p140mDia, a mammalian homolog of Drosophila diaphanous, is a target protein for Rho small GTPase and is a ligand for profilin.

Rho small GTPase regulates cell morphology, adhesion and cytokinesis through the actin cytoskeleton. We have identified a protein, p140mDia, as a downstream effector of Rho. It is a mammalian homolog of Drosophila diaphanous, a protein required for cytokinesis, and belongs to a family of formin-related proteins containing repetitive polyproline stretches. p140mDia binds selectively to the GTP-bound form of Rho and also binds to profilin. p140mDia, profilin and RhoA are co-localized in the spreading lamellae of cultured fibroblasts. They are also co-localized in membrane ruffles of phorbol ester-stimulated sMDCK2 cells, which extend these structures in a Rho-dependent manner. The three proteins are recruited around phagocytic cups induced by fibronectin-coated beads. Their recruitment is not induced after Rho is inactivated by microinjection of botulinum C3 exoenzyme. Overexpression of p140mDia in COS-7 cells induced homogeneous actin filament formation. These results suggest that Rho regulates actin polymerization by targeting profilin via p140mDia beneath the specific plasma membranes.     

Production of nisin Z using <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1 from hydrolyzed sago starch

A membrane bioreactor for production of nisin Z was constructed using Lactococcus lactis IO-1 in continuous culture using hydrolyzed sago starch as carbon source. A strategy used to enhance the productivity of nisin Z was to maintain the cells in a continuous growth at high cell concentration. This resulted in a volumetric productivity of nisin Z, as 50,000 IU l⁻¹ h⁻¹ using a cell concentration of 15 g l⁻¹, 30^°C, pH 5.5 and a dilution rate of 1.24 h⁻¹. Adding 10 g l⁻¹ YE and 2 g l⁻¹ polypeptone, other inducers were unnecessary to maintain production of nisin. The operating conditions of the reactor removed nisin and lactate, thus minimizing their effects which allowed the maintenance of cells in continuous exponential growth phase mode with high metabolic activity.     

mDia2 induces the actin scaffold for the contractile ring and stabilizes its position during cytokinesis in NIH 3T3 cells

mDia proteins are mammalian homologues of Drosophila diaphanous and belong to the formin family proteins that catalyze actin nucleation and polymerization. Although formin family proteins of nonmammalian species such as Drosophila diaphanous are essential in cytokinesis, whether and how mDia proteins function in cytokinesis remain unknown. Here we depleted each of the three mDia isoforms in NIH 3T3 cells by RNA interference and examined this issue. Depletion of mDia2 selectively increased the number of binucleate cells, which was corrected by coexpression of RNAi-resistant full-length mDia2. mDia2 accumulates in the cleavage furrow during anaphase to telophase, and concentrates in the midbody at the end of cytokinesis. Depletion of mDia2 induced contraction at aberrant sites of dividing cells, where contractile ring components such as RhoA, myosin, anillin, and phosphorylated ERM accumulated. Treatment with blebbistatin suppressed abnormal contraction, corrected localization of the above components, and revealed that the amount of F-actin at the equatorial region during anaphase/telophase was significantly decreased with mDia2 RNAi. These results demonstrate that mDia2 is essential in mammalian cell cytokinesis and that mDia2-induced F-actin forms a scaffold for the contractile ring and maintains its position in the middle of a dividing cell.     

Identification of new amine acceptor protein substrate candidates of transglutaminase in rat liver extract: use of 5-(biotinamido) pentylamine as a probe

Transglutaminases (TGs) are a family of enzymes that catalyze Ca(2+)-dependent post-translational modification of proteins by introducing protein-protein crosslinks (between specific glutamine and lysine residues), amine incorporation, and site-specific deamidation. In this study, new amine acceptor protein substrates of TG were isolated from rat liver extract and identified using 5-(biotinamido) pentylamine, a biotinylated primary amine substrate, as a probe. TG protein substrate candidates labeled with biotin by endogenous TG activity were isolated and recovered by avidin column chromatography. Proteins with molecular masses of 40, 42, and 45 kDa were the main components of the labeled proteins. Determination of their partial amino acid sequences and immunoblotting analyses were done to identify them. The 45-kDa protein was identical with betaine-homocysteine S-methyltransferase (EC 2.2.2.5), which was identified in our previous study. The 40- and 42-kDa proteins were identified as arginase-I (EC 3.5.3.1) and fructose-1,6-bisphosphatase (EC 3.1.3.11) respectively. TG catalyzed incorporation of 5-(biotinamido) pentylamine into both arginase-I and fructose-1,6-bisphosphatase purified from rat liver was confirmed in vitro. These results suggest that these two enzymes are the new protein substrate candidates of TG and that they can be modified post-translationally by cellular TG.     

AtObgC, a plant ortholog of bacterial Obg, is a chloroplast-targeting GTPase essential for early embryogenesis

Obg is a ribosome-associated GTPase essential for bacterial viability and is conserved in most organisms, from bacteria to eukaryotes. Obg is also expressed in plants, which predicts an important role for this molecule in plant viability; however, the functions of the plant Obg homologs have not been reported. Here, we first identified Arabidopsis AtObgC as a plant chloroplast-targeting Obg and elucidated its molecular biological and physiological properties. AtObgC encodes a plant-specific Obg GTPase that contains an N-terminal region for chloroplast targeting and has intrinsic GTP hydrolysis activity. A targeting assay using a few AtObgC N-terminal truncation mutants revealed that AtObgC localizes to chloroplasts and its transit peptide consists of more than 50 amino acid residues. Interestingly, GFP-fused full-length AtObgC exhibited a punctate staining pattern in chloroplasts of Arabidopsis protoplasts, which suggests a dimerization or multimerization of AtObgC. Moreover, its Obg fold was indispensable for the generation of the punctate staining pattern, and thus, was supposed to be important for such oligomerization of AtObgC by mediating the protein–protein interaction. In addition, the T-DNA insertion AtObgC null mutant exhibited an embryonic lethal phenotype that disturbed the early stage of embryogenesis. Altogether, our results provide a significant implication that AtObgC as a chloroplast targeting GTPase plays an important role at the early embryogenesis by exerting its function in chloroplast protein synthesis.     

Tiny keratotic brown lesions on the interdigital web between the toes of a healthy man caused by Curvularia species infection and a review of cutaneous Curvularia infections

A 74-year-old healthy Japanese man had noticed an itchy lesion with two tiny thick brown scales on the fourth interdigital web of his right foot 3 months earlier. The brown fungus isolated from the scales had demonstrated brown, ellipsoidal, obovoid or clavate, 3-septate conidia. Morphologically, the fungus was identified as Curvularia sp., and was differentiated from other human pathogenic species of the genus, such as C. lunata, C. pallescens, C. trifolii, C. clavata, and C. brachyspora, by molecular analysis based on the DNA sequence data. The fungus grows well on keratotic materials (hairs, nails, and callus), which indicates that it might have the ability to infect the skin surface.     

Analysis of a range of catabolic mutants provides evidence that phytanoyl-coenzyme A does not act as a substrate of the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase complex in Arabidopsis during dark-induced senescence

The process of dark-induced senescence in plants is not fully understood, however, the functional involvement of an electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO), has been demonstrated. Recent studies have revealed that the enzymes isovaleryl-coenzyme A (CoA) dehydrogenase and 2-hydroxyglutarate dehydrogenase act as important electron donors to this complex. In addition both enzymes play a role in the breakdown of cellular carbon storage reserves with isovaleryl-CoA dehydrogenase being involved in degradation of the branched-chain amino acids, phytol, and lysine while 2-hydroxyglutarate dehydrogenase is exclusively involved in lysine degradation. Given that the chlorophyll breakdown intermediate phytanoyl-CoA accumulates dramatically both in knockout mutants of the ETF/ETFQO complex and of isovaleryl-CoA dehydrogenase following growth in extended dark periods we have investigated the direct importance of chlorophyll breakdown for the supply of carbon and electrons during this process. For this purpose we isolated three independent Arabidopsis (Arabidopsis thaliana) knockout mutants of phytanoyl-CoA 2-hydroxylase and grew them under the same extended darkness regime as previously used. Despite the fact that these mutants accumulated phytanoyl-CoA and also 2-hydroxyglutarate they exhibited no morphological changes in comparison to the other mutants previously characterized. These results are consistent with a single entry point of phytol breakdown into the ETF/ETFQO system and furthermore suggest that phytol is not primarily metabolized by this pathway. Furthermore analysis of isovaleryl-CoA dehydrogenase/2-hydroxyglutarate dehydrogenase double mutants generated here suggest that these two enzymes essentially account for the entire electron input via the ETF complex.     

Stable cesium uptake and accumulation capacities of five plant species as influenced by bacterial inoculation and cesium distribution in the soil

The effects of inoculation with Bacillus and Azospirillum strains on growth and cesium accumulation of five plant species, Komatsuna, Amaranth, sorghum, common millet and buckwheat, grown on cesium-spiked soil were assessed for potential use in cesium remediation. Pot experiments were performed using “artificially” Cs-contaminated soil. Three treatments were applied based on Cs location in the soil. For a soil height of 15 cm in the pots, Cs was added as follows: in the top five cm to imitate no ploughing condition; in the bottom five cm simulating inverted ploughing; and uniformly distributed Cs reproducing normal plowing. Generally, inoculation of Cs-exposed plants significantly enhanced growth and tolerance to this element. Transfer factor (ratio of Cs concentration in the plant tissues to that in surrounding soil) was strongly influenced by Cs distribution, with higher values in the top-Cs treatment. Within this treatment, inoculation of Komatsuna with Bacillus and Azospirillum strains resulted in the greatest transfer factors of 6.55 and 6.68, respectively. Cesium content in the shoots was high in the Azospirillum-inoculated Komatsuna, Amaranth, and buckwheat, i.e., 1,830, 1,220, and 1,030 µg per pot, respectively (five plants were grown in each pot). Therefore, inoculation of Komatsuna and Amaranth with the strains tested here could be effective in enhancing Cs accumulation. The decrease of Cs transfer under uniform- and bottom-Cs treatments would suggest that countermeasures aiming at decreasing the transfer of Cs could rely on ploughing practices.