the bacteria and that small soluble molecules, probably amino acids, inhibit development during the first few hours and subsequently the cells become unable to react to the molecules and commit to development. We propose that commitment serves as a checkpoint that ensures the completion of cooperative aggregation of developing Dictyostelium cells once it has begun, dampening the response to nutritional cues that might inappropriately block development.     

Computational chemical analysis of Ru(II)‐Pheox–catalyzed highly enantioselective intramolecular cyclopropanation reactions

Computational chemical analysis of Ru(II)‐Pheox–catalyzed highly enantioselective intramolecular cyclopropanation reactions was performed using density functional theory (DFT). In this study, cyclopropane ring–fused γ‐lactones, which are 5.8 kcal/mol more stable than the corresponding minor enantiomer, are obtained as the major product. The results of the calculations suggest that the enantioselectivity of the Ru(II)‐Pheox–catalyzed intramolecular cyclopropanation reaction is affected by the energy differences between the starting structures 5l and 5i . The reaction pathway was found to be a stepwise mechanism that proceeds through the formation of a metallacyclobutane intermediate. This is the first example of a computational chemical analysis of enantioselective control in an intramolecular carbene‐transfer reaction using C₁‐symmetric catalysts.     

Molecular cloning and characterization of FRAT2, encoding a positive regulator of the WNT signaling pathway

FRAT1 positively regulates the WNT signaling pathway by stabilizing beta-catenin through the association with glycogen synthase kinase-3beta. Here, we have cloned FRAT2 cDNAs, spanning the complete coding sequence, from a human fetal lung cDNA library. FRAT2 encoded 233 amino-acid protein, which showed 77.3% total amino-acid identity with FRAT1. FRAT2 and FRAT1 were more homologous in the acidic domain (96% identity), the proline-rich domain (92% identity), and the GSK-3beta binding domain (100% identity). The FRAT2 gene was mapped to human chromosome 10q24.1. The FRAT2 mRNA of 2.4-kb in size was relatively highly expressed in MKN45 (gastric cancer), HeLa S3 (cervical cancer), and K-562 (chronic myelogenous leukemia). Xenopus axis duplication assay revealed that the wild-type FRAT2 mRNA, but not the mutant FRAT2 mRNA lacking the acidic domain and the proline-rich domain, has the capacity to induce the secondary axis. These results indicate that FRAT2, just like FRAT1, functions as a positive regulator of the WNT signaling pathway. Thus, up-regulation of FRAT2 in human cancer might be implicated in carcinogenesis through activation of the WNT signaling pathway.     

Two new modes of smooth muscle myosin regulation by the interaction between the two regulatory light chains, and by the S2 domain

Previous studies indicated that single-headed smooth muscle myosin and S1 (a single head fragment) are not regulated through phosphorylation of the regulatory light chain (RLC). To investigate the importance of the double-headedness of myosin and of the S2 region for the phosphorylation-dependent regulation, we made three types of recombinant mutant smooth muscle HMMs with one intact head and an N-terminally truncated head. The truncated head of Delta MD lacked the motor domain, that of Delta(MD+ELC) lacked the motor and essential light chain binding domains, and single-headed HMM had one intact head alone. The basal ATPase activities of the three mutants decreased as the KCl concentration became less than 0.1 M. Such a decrease was not observed for S1, which had no S2 region, suggesting that S2 is necessary for this myosin behavior. This activity decrease also disappeared when RLCs of Delta MD and Delta(MD+ELC), but that of single-headed HMM, were phosphorylated. When their RLCs were unphosphorylated, the three mutants exhibited similar actin-activated ATPase levels. However, when they were phosphorylated, the actin-activated ATPase activities of Delta MD and Delta(MD+ELC) increased to the S1 level, while that of single-headed HMM remained unchanged. Even in the phosphorylated state, the actin-activated ATPase activities of the three mutants and S1 were much lower than that of wild-type HMM. We propose that S2 has an inhibitory function that is canceled by an interaction between two phosphorylated RLCs. We also propose that a cooperative interaction between two motor domains is required for a higher level of actin activation.     

Rho-kinase--mediated contraction of isolated stress fibers

It is widely accepted that actin filaments and the conventional double-headed myosin interact to generate force for many types of nonmuscle cell motility, and that this interaction occurs when the myosin regulatory light chain (MLC) is phosphorylated by MLC kinase (MLCK) together with calmodulin and Ca(2+). However, recent studies indicate that Rho-kinase is also involved in regulating the smooth muscle and nonmuscle cell contractility. We have recently isolated reactivatable stress fibers from cultured cells and established them as a model system for actomyosin-based contraction in nonmuscle cells. Here, using isolated stress fibers, we show that Rho-kinase mediates MLC phosphorylation and their contraction in the absence of Ca(2+). More rapid and extensive stress fiber contraction was induced by MLCK than was by Rho-kinase. When the activity of Rho-kinase but not MLCK was inhibited, cells not only lost their stress fibers and focal adhesions but also appeared to lose cytoplasmic tension. Our study suggests that actomyosin-based nonmuscle contractility is regulated by two kinase systems: the Ca(2+)-dependent MLCK and the Rho-kinase systems. We propose that Ca(2+) is used to generate rapid contraction, whereas Rho-kinase plays a major role in maintaining sustained contraction in cells.     

Analysis of molecular interactions of the p53-family p51(p63) gene products in a yeast two-hybrid system: homotypic and heterotypic interactions and association with p53-regulatory factors

p51 in the p53 tumor suppressor family, also referred to as p63, encodes multiple isoforms including p51A (TAp63gamma) and p51B (TAp63alpha). The p53 protein forms a tetramer, and its stability and activity are regulated by molecular association with viral and cellular proteins and by biochemical modifications. Using a yeast two-hybrid system, the p51A and p51B isoforms were examined for homotypic and heterotypic interactions in the p53 family proteins and for their affinity to the p53-regulatory factors. Results indicate a homotypic interaction dependent on the presumed oligomerization domain of the p51 proteins. The possibility of a weak heterotypic interaction between p51 and p73 proteins was suggested, while association between p51 and p53 appeared improbable. Furthermore, unlike p53, the p51 proteins failed to display an affinity to SV40 large T antigen or MDM2-family proteins. Having several features in common with p53, the p51 proteins may function in biological processes apart from p53.     

Functional roles of ionic and hydrophobic surface loops in smooth muscle myosin: their interactions with actin

This investigation ascertains whether, in (smooth muscle) myosin, certain residues engage in functional interactions with their actin conjugates in an actomyosin complex. Such interactions have been postulated from putting together crystallographic models of the two proteins [Rayment, I., Rypniewski, W. R., Schmidt-B?se, K., Smith, R., Tomchick, D. R., Benning, M. M., Winkelmann, D. A., Wesenberg, G., and Holden, H. M. (1993) Science 261, 50-58]. Here, in several instances, we ask whether mutation of a particular residue significantly impairs a function, and find that the answers are largely rationalized by the original postulation. Additionally, a novel element emerges from our investigation. To assess function, we test the wild type and mutant systems as they perform in the steady state of ATP degradation. In doing so, we assume, as usual, that degradation proceeds from an early stage in which the complex forms (and is described by parameter K(app)) to a later stage during which the product leaves the complex (and is described by parameter V(max)). Interestingly, certain defects induced by the mutations are associated with changes in K(app), and other defects are associated with changes in V(max), suggesting that our procedure at least roughly distinguishes between events according to the time in the degradation at which they occur. In this framework, we suggest that (1) in the actin-myosin association phase, cationic residues Lys-576 and Lys-578 interact with anionic residues of the so-called second actin, and (2) in the product leaving phase, hydrophobic residues Trp-546, Phe-547, and Pro-548, as well as the Thr-532/Asn-533/Pro-534/Pro-535 sequence, sever connections with the so-called first actin. The role of Glu-473 is also examined.     

Isolation of a new potent cytotoxic pigment along with indigotin from the pathogenic basidiomycetous fungus Schizophyllum commune

An indole derivative, schizocommunin, was isolated along with indigotin (indigo), indirubin, isatin, and tryptanthrin, from the liquid culture medium in which a culture of Schizophyllum commune, isolated from the bronchus of a human patient with allergic bronchopulmonary mycosis, had been grown. The structure of schizocommunin was established by spectroscopic investigation. Schizocommunin showed the strong cytotoxicity against murine lymphoma cells. The assignments of the ¹H- and ¹³C-NMR signals of indigotin were also listed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Oligomerization of N-tosylindole with aluminum chloride

The reactivity of N-tosylindole (4) in the presence of aluminum chloride was studied, and two types of oligomerization of 4 were observed. One type was condensation between both pyrrole parts (dimers 5 and 6 and trimer 7) and the other was between a pyrrole part and a benzene part of each indole nucleus (dimers 8 and 9).     

Crystallization and MAD data collection of high-molecular weight cytochrome c from Desulfovibrio vulgaris Miyazaki F

Hexadecaheme high molecular weight cytochrome c from a sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F has been successfully purified and crystallized. X-ray diffraction data have been collected by the multiple wavelength anomalous dispersion method. The crystal belongs to the space group P2(1)2(1)2(1) with unit-cell parameters a=60.42, b=84.29 and c=144.16 A and contains one molecule per asymmetric unit.