Photosynthesis-dependent Ca2+ influx and functional diversity between phospholipases in the formation of cell polarity in migrating cells of red algae

Unicellular spore cells, designated as monospores (also called archeospores), are well known as migrating plant cells, in which establishment of the anterior-posterior axis directs asymmetrical distribution of F-actin. Since the mechanisms of cell polarity formation are not yet fully elucidated in monospores, we investigated the roles of phosphoinositide signaling systems and Ca²⁺ mobilization in migration. Although we have already found the critical involvement of phosphatidylinositol 3-kinase in the establishment of cell polarity, we recently demonstrated the important roles of extracellular Ca²⁺ influx, phospholipase C (PLC) and phospholipase D (PLD). The remarkable characteristics of these factors are that Ca²⁺ influx depends on photosynthetic activity and that PLC and PLD play roles in the establishment and maintenance of cell polarity, respectively. These findings could provide new insight into the regulation of migration in eukaryotic cells.Key words: Ca²⁺ influx, cell polarity, phospholipase C, phospholipase D, photosynthesis, Porphyra yezoensisMonospores are responsible for asexual and clonal propagation of the marine multicellular red algae Porphyra and have an exceptional characteristic as migrating plant cells.^1–5 Monospores possess a round shape just after release from gametophytic blades (Fig. 1A and B), then undergo morphological change during migration. The establishment of cell polarity leads to the determination of anterior-posterior axis and asymmetrical localization of F-actin (Fig. 1C). After migration, monospores adhere to the substratum in which the apical-basal axis has been established for further development (Fig. 1D). Asymmetrical distribution of F-actin is also found in chemotaxic migration of Dictyostelium cells and leukocytes.^6,7 In these cells, reciprocal local accumulation of phosphoinositides, such as phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P₃] at the leading edge and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂] at the trailing side, is critical for the establishment of cell polarity. Phosphatidylinositol 3-kinase (PI3K) and PtdIns(3,4,5)P₃-specific D-3-phosphatase PTEN have been identified as key modulators in the establishment of cell polarity, bringing asymmetrical distribution of these two phosphoinositides in plasma membranes.^6,8 Similarly, we found the involvement of PI3K activity in the establishment of cell polarity in migrating monospores,³ suggesting the evolutional conservation of the function of PI3K in migrating eukaryotic cells. On the other hand, the importance of cell wall synthesis has been found in the maintenance of the cell polarity during monospore migration⁴ as reported in Fucus zygotes.^9,10 Therefore, the establishment and maintenance of cell polarity are thought to be regulated separately in monospores of P. yezoensis. In this addendum, further evidence of differential regulation of cell polarity formation by extracellular Ca²⁺ influx and phospholipases in migrating monospores of red algae is documented according to our recent report.⁵Open in a separate windowFigure 1Establishment and maintenance of cell polarity in monospores from the red alga P. yezoensis. (A) Discharge of unicellular monospores from a multicellular gametophytic blade of P. yezoensis strain TU-1. Scale bar = 20 µm. (B–D) Asymmetrical distribution of F-actin during early development of monospores. F-actin was stained with alex Flour 488 phalloidin. (B) Discharged monospore. (C) Migrating monospore. (D) Adhering monospore. Upper and lower photos in each panel show bright-field and fluorescent images, respectively. Arrow in (C) indicates the direction of migration. Scale bars = 5 µm. (e) Schematic representation of our working hypothesis about the formation of cell polarity required for monospore migration. Photosynthesis-dependent [Ca²⁺]_cyt increase regulates PLC and PI3K for the establishment of cell polarity, while PLD is required for the maintenance of the established cell polarity. DG, diacylgycerol; IP3, inositol-1,4,5-trisphosphate; IP3r, IP3 receptor; PC, phosphatidylcholine.     

Consecutive monitoring of lifelong production of conidia by individual conidiophores of Blumeria graminis f. sp. hordei on barley leaves by digital microscopic techniques with electrostatic micromanipulation

Conidial formation and secession by living conidiophores of Blumeria graminis f. sp. hordei on barley leaves were consecutively monitored using a high-fidelity digital microscopic technique combined with electrostatic micromanipulation to trap the released conidia. Conidial chains formed on conidiophores through a series of septum-mediated division and growth of generative cells. Apical conidial cells on the conidiophores were abstricted after the conidial chains developed ten conidial cells. The conidia were electrically conductive, and a positive charge was induced in the cells by a negatively polarized insulator probe (ebonite). The electrostatic force between the conidia and the insulator was used to attract the abstricted conidia from the conidiophores on leaves. This conidium movement from the targeted conidiophore to the rod was directly viewed under the digital microscope, and the length of the interval between conidial septation and secession, the total number of the conidia produced by a single conidiophore, and the modes of conidiogenesis were clarified. During the stage of conidial secession, the generative cells pushed new conidial cells upwards by repeated division and growth. The successive release of two apical conidia was synchronized with the successive septation and growth of a generative cell. The release ceased after 4-5 conidia were released without division and growth of the generative cell. Thus, the life of an individual conidiophore (from the erection of the conidiophore to the release of the final conidium) was shown to be 107 h and to produce an average of 33 conidia. To our knowledge, this is the first report on the direct estimation of life-long conidial production by a powdery mildew on host leaves.     

2-(3-Amino-3-deoxy-beta-D-xylofuranosyl)thiazole-4-carboxamide: a new tiazofurin analogue with potent antitumour activity

A new tiazofurin analogue, 2-(3-amino-3-deoxy-beta-d-xylofuranosyl)thiazole-4-carboxamide (3), was synthesized starting from d-glucose and evaluated for its in vitro antiproliferative activity against a panel of human tumour cell lines. Compound 3 exhibited the most powerful cytotoxicity against K562 cells, being approximately 100-fold more potent than tiazofurin. This analogue was also active against Jurkat, HT-29 and HeLa malignant cells, with respective IC(50) values being ca. 2-, 27- and 17-fold lower than those observed for tiazofurin. Remarkably, compound 3 did not exhibit any significant cytotoxicity towards normal foetal lung MRC-5 cell line.     

Decomposed pairwise regression analysis of genetic and geographic distances reveals a metapopulation structure of stream-dwelling Dolly Varden charr

Isolation by distance is usually tested by the correlation of genetic and geographic distances separating all pairwise populations' combinations. However, this method can be significantly biased by only a few highly diverged populations and lose the information of individual population. To detect outlier populations and investigate the relative strengths of gene flow and genetic drift for each population, we propose a decomposed pairwise regression analysis. This analysis was applied to the well-described one-dimensional stepping-stone system of stream-dwelling Dolly Varden charr ( Salvelinus malma ). When genetic and geographic distances were plotted for all pairs of 17 tributary populations, the correlation was significant but weak ( r ² = 0.184). Seven outlier populations were determined based on the systematic bias of the regression residuals, followed by Akaike's information criteria. The best model, 10 populations included, showed a strong pattern of isolation by distance ( r ² = 0.758), suggesting equilibrium between gene flow and genetic drift in these populations. Each outlier population was also analysed by plotting pairwise genetic and geographic distances against the 10 nonoutlier populations, and categorized into one of the three patterns: strong genetic drift, genetic drift with a limited gene flow and a high level of gene flow. These classifications were generally consistent with a priori predictions for each population (physical barrier, population size, anthropogenic impacts). Combined the genetic analysis with field observations, Dolly Varden in this river appeared to form a mainland-island or source-sink metapopulation structure. The generality of the method will merit many types of spatial genetic analyses.     

Optimization of sulfonamide derivatives as highly selective EP1 receptor antagonists

A series of 4-[(2-{isobutyl[(5-methyl-2-furyl)sulfonyl]amino}phenoxy)methyl]benzoic acids and 4-({2-[isobutyl(1,3-thiazol-2-ylsulfonyl)amino]phenoxy}methyl)benzoic acids were synthesized and evaluated for their EP receptor affinities and EP1 receptor antagonist activities. Further structural optimization was carried out to reduce inhibitory activity against hepatic cytochrome P450 isozymes, which could represent a harmful potential drug interaction. Selected compounds were also evaluated for their binding affinities to hTP, hDP, mFP, and hIP, and for their hEP1 receptor antagonist activities. The results of structure-activity relationship studies are also presented.     

Quantitative evaluation of each catalytic subsite of cathepsin B for inhibitory activity based on inhibitory activity-binding mode relationship of epoxysuccinyl inhibitors by X-ray crystal structure analyses of complexes

To quantitatively estimate the inhibitory effect of each substrate-binding subsite of cathepsin B (CB), a series of epoxysuccinyl derivatives with different functional groups bound to both carbon atoms of the epoxy ring were synthesized, and the relationship between their inhibitory activities and binding modes at CB subsites was evaluated by the X-ray crystal structure analyses of eight complexes. With the common reaction in which the epoxy ring of inhibitor was opened to form a covalent bond with the SgammaH group of the active center Cys29, the observed binding modes of the substituents of inhibitors at the binding subsites of CB enabled the quantitative assessment of the inhibitory effect of each subsite. Although the single blockage of S1' or S2' subsite exerts only the inhibitory effect of IC50 = approximately 24 microM (k2 = approximately 1250 M(-1) s(-1)) or approximately 15 microM (k2 = approximately 1800 M(-1) s(-1)), respectively, the synchronous block of both subsites leads to IC50 = approximately 23 nM (k2 = 153,000 - 185,000 M(-1) s(-1)), under the condition that (i) the inhibitor possesses a P1' hydrophobic residue such as Ile and a P2' hydrophobic residue such as Ala, Ile or Pro, and (ii) the C-terminal carboxyl group of a P2' residue is able to form paired hydrogen bonds with the imidazole NH of His110 and the imidazole N of His111 of CB. The inhibitor of a Pn' > or = 3' substituent was not potentiated by collision with the occluding loop. On the other hand, it was suggested that the inhibitory effects of Sn subsites are independent of those of Sn' subsites, and the simultaneous blockage of the funnel-like arrangement of S2 and S3 subsites leads to the inhibition of IC50 = approximately 40 nM (k2 = approximately 66,600 M(-1) s(-1)) regardless of the lack of Pn' substituents. Here we present a systematic X-ray structure-based evaluation of structure-inhibitory activity relationship of each binding subsite of CB, and the results provide the structural basis for designing a more potent CB-specific inhibitor.     

Silencing of hepatitis A virus infection by small interfering RNAs

Infection by hepatitis A virus (HAV) can cause acute hepatitis and, rarely, fulminant liver failure, in particular in patients chronically infected with hepatitis C virus. Based on our previous observation that small interfering RNAs (siRNAs) can silence translation and replication of the firefly luciferase-encoding HAV replicon, we now exploited this technology to demonstrate the effect of siRNAs on viral infection in Huh-7 cells. Freshly and persistently infected cells were transfected with siRNAs targeting various sites in the HAV nonstructural genes. Compared to a single application, consecutive siRNA transfections targeting multiple sequences in the viral genome resulted in a more efficient and sustained silencing effect than a single transfection. In most instances, multiple applications of a single siRNA led to the emergence of viral escape mutants with mutated target sites that rendered these genomes resistant to RNA interference (RNAi). Efficient and sustained suppression of the viral infectivity was achieved after consecutive applications of an siRNA targeting a computer-predicted hairpin structure. This siRNA holds promise as a therapeutic tool for severe courses of HAV infection. In addition, the results provide new insight into the structural bases for sequence-specific RNAi.