A new FtsZ-interacting protein, YlmF, complements the activity of FtsA during progression of cell division in Bacillus subtilis

The assembly of ring-like structures, composed of FtsZ proteins (i.e. the Z ring), is the earliest and most essential process in bacterial cytokinesis. It has been shown that this process is directly regulated by the FtsZ-binding proteins, FtsA, ZapA, and EzrA, in Bacillus subtilis. In this study, protein complexes that are involved in Z-ring formation were chemically cross-linked in vivo, purified by affinity chromatography, and analysed by mass spectrometry. Analysis of the results identified YlmF as a new component of the FtsZ complex. Yeast two-hybrid analysis and fluorescence microscopy of YFP-YlmF in B. subtilis cells indicated YlmF localizes to the division site in an FtsZ-dependent manner. A single disruption of YlmF resulted in a slight elongation of cells; however, simultaneous inactivation of both YlmF and FtsA showed synthetic lethality caused by complete blockage of cell division due to the defect in Z-ring formation. In contrast, the ftsA-null mutant phenotype, caused by inefficient Z-ring formation, could be complemented by overexpression of YlmF. These results suggest that YlmF has an overlapping function with FtsA in stimulating the formation of Z rings in B. subtilis.     

Molecular cloning and characterization of a novel human beta1,3-glucosyltransferase, which is localized at the endoplasmic reticulum and glucosylates O-linked fucosylglycan on thrombospondin type 1 repeat domain

Protein O-linked fucosylation is an unusual glycosylation associated with many important biological functions such as Notch signaling. Two fucosylation pathways synthesizing O-fucosylglycans have been reported on cystein-knotted proteins, that is, on epidermal growth factor-like (EGF-like) domains and on thrombospondin Type 1 repeat (TSR) domains. We report here the molecular cloning and characterization of a novel beta1,3-glucosyltransferase (beta3Glc-T) that synthesizes a Glcbeta1,3Fucalpha- structure on the TSR domain. We found a novel glycosyltransferase gene with beta1,3-glycosyltransferase (beta3GT) motifs in databases. The recombinant enzyme expressed in human embryonic kidney 293T (HEK293T) cells exhibited glucosyltransferase activity toward fucose-alpha-para-nitrophenyl (Fucalpha-pNp). Thin-layer chromatography (TLC) analysis revealed that the product of the recombinant enzyme migrated to the same position as did the product of endogenous beta3Glc-T of Chinese hamster ovary (CHO) cells. The two products could be digested by beta-glucosidase from almond and by exo-1,3-beta-glucanase from Trichoderma sp. These results strongly suggested that the product has the structure of Glcbeta1-3Fuc. Therefore, we named this novel enzyme beta3Glc-T. Immunostaining revealed that FLAG-tagged beta3Glc-T is an enzyme residing in the endoplasmic reticulum (ER) via retention signal, "REEL," which is a KDEL-like sequence, at the C-terminus. The TSR domain expressed in Escherichia coli was first fucosylated by the recombinant protein O-fucosyltransferase 2 (POFUT2), after which it became an acceptor substrate for the recombinant beta3Glc-T, which could apparently transfer Glc to the fucosylated TSR domain. Our results suggest that a novel glycosyltransferase, beta3Glc-T, contributes to the elongation of O-fucosylglycan and that this occurs specifically on TSR domains.     

Large-scale histological analysis of leaf mutants using two simple leaf observation methods: identification of novel genetic pathways governing the size and shape of leaves

Observations of cellular organization are essential in understanding the mechanisms underlying leaf morphogenesis. These observations require several preparative steps, such as fixation and clearing of organs, and such procedures are time-consuming and labor-intensive for large-scale analyses. Thus, we have developed simple methods for the observation of leaf epidermal and mesophyll cells. To visualize the epidermis, a gel cast was made of the leaf surface, which was then observed under a light microscope. To visualize the leaf mesophyll cells, leaves were immersed in a solution containing Triton X-100, briefly centrifuged, and then viewed under a light microscope. These methods allowed us to conduct a histological phenome analysis for a large number of known and newly isolated leaf-shape/size mutants of Arabidopsis thaliana by measuring various parameters, including cell number, size, and distribution of cells within a leaf blade. Mutants showed changes in leaf size caused by specific increases or decreases in the number and/or size of cells. In addition, altered cell distributions in the leaf blade were observed, resulting from increases or decreases in the number of cells along the proximo-distal or medio-lateral axis, or recruitment of cells along a particular axis at the expense of other leaf parts. These results provide a phenomic view of the cellular behavior involved in organ size control and leaf-shape patterning.     

Resonance Raman characterization of archaeal and bacterial Rieske protein variants with modified hydrogen bond network around the [2Fe-2S] center

The rate of quinol oxidation by cytochrome bc(1)/b(6)f complex is in part associated with the redox potential (E(m)) of its Rieske [2Fe-2S] center, for which an approximate correlation with the number of hydrogen bonds to the cluster has been proposed. Here we report comparative resonance Raman (RR) characterization of bacterial and archaeal high-potential Rieske proteins and their site-directed variants with a modified hydrogen bond network around the cluster. Major differences among their RR spectra appear to be associated in part with the presence or absence of Tyr-156 (in the Rhodobacter sphaeroides numbering) near one of the Cys ligands to the cluster. Elimination of the hydrogen bond between the terminal cysteinyl sulfur ligand (S(t)) and Tyr-Oeta (as with the Y156W variant, which has a modified histidine N(epsilon) pK(a,ox)) induces a small structural bias of the geometry of the cluster and the surrounding protein in the normal coordinate system, and significantly affects some Fe-S(b/t) stretching vibrations. This is not observed in the case of the hydrogen bond between the bridging sulfide ligand (S(b)) and Ser-Ogamma, which is weak and/or unfavorably oriented for extensive coupling with the Fe-S(b/t) stretching vibrations.     

Production of trans-10, cis-12 Conjugated Linoleic Acid in Rice

Conjugated linoleic acid (CLA) has anti-carcinogenic and anti-atherosclerosis activity, and modulatory effects on the immune system and lipid metabolism. To produce a transgenic rice plant that can accumulate CLA, a linoleate isomerase gene that can convert linoleic acid to trans-10, cis-12 CLA was introduced and expressed under the control of seed-specific promoters from the oleosin and globulin genes. The fatty acid composition of the transgenic rice grain was analyzed by gas chromatography. Although there was no clear difference in the fatty acid composition between seeds from transformed versus untransformed plants, a peak of trans-10, cis-12 CLA methyl ester, which was not present in seeds from untransformed plants, was found in transformed plants. The trans-10, cis-12 CLA comprised an average of 1.3% (w/w) of the total fatty acids in seeds carrying the oleosin promoter in comparison to 0.01% (w/w) in seeds carrying the globulin promoter. In addition, approximately 70 and 28% of the total amount of the CLA isomer were present in the triacylglycerol and free fatty acid fractions, respectively. These results demonstrate the ability to produce fatty acid components of vegetable oils with novel physiological activities in crops.     

Transport of a hydrophobic biosynthetic precursor by lipophorin in the hemolymph of a geometrid female moth which secretes an epoxyalkenyl sex pheromone

Previous experiments with a geometrid species, Ascotis selenaria cretacea, have suggested that a pheromonal C19 3,4-epoxy-6,9-diene is biosynthesized from the corresponding 3,6,9-triene produced outside a pheromone gland and transported to it via hemolymph after association with lipophorin. In order to clarify this transport, high-density lipophorin (HDLp) in the female moths showing two bands (apoLp I with ca. 250 kDa and apoLp II with ca. 80 kDa) on an SDS-PAGE was purified by KBr equilibrium density-gradient ultracentrifugation, and the association of the triene was confirmed by GC-MS analysis of a solvent extract from the isolated protein. Next, the role of HDLp was revealed by a topical application of the deuterated trienyl precursor to the abdomens of the females. The trienyl precursor was associated with HDLp. In their pheromone glands, the triene and the deuterated epoxy pheromone were detected, indicating movement of the triene via the hemolymph. Experiments with male moths of A. s. cretacea and female moths of Bombyx mori showed the same association of HDLp with the triene topically applied. This result suggested that the adult females of A. s. cretacea did not develop HDLp specialized in the triene transport. Furthermore, the topical application of a mixture including the trienyl precursor and two other related hydrocarbons showed equal amounts of association by HDLp but selective delivery of the precursor to pheromone glands in the A. s. cretacea females.     

Multidrug-resistance-associated protein plays a protective role in menadione-induced oxidative stress in endothelial cells

AimsMenadione, a redox-cycling quinone known to cause oxidative stress, binds to reduced glutathione (GSH) to form glutathione S-conjugate. Glutathione S-conjugates efflux is often mediated by multidrug-resistance-associated protein (MRP). We investigated the effect of a transporter inhibitor, MK571 (3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid), on menadione-induced oxidative stress in bovine aortic endothelial cells (BAECs).Main methodsBAECs were treated with menadione and MK571, and cell viability was measured. Modulation of intracellular GSH levels was performed with buthionine sulfoximine and GSH ethyl ester treatments. Intracellular superoxide was estimated by dihydroethidium oxidation using fluorescence microscopy or flow cytometry. Expression of MRP was determined by flow cytometry using phycoerythrin-conjugated anti-MRP monoclonal antibody.Key findingsIntracellular GSH depletion by buthionine sulfoximine promoted the loss of viability of BAECs exposed to menadione. Exogenous GSH, which does not permeate the cell membrane, or GSH ethyl ester protected BAECs against the loss of viability induced by menadione. The results suggest that GSH binds to menadione outside the cells as well as inside. Pretreatment of BAECs with MK571 dramatically increased intracellular levels of superoxide generated from menadione, indicating that menadione may accumulate in the intracellular milieu. Finally, we found that MK571 aggravated menadione-induced toxicity in BAECs and that MRP levels were increased in menadione-treated cells.SignificanceWe conclude that MRP plays a vital role in protecting BAECs against menadione-induced oxidative stress, presumably due to its ability to transport glutathione S-conjugate.     

Modulation of the Poly(ADP-ribosyl)ation Reaction via the Arabidopsis ADP-Ribose/NADH Pyrophosphohydrolase,AtNUDX7, Is Involved in the Response to Oxidative Stress

Here, we assessed modulation of the poly(ADP-ribosyl)ation (PAR) reaction by an Arabidopsis (Arabidopsis thaliana) ADP-ribose (Rib)/NADH pyrophosphohydrolase, AtNUDX7 (for Arabidopsis Nudix hydrolase 7), in AtNUDX7-overexpressed (Pro_35S:AtNUDX7) or AtNUDX7-disrupted (KO-nudx7) plants under normal conditions and oxidative stress caused by paraquat treatment. Levels of NADH and ADP-Rib were decreased in the Pro_35S:AtNUDX7 plants but increased in the KO-nudx7 plants under normal conditions and oxidative stress compared with the control plants, indicating that AtNUDX7 hydrolyzes both ADP-Rib and NADH as physiological substrates. The Pro_35S:AtNUDX7 and KO-nudx7 plants showed increased and decreased tolerance, respectively, to oxidative stress compared with the control plants. Levels of poly(ADP-Rib) in the Pro_35S:AtNUDX7 and KO-nudx7 plants were markedly higher and lower, respectively, than those in the control plants. Depletion of NAD⁺ and ATP resulting from the activation of the PAR reaction under oxidative stress was completely suppressed in the Pro_35S:AtNUDX7 plants. Accumulation of NAD⁺ and ATP was observed in the KO-nudx7- and 3-aminobenzamide-treated plants, in which the PAR reaction was suppressed. The expression levels of DNA repair factors, AtXRCC1 and AtXRCC2 (for x-ray repair cross-complementing factors 1 and 2), paralleled that of AtNUDX7 under both normal conditions and oxidative stress, although an inverse correlation was observed between the levels of AtXRCC3, AtRAD51 (for Escherichia coli RecA homolog), AtDMC1 (for disrupted meiotic cDNA), and AtMND1 (for meiotic nuclear divisions) and AtNUDX7. These findings suggest that AtNUDX7 controls the balance between NADH and NAD⁺ by NADH turnover under normal conditions. Under oxidative stress, AtNUDX7 serves to maintain NAD⁺ levels by supplying ATP via nucleotide recycling from free ADP-Rib molecules and thus regulates the defense mechanisms against oxidative DNA damage via modulation of the PAR reaction.Reactive oxygen species (ROS) are by-products of normal metabolic processes, including chloroplastic, mitochondrial, and plasma membrane-linked electron transport systems, in all aerobic organisms (Gutteridge and Halliwell, 1989). Although the production and destruction of ROS are in balance, the imposition of biotic and abiotic stressful conditions can give rise to excess concentrations of ROS, leading to an imbalance of production and scavenging mechanisms (Mittler, 2002; Mullineaux and Karpinski, 2002; Kroj et al., 2003; Mahalingam et al., 2003). Excess ROS, leading to oxidative stress, can damage organelles, oxidize proteins, nick DNA (single-base DNA damage), deplete antioxidant levels, and ultimately trigger cell death (Gutteridge and Halliwell, 1989). Recently, ROS have been recognized as important signaling molecules that control diverse signaling pathways involved in a variety of cellular responses such as programmed cell death, pathogen defense, and hormone signaling (Foyer and Noctor, 2005; Kwak et al., 2006; Torres et al., 2006). In addition, oxidative stress causes dramatic inhibition of the tricarboxylic acid cycle and large sectors of amino acid metabolism followed by backing up of glycolysis and diversion of carbon into the oxidative pentose phosphate pathway (Baxter et al., 2007). Therefore, organisms have developed efficient systems to keep ROS levels in check and repair damage from attack by ROS.Among various defense systems against attack by ROS, the poly(ADP-ribosyl)ation (PAR) of proteins by poly(ADP-Rib)polymerase (PARP), by which branched polymers of ADP-Rib are attached using β-NAD⁺ to a specific amino acid residue of an acceptor protein, is a posttranslational modification for responding early to DNA damage, such as single-strand DNA break and resealing, caused by oxidative stress and, thus, is crucial for genomic integrity and cell survival (Qin et al., 2008). PARP detects DNA strand breaks and converts the damage into intracellular signals that can activate DNA repair programs or cell death, according to the severity of the injury, via the PAR reaction of nuclear proteins involved in chromatin architecture and DNA metabolism and interacts with the x-ray repair cross complementing factor 1 (XRCC1), an adaptor protein that also has two interfaces with two important single-strand DNA break (SSB) repair (SSBR)/base excision repair (BER) enzymes: DNA ligase and DNA polymerase β (Caldecott et al., 1995, 1996; Kubota et al., 1996; Masson et al., 1998). DNA polymerase β fills the single nucleotide gap, preparing the strand for ligation by a complex of DNA ligase III and XRCC1 (Winters et al., 1999; Thompson and West, 2000). Thereby, the fast recruitment of SSBR/BER factors is archived in the site of the lesion. Modifications of proteins with poly(ADP-Rib) are reversed by poly(ADP-Rib) glycohydrolase (PARG), by which ADP-Rib polymers are hydrolyzed to free ADP-Rib, since incorrect signal transduction is caused by excessive accumulation of poly(ADP-Rib) modification (Davidovic et al., 2001). However, it has been reported that a massive PAR reaction results in the overconsumption of NAD⁺ and ATP and, ultimately, in energy depletion causing necrotic cell death (Ha and Snyder, 1999; Virág and Szabó, 2002; De Block et al., 2005).Nudix (for nucleoside diphosphates linked to some moiety X) hydrolases catalyze the hydrolysis of intact and oxidatively damaged nucleoside diphosphates and triphosphates, nucleotide sugars, coenzymes, dinucleoside polyphosphates, and RNA caps in various organisms such as bacteria, yeast, algae, nematodes, vertebrates, and plants (Bessman et al., 1996; Xu et al., 2004; Kraszewska, 2008). We have previously reported the characteristics of cytosolic Nudix hydrolases (AtNUDX1–AtNUDX11) in Arabidopsis (Arabidopsis thaliana; Ogawa et al., 2005). Among them, the recombinant AtNUDX7 showed high affinity for ADP-Rib and NADH as substrates in vitro, converting NADH to a reduced form of nicotinamide mononucleotide (NMNH) plus AMP and ADP-Rib to AMP plus Rib 5-P (Ogawa et al., 2005). AtNUDX7 was expressed more strongly in leaf than in stem and root. Therefore, the enzyme might be involved in nucleotide recycling relating to the metabolism of NADH and/or poly(ADP-Rib).Recent studies revealed that the actions of AtNUDX7 (At4g12720) are closely related to immune responses to pathogens. Knockout of AtNUDX7 (KO-nudx7) in Arabidopsis plants led to deleterious inference for cells, such as microscopic cell death, constitutive expression of pathogenesis-related genes, resistance to bacterial pathogens, and accumulation of NADH (Jambunathan and Mahalingam, 2006). Furthermore, AtNUDX7 exerted a negative regulatory effect on EDS1 signaling, which controls the activation of defenses and programmed cell death conditioned by intracellular Toll-related immune receptors that recognized specific pathogen effectors (Bartsch et al., 2006). More recently, Ge et al. (2007) reported that KO-nudx7 plants show heightened defense responses, which are both dependent on and independent of the accumulation of NPR1 and salicylic acid, to pathogenic attack. On the other hand, Adams-Phillips et al. (2008) reported that KO-nudx7 plants exhibit a reduced hypersensitive-response phenotype, although the growth of both virulent and avirulent pathogens is suppressed in the plants. These findings support the hypothesis that regulation of the metabolism of NADH and/or ADP-Rib by Nudix hydrolases is important for stress-related defense systems in higher plants. However, the direct actions of the enzymes on stress responses are not established yet.In this study, to assess the functions of Arabidopsis Nudix hydrolases having ADP-Rib and NADH pyrophosphohydrolase activities under normal conditions and oxidative stress, we analyzed the effect of the overexpression or disruption of AtNUDX7 on levels of ADP-Rib, NAD(H), and ATP as well as PAR activity and oxidative stress tolerance in Arabidopsis. The evidence presented here suggests that AtNUDX7 serves to balance between NADH and NAD⁺ by NADH turnover under normal conditions. In addition, AtNUDX7 functions in the maintenance of NAD⁺ levels by supplying ATP via nucleotide recycling from free ADP-Rib molecules and the modulation of the PAR reaction, thereby regulating the DNA repair pathways, in response to oxidative stress.     

A Novel Nuclear-Encoded Protein, NDH-Dependent Cyclic Electron Flow 5, is Essential for the Accumulation of Chloroplast NAD(P)H Dehydrogenase Complexes

The chloroplast NAD(P)H dehydrogenase (NDH) complex, which reducesplastoquinones in thylakoid membranes, is involved in PSI cyclicelectron flow and chlororespiration. In addition to land plants,the NDH complex is conserved in cyanobacteria. In this study,we identified a novel NDH-related gene of Arabidopsis, NDH-dependentcyclic electron flow 5 (NDF5, At1g55370). Post-illuminationincreases in chlorophyll fluorescence were absent in ndf5 mutantplants, which indicated that NDF5 is essential for NDH activity.Sequence analysis did not reveal any known functional motifsin NDF5, but there was some homology in amino acid sequencebetween NDF5 and NDF2, a known NDH subunit. NDF5 and NDF2 homologswere present in higher plants, but not cyanobacteria. A singlehomolog, which had similarity to both NDF5 and NDF2, was identifiedin the moss Physcomitrella patens. Immunoblot analysis showedthat NDF5 localizes to membrane fractions of chloroplasts. Thestability of NdhH, a subunit of the NDH complex, as well asNDF5 and NDF2, was decreased in ndf5, ndf2 and double ndf2/ndf5mutants, resulting in a loss of NDH activity in these mutants.These results indicated that both NDF5 and NDF2 have essentialfunctions in the stabilization of the NDH complex. We proposethat NDF5 and NDF2 were acquired by land plants during evolution,and that in higher plants both NDF5 and NDF2 are critical toregulate NDH activity and each other's protein stability, aswell as the stability of additional NDH subunits.