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Triple Mutant (triple + mutant)
Selected AbstractsConversion of a glutamate dehydrogenase into methionine/norleucine dehydrogenase by site-directed mutagenesisFEBS JOURNAL, Issue 22 2001Xing-Guo Wang In earlier attempts to shift the substrate specificity of glutamate dehydrogenase (GDH) in favour of monocarboxylic amino-acid substrates, the active-site residues K89 and S380 were replaced by leucine and valine, respectively, which occupy corresponding positions in leucine dehydrogenase. In the GDH framework, however, the mutation S380V caused a steric clash. To avoid this, S380 has been replaced with alanine instead. The single mutant S380A and the combined double mutant K89L/S380A were satisfactorily overexpressed in soluble form and folded correctly as hexameric enzymes. Both were purified successfully by Remazol Red dye chromatography as routinely used for wild-type GDH. The S380A mutant shows much lower activity than wild-type GDH with glutamate. Activities towards monocarboxylic substrates were only marginally altered, and the pH profile of substrate specificity was not markedly altered. In the double mutant K89L/S380A, activity towards glutamate was undetectable. Activity towards l -methionine, l -norleucine and l -norvaline, however, was measurable at pH 7.0, 8.0 and 9.0, as for wild-type GDH. Ala163 is one of the residues that lines the binding pocket for the side chain of the amino-acid substrate. To explore its importance, the three mutants A163G, K89L/A163G and K89L/S380A/A163G were constructed. All three were abundantly overexpressed and showed chromatographic behaviour identical with that of wild-type GDH. With A163G, glutamate activity was lower at pH 7.0 and 8.0, but by contrast higher at pH 9.0 than with wild-type GDH. Activities towards five aliphatic amino acids were remarkably higher than those for the wild-type enzyme at pH 8.0 and 9.0. In addition, the mutant A163G used l -aspartate and l -leucine as substrates, neither of which gave any detectable activity with wild-type GDH. Compared with wild-type GDH, the A163 mutant showed lower catalytic efficiencies and higher Km values for glutamate/2-oxoglutarate at pH 7.0, but a similar kcat/Km value and lower Km at pH 8.0, and a nearly 22-fold lower S0.5 (substrate concentration giving half-saturation under conditions where Michaelis,Menten kinetics does not apply) at pH 9.0. Coupling the A163G mutation with the K89L mutation markedly enhanced activity (100,1000-fold) over that of the single mutant K89L towards monocarboxylic amino acids, especially l -norleucine and l -methionine. The triple mutant K89L/S380A/A163G retained a level of activity towards monocarboxylic amino acids similar to that of the double mutant K89L/A163G, but could no longer use glutamate as substrate. In terms of natural amino-acid substrates, the triple mutant represents effective conversion of a glutamate dehydrogenase into a methionine dehydrogenase. Kinetic parameters for the reductive amination reaction are also reported. At pH 7 the triple mutant and K89L/A163G show 5 to 10-fold increased catalytic efficiency, compared with K89L, towards the novel substrates. In the oxidative deamination reaction, it is not possible to estimate kcat and Km separately, but for reductive amination the additional mutations have no significant effect on kcat at pH 7, and the increase in catalytic efficiency is entirely attributable to the measured decrease in Km. At pH 8 the enhancement of catalytic efficiency with the novel substrates was much more striking (e.g. for norleucine ,,2000-fold compared with wild-type or the K89L mutant), but it was not established whether this is also exclusively due to more favourable Michaelis constants. [source] The single Cdk1-G1 cyclin of Cryptococcus neoformans is not essential for cell cycle progression, but plays important roles in the proper commitment to DNA synthesis and bud emergence in this yeastFEMS YEAST RESEARCH, Issue 5 2010Eric V. Virtudazo Abstract The cell cycle pattern of the pathogenic basidiomycetous yeast Cryptococcus neoformans differs from that of the ascomycetous budding yeast Saccharomyces cerevisiae. To clarify the cell cycle control mechanisms at the molecular level, homologues of cell cycle control genes in C. neoformans were cloned and analyzed. Here, we report on the cloning and characterization of genes coding for CDK1 cyclin homologues, in particular, the C. neoformans G1 cyclin. We have identified three putative CDK1 cyclin homologues and two putative CDK5 (PHO85) cyclin homologues from the genome. Complementation tests in an S. cerevisiae G1 cyclin triple mutant confirmed that C. neoformans CLN1 is able to complement S. cerevisiae G1 cyclin deficiency, demonstrating that it is a G1 cyclin homologue. Interestingly, cells deleted of the single Cdk1-G1 cyclin were viable, demonstrating that this gene is not essential. However, it exhibited aberrant budding and cell division and a clear delay in the initiation of DNA synthesis as well as an extensive delay in budding. The fact that the mutant managed to traverse the G1 to M phase may be due to the activities of Pho85-related G1 cyclins. Also, that C. neoformans had only a single Cdk1-G1 cyclin highlighted the importance of keeping in order the commitment to the initiation of DNA synthesis first and then that of budding, as discussed. [source] Sfrp1, Sfrp2, and Sfrp5 regulate the Wnt/,-catenin and the planar cell polarity pathways during early trunk formation in mouseGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 2 2008Wataru Satoh The secreted frizzled-related protein gene family encodes proteins that regulate Wnt signaling. Msx1 in situ hybridization of 9.5 days post coitus mouse embryos showing normal neural tube development in an Sfrp1; Sfrp2 double mutant (left) but severe neural tube defects in a Looptail (Lp/+); Sfrp1; Sfrp2 triple mutant (right). These findings suggest that Sfrps regulate the Wnt planar cell polarity pathway. See Satoh et al. in this issue. [source] Comparative study of the physiological roles of three peroxidases (NADH peroxidase, Alkyl hydroperoxide reductase and Thiol peroxidase) in oxidative stress response, survival inside macrophages and virulence of Enterococcus faecalisMOLECULAR MICROBIOLOGY, Issue 5 2007Stephanie La Carbona Summary The opportunistic pathogen Enterococcus faecalis is well equipped with peroxidatic activities. It harbours three loci encoding a NADH peroxidase, an alkyl hydroperoxide reductase and a protein (EF2932) belonging to the AhpC/TSA family. We present results demonstrating that ef2932 does encode a thiol peroxidase (Tpx) and show that it is part of the regulon of the hydrogen peroxide regulator HypR. Characterization of unmarked deletion mutants showed that all three peroxidases are important for the defence against externally provided H2O2. Exposure to internal generated H2O2 by aerobic growth on glycerol, lactose, galactose or ribose showed that Npr was absolutely required for aerobic growth on glycerol and optimal growth on the other substrates. Growth on glycerol was also dependent on Ahp. Addition of catalase restored growth of the mutants, and therefore, extracellular H2O2 concentrations have been determined. This showed that the time point of growth arrest of the ,npr mutant correlated with the highest H2O2 concentration measured. Analysis of the survival of the different strains inside peritoneal macrophages revealed that Tpx was the most important antioxidant activity for protecting the cells against the hostile phagocyte environment. Finally, the ,tpx and the triple mutant showed attenuated virulence in a mouse peritonitis model. [source] Proinflammatory signalling stimulated by the type III translocation factor YopB is counteracted by multiple effectors in epithelial cells infected with Yersinia pseudotuberculosisMOLECULAR MICROBIOLOGY, Issue 5 2003Gloria I. Viboud Summary Type III secretion systems are used by several pathogens to translocate effector proteins into host cells. Yersinia pseudotuberculosis delivers several Yop effectors (e.g. YopH, YopE and YopJ) to counteract signalling responses during infection. YopB, YopD and LcrV are components of the translocation machinery. Here, we demonstrate that a type III translocation protein stimulates proinflammatory signalling in host cells, and that multiple effector Yops counteract this response. To examine proinflammatory signalling by the type III translocation machinery, HeLa cells infected with wild-type or Yop,Y. pseudotuberculosis strains were assayed for interleukin (IL)-8 production. HeLa cells infected with a YopEHJ, triple mutant released significantly more IL-8 than HeLa cells infected with isogenic wild-type, YopE,, YopH, or YopJ, bacteria. Complementation analysis demonstrated that YopE, YopH or YopJ are sufficient to counteract IL-8 production. IL-8 production required YopB, but did not require YopD, pore formation or invasin-mediated adhesion. In addition, YopB was required for activation of nuclear factor kappa B, the mitogen-activated protein kinases ERK and JNK and the small GTPase Ras in HeLa cells infected with the YopEHJ, mutant. We conclude that interaction of the Yersinia type III translocator factor YopB with the host cell triggers a proinflammatory signalling response that is counteracted by multiple effectors in host cells. [source] Effects of Three Characteristic Amino Acid Residues of Pharaonis Phoborhodopsin on the Absorption Maximum ,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2000Kazumi Shimono ABSTRACT Phoborhodopsin (pR or sensory rhodopsin II, sRII) or pharaonis phoborhodopsin (ppR or pharaonis sensory rhodopsin II, psRII) has a unique absorption maximum (,max) compared with three other archaeal rhodopsins: ,max of pR or ppR at ca 500 nm and others at 560,590 nm. Alignment of amino acid sequences revealed three sites characteristic of the shorter wavelength,absorbing pigments. The amino acids of these three sites are conserved completely among archaeal rhodopsins having longer ,max, and are different from those of pR or ppR. We replaced these amino acids of ppR with amino acids corresponding to those of bacteriorhodopsin, Val-108 to Met, Gly-130 to Ser and Thr-204 to Ala. The ,max of V108M mutant was 502 nm with a slight redshift. G130S and T204A mutants had ,max of 503 and 508 nm, respectively. Thus, each site contributes only a small effect to the color tuning. We then constructed three double mutants and one triple mutant. The opsin-shifts of these mutants suggest that Val-108 and Thr-204 or Gly-130 are synergistic, and that Gly-130 and Thr-204 work additively. Even in the triple mutant, the ,max was 515 nm, an opsin-shift only ca 30% of the shift value from 500 to 560 nm. This means that there is another yet unidentified factor responsible for the color tuning. [source] Substitutions of prolines examine their role in kinetic trap formation of the caspase recruitment domain (CARD) of RICKPROTEIN SCIENCE, Issue 3 2006Yun-Ru Chen Abstract Caspase recruitment domains (CARDs) are small helical protein domains that adopt the Greek key fold. For the two CARDs studied to date, RICK-CARD and caspase-1-CARD (CP1-CARD), the proteins unfold by an apparent two-state process at equilibrium. However, the folding kinetics are complex for both proteins and may contain kinetically trapped species on the folding pathway. In the case of RICK-CARD, the time constants of the slow refolding phases are consistent with proline isomerism. RICK-CARD contains three prolines, P47 in turn 3, and P85 and P87. The latter two prolines constitute a nonconserved PxP motif in helix 6. To examine the role of the prolines in the complex folding kinetics of RICK-CARD, we generated seven proline-to-alanine mutants, including three single mutants, three double mutants, and one triple mutant. We examined the spectroscopic properties, equilibrium folding, binding to CP1-CARD, and folding kinetics. The results show that P85 is critical for maintaining the function of the protein and that all mutations decrease the stability. Results from single mixing and sequential mixing stopped-flow studies strongly suggest the presence of parallel folding pathways consisting of at least two unfolded populations. The mutations affect the distribution of the two unfolded species, thereby affecting the population that folds through each channel. The two conformations also are present in the triple mutant, demonstrating that interconversion between them is not due to prolyl isomerism. Overall, the data show that the complex folding pathway, especially formation of kinetically trapped species, is not due to prolyl isomerism. [source] Arabidopsis thaliana class-II TGA transcription factors are essential activators of jasmonic acid/ethylene-induced defense responsesTHE PLANT JOURNAL, Issue 2 2010Mark Zander Summary The three closely related Arabidopsis basic leucine zipper (bZIP) transcription factors TGA2, TGA5 and TGA6 are required for the establishment of the salicylic acid (SA)-dependent plant defense response systemic acquired resistance, which is effective against biotrophic pathogens. Here we show that the same transcription factors are essential for the activation of jasmonic acid (JA)- and ethylene (ET)-dependent defense mechanisms that counteract necrotrophic pathogens: the tga256 triple mutant is impaired in JA/ET-induced PDF1.2 and b-CHI expression, which correlates with a higher susceptibility against the necrotroph Botrytis cinerea. JA/ET induction of the trans -activators ERF1 and ORA59, which act upstream of PDF1.2, was slightly increased (ERF1) or unaffected (ORA59). PDF1.2 expression can be restored in the tga256 mutant by increased expression of ORA59, as observed in the tga256 jin1 quadruple mutant, which lacks the transcription factor JIN1/AtMYC2 that functions as a negative regulator of the JA/ET-dependent anti-fungal defense program. Whereas JA/ET-induced PDF1.2 expression is strongly suppressed by SA in wild-type plants, no negative effect of SA on PDF1.2 expression was observed in the tga256 jin1 quadruple mutant. These results imply that the antagonistic effects of TGA factors and JIN1/AtMYC2 on the JA/ET pathway are necessary to evoke the SA-mediated suppression of JA/ET-induced defense responses. [source] The AT-hook-containing proteins SOB3/AHL29 and ESC/AHL27 are negative modulators of hypocotyl growth in ArabidopsisTHE PLANT JOURNAL, Issue 1 2008Ian H. Street Summary SOB3, which encodes a plant-specific AT-hook motif containing protein, was identified from an activation-tagging screen for suppressors of the long-hypocotyl phenotype of a weak phyB allele, phyB-4. sob3-D (suppressor of phyB-4#3 dominant) overexpressing seedlings have shorter hypocotyls, and as adults develop larger flowers and leaves, and are delayed in senescence compared with wild-type plants. At the nucleotide level, SOB3 is closely related to ESCAROLA (ESC), which was identified in an independent activation-tagging screen. ESC overexpression also suppresses the phyB-4 long-hypocotyl phenotype, and confers an adult morphology similar to sob3-D, suggesting similar functions. Analysis of transgenic plants harboring SOB3:SOB3-GUS or ESC:ESC-GUS translational fusions, driven by their endogenous promoter regions, showed GUS activity in the hypocotyl and vasculature tissue in light- and dark-grown seedlings. A loss-of-function SOB3 allele (sob3-4) was generated through an ethyl methanesulfonate intragenic suppressor screen of sob3-D phyB-4 plants, and this allele was combined with a predicted null allele, disrupting ESC (esc-8), to examine potential genetic interactions. The sob3-4 esc-8 double mutant had a long hypocotyl in multiple fluence rates of continuous white, far-red, red and blue light. sob3-4 esc-8 phyB-9 and sob3-4 esc-8 cry-103 triple mutants also had longer hypocotyls than photoreceptor single mutants. In contrast, the sob3-4 esc-8 phyA-211 triple mutant was the same length as phyA-211 single mutants. Taken together, these data indicate that SOB3 and ESC act redundantly to modulate hypocotyl growth inhibition in response to light. [source] Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedlingTHE PLANT JOURNAL, Issue 4 2007Ralf Stracke Summary The genes MYB11, MYB12 and MYB111 share significant structural similarity and form subgroup 7 of the Arabidopsis thaliana R2R3-MYB gene family. To determine the regulatory potential of these three transcription factors, we used a combination of genetic, functional genomics and metabolite analysis approaches. MYB11, MYB12 and MYB111 show a high degree of functional similarity and display very similar target gene specificity for several genes of flavonoid biosynthesis, including CHALCONE SYNTHASE, CHALCONE ISOMERASE, FLAVANONE 3-HYDROXYLASE and FLAVONOL SYNTHASE1. Seedlings of the triple mutant myb11 myb12 myb111, which genetically lack a complete subgroup of R2R3-MYB genes, do not form flavonols while the accumulation of anthocyanins is not affected. In developing seedlings, MYB11, MYB12 and MYB111 act in an additive manner due to their differential spatial activity; MYB12 controls flavonol biosynthesis mainly in the root, while MYB111 controls flavonol biosynthesis primarily in cotyledons. We identified and confirmed additional target genes of the R2R3-MYB subgroup 7 factors, including the UDP-glycosyltransferases UGT91A1 and UGT84A1, and we demonstrate that the accumulation of distinct and structurally identified flavonol glycosides in seedlings correlates with the expression domains of the different R2R3-MYB factors. Therefore, we refer to these genes as PFG1,3 for ,PRODUCTION OF FLAVONOL GLYCOSIDES'. [source] Negative regulation of defense responses in Arabidopsis by two NPR1 paralogsTHE PLANT JOURNAL, Issue 5 2006Yuelin Zhang Summary NPR1 is required for systemic acquired resistance, and there are five NPR1 paralogs in Arabidopsis. Here we report knockout analysis of two of these, NPR3 and NPR4. npr3 single mutants have elevated basal PR-1 expression and the npr3 npr4 double mutant shows even higher expression. The double mutant plants also display enhanced resistance against virulent bacterial and oomycete pathogens. This enhanced disease resistance is partially dependent on NPR1, can be in part complemented by either wild-type NPR3 or NPR4, and is not associated with an elevated level of salicylic acid. NPR3 and NPR4 interact with TGA2, TGA3, TGA5 and TGA6 in yeast two-hybrid assays. Using bimolecular fluorescence complementation analysis, we show that NPR3 interacts with TGA2 in the nucleus of onion epidermal cells and Arabidopsis mesophyll protoplasts. Combined with our previous finding that basal PR-1 levels are also elevated in the tga2 tga5 tga6 triple mutant, we propose that NPR3 and NPR4 negatively regulate PR gene expression and pathogen resistance through their association with TGA2 and its paralogs. [source] The effect of a proline residue on the rate of growth and the space group of ,-spectrin SH3-domain crystalsACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2009Ana Cámara-Artigas ,-Spectrin SH3-domain (Spc-SH3) crystallization is characterized by very fast growth of the crystals in the presence of ammonium sulfate as a precipitant agent. The origin of this behaviour can be attributed to the presence of a proline residue that participates in a crystal contact mimicking the binding of proline-rich sequences to SH3 domains. This residue, Pro20, is located in the RT loop and is the main contact in one of the interfaces present in the orthorhombic Spc-SH3 crystal structures. In order to understand the molecular interactions that are responsible for the very fast crystal growth of the wild-type (WT) Spc-SH3 crystals, the crystal structure of a triple mutant in which the residues Ser19-Pro20-Arg21 in the RT loop have been replaced by Gly19-Asp20-Ser21 (GDS Spc-SH3 mutant) has been solved. The removal of the critical proline residue results in slower nucleation of the Spc-SH3 crystals and a different arrangement of the protein molecules in the unit cell, leading to a crystal that belongs to the tetragonal space group P41212, with unit-cell parameters a = b = 42.231, c = 93.655,Å, and that diffracts to 1.45,Å resolution. For both WT Spc-SH3 and the GDS mutant, light-scattering experiments showed that a dimer was formed in solution within a few minutes of the addition of 2,M ammonium sulfate at pH 6.5 and allowed the proposal of a mechanism for the nucleation and crystal growth of Spc-SH3 in which the Pro20 residue plays a key role in the rate of crystal growth. [source] Structure of a highly stable mutant of human fibroblast growth factor 1ACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2009Anna Szlachcic Fibroblast growth factors (FGFs) are involved in diverse cellular processes such as cell migration, angiogenesis, osteogenesis, wound healing and embryonic and foetal development. Human acidic fibroblast growth factor (FGF-1) is the only member of the FGF family that binds with high affinity to all four FGF receptors and thus is considered to be the human mitogen with the broadest specificity. However, pharmacological applications of FGF-1 are limited owing to its low stability. It has previously been reported that the introduction of single mutations can significantly improve the stability of FGF-1 and its resistance to proteolytic degradation. Here, the structure of the Q40P/S47I/H93G triple mutant of FGF-1, which exhibits much higher stability, a prolonged half-life and enhanced mitogenic activity, is presented. Compared with the wild-type structure, three localized conformational changes in the stable triple mutant were observed, which is in agreement with the perfect energetic additivity of the single mutations described in a previous study. The huge change in FGF-1 stability (the denaturation temperature increased by 21.5,K, equivalent to ,,Gden = 24.3,kJ,mol,1) seems to result from the formation of a short 310 -helix (position 40), an improvement in the propensity of amino acids to form ,-sheets (position 47) and the rearrangement of a local hydrogen-bond network (positions 47 and 93). [source] The structure of a triple mutant of pI258 arsenate reductase from Staphylococcus aureus and its 5-thio-2-nitrobenzoic acid adductACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2004Joris Messens Structural insights into formation of the complex between the ubiquitous thiol,disulfide oxidoreductase thioredoxin and its oxidized substrate are under-documented owing to its entropical instability. In vitro, it is possible via a reaction with 5,5,-dithiobis-(2-nitrobenzoic acid) to make a stable mixed-disulfide complex between thioredoxin from Staphylococcus aureus and one of its substrates, oxidized pI258 arsenate reductase (ArsC) from S. aureus. In the absence of the crystal structure of an ArsC,thioredoxin complex, the structures of two precursors of the complex, the ArsC triple mutant ArsC C10SC15AC82S and its 5-thio-2-nitrobenzoic acid (TNB) adduct, were determined. The ArsC triple mutant has a structure very similar to that of the reduced form of wild-type ArsC, with a folded redox helix and a buried catalytic Cys89. In the adduct form, the TNB molecule is buried in a hydrophobic pocket and the disulfide bridge between TNB and Cys89 is sterically inaccessible to thioredoxin. In order to form a mixed disulfide between ArsC and thioredoxin, a change in the orientation of the TNB,Cys89 disulfide in the structure is necessary. [source] A thermostable triple mutant of pyranose 2-oxidase from Trametes multicolor with improved properties for biotechnological applicationsBIOTECHNOLOGY JOURNAL, Issue 4 2009Oliver Spadiut Abstract In order to increase the thermal stability and the catalytic properties of pyranose oxidase (P2Ox) from Trametes multicolor toward its poor substrate D-galactose and the alternative electron acceptor 1,4-benzoquinone (1,4-BQ), we designed the triple-mutant T169G/E542K/V546C. Whereas the wild-type enzyme clearly favors D-glucose as its substrate over D-galactose [substrate selectivity (kcat/KM)Glc/(kcat/KM)Gal = 172], the variant oxidizes both sugars equally well [(kcat/KM)Glc/(kcat/KM)Gal = 0.69], which is of interest for food biotechnology. Furthermore, the variant showed lower KM values and approximately ten-fold higher kcat values for 1,4-BQ when D-galactose was used as the saturating sugar substrate, which makes this enzyme particularly attractive for use in biofuel cells and enzyme-based biosensors. In addition to the altered substrate specificity and reactivity, this mutant also shows significantly improved thermal stability. The half life time at 60°C was approximately 10 h, compared to 7.6 min for the wild-type enzyme. We performed successfully small-scale bioreactor pilot conversion experiments of D -glucose/D -galactose mixtures at both 30 and 50°C, showing the usefulness of this P2Ox variant in biocatalysis as well as the enhanced thermal stability of the enzyme. Moreover, we determined the crystal structure of the mutant in its unligated form at 1.55 Å resolution. Modeling D-galactose in position for oxidation at C2 into the mutant active site shows that substituting Thr for Gly at position 169 favorably accommodates the axial C4 hydroxyl group that would otherwise clash with Thr169 in the wild-type. [source] Active FKHRL1 overcomes imatinib resistance in chronic myelogenous leukemia-derived cell lines via the production of tumor necrosis factor-related apoptosis-inducing ligandCANCER SCIENCE, Issue 12 2007Satoru Kikuchi FKHRL1 (also called FOXO3a) is a member of the Forkhead Box, class O (FOXO) subfamily of forkhead transcription factors and functions downstream of Bcr,Abl tyrosine kinase as a phosphorylated inactive form in chronic myelogenous leukemia (CML). The Bcr,Abl tyrosine kinase inhibitor imatinib induces cell cycle arrest and subsequent apoptosis via the conversion of FKHRL1 from the phosphorylated inactive form to the dephosphorylated active form in CML-derived cell lines. In the present study, we examined whether active FKHRL1 can overcome resistance to imatinib. To this end, we generated a 4-hydroxytamoxifen-inducible active FKHRL1 (FKHRL1-TM; a triple mutant of FKHRL1 in which all three Akt phosphorylation sites have been mutated),estrogen receptor fusion protein expression system in CML-derived imatinib-resistant cell lines. 4-Hydroxytamoxifen inhibited cell growth and cell cycle progression, and subsequently induced apoptosis, accompanied by upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Thus, active FKHRL1 antagonized deregulated proliferation and induced apoptosis in these cell lines. In addition, imatinib-resistant cells underwent apoptosis after transfection with full-length TRAIL cDNA. Collectively, our results suggest that active FKHRL1 can overcome imatinib resistance in CML cells, in part via TRAIL production. (Cancer Sci 2007; 98: 1949,1958) [source] Drosophila CtBP regulates proliferation and differentiation of eye precursors and complexes with Eyeless, Dachshund, Dan, and Danr during eye and antennal developmentDEVELOPMENTAL DYNAMICS, Issue 9 2010Chinh Q. Hoang Abstract Specification factors regulate cell fate in part by interacting with transcriptional co-regulators like CtBP to regulate gene expression. Here, we demonstrate that CtBP forms a complex or complexes with the Drosophila melanogaster Pax6 homolog Eyeless (Ey), and with Distal antenna (Dan), Distal antenna related (Danr), and Dachshund to promote eye and antennal specification. Phenotypic analysis together with molecular data indicate that CtBP interacts with Ey to prevent overproliferation of eye precursors. In contrast, CtBP,dan,danr triple mutant adult eyes have significantly fewer ommatidia than CtBP single or dan,danr double mutants, suggesting that the CtBP/Dan/Danr complex functions to recruit ommatidia from the eye precursor pool. Furthermore, CtBP single and to a greater extent CtBP,dan,danr triple mutants affect the establishment and maintenance of the R8 precursor, which is the founding ommatidial cell. Thus, CtBP interacts with different eye specification factors to regulate gene expression appropriate for proliferative vs. differentiative stages of eye development. Developmental Dynamics 239:2367,2385, 2010. © 2010 Wiley-Liss, Inc. [source] Three independent signalling pathways repress motility in Pseudomonas fluorescens F113MICROBIAL BIOTECHNOLOGY, Issue 4 2009Ana Navazo Summary Motility is one of the most important traits for rhizosphere colonization by pseudomonads. Despite this importance, motility is severely repressed in the rhizosphere-colonizing strain Pseudomonas fluorescens F113. This bacterium is unable to swarm under laboratory conditions and produce relatively small swimming haloes. However, phenotypic variants with the ability to swarm and producing swimming haloes up to 300% larger than the wild-type strain, arise during rhizosphere colonization. These variants harbour mutations in the genes encoding the GacA/GacS two-component system and in other genes. In order to identify genes and pathways implicated in motility repression, we have used generalized mutagenesis with transposons. Analysis of the mutants has shown that besides the Gac system, the Wsp system and the sadB gene, which have been previously implicated in cyclic di-GMP turnover, are implicated in motility repression: mutants in the gacS, sadB or wspR genes can swarm and produce swimming haloes larger than the wild-type strain. Epistasis analysis has shown that the pathways defined by each of these genes are independent, because double and triple mutants show an additive phenotype. Furthermore, GacS, SadB and WspR act at different levels. Expression of the fleQ gene, encoding the master regulator of flagella synthesis is higher in the gacS - and sadB - backgrounds than in the wild-type strain and this differential expression is reflected by a higher secretion of the flagellin protein FliC. Conversely, no differences in fleQ expression or FliC secretion were observed between the wild-type strain and the wspR - mutant. [source] Functional redundancy in the Arabidopsis Cathepsin B gene family contributes to basal defence, the hypersensitive response and senescenceNEW PHYTOLOGIST, Issue 2 2009Hazel McLellan Summary ,,Cysteine proteases are required for programmed cell death (PCD) in animals. Recent work in Nicotiana benthamiana has implicated cathepsin B-like cysteine proteases in the hypersensitive response (HR) in plants, a form of PCD involved in disease resistance. Here, we investigate the function and regulation of Cathepsin B (CathB) genes in plant defence, and in both pathogen-inducible and developmental forms of PCD. ,,Single, double and triple knockout mutants were isolated for the three Arabidopsis thaliana AtCathB genes. ,,AtCathB genes were redundantly required for full basal resistance against the virulent bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. By contrast, AtCathB genes were not required for R gene-mediated resistance to Pst DC3000 expressing AvrB or AvrRps4. Neither did they contribute to PCD triggered by AvrRps4, although they were crucial for the full development of PCD during HR triggered by AvrB. Cathepsin B has also been proposed to play a positive regulatory role in senescence. Atcathb triple mutants showed a delay in senescence and a seven-fold decrease in accumulation of senescence marker gene SAG12. ,,Our results demonstrate a redundant function for AtCathB genes in basal defence as well as a potential regulatory role in distinct forms of plant PCD. [source] |