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Normal Growth Conditions (normal + growth_condition)
Selected AbstractsRole of the two type II myosins, Myo2 and Myp2, in cytokinetic actomyosin ring formation and function in fission yeastCYTOSKELETON, Issue 3 2003Daniel P. Mulvihill Abstract The formation and contraction of a cytokinetic actomyosin ring (CAR) is essential for the execution of cytokinesis in fission yeast. Unlike most organisms in which its composition has been investigated, the fission yeast CAR contains two type II myosins encoded by the genes myo2+ and myp2+. myo2+ is an essential gene whilst myp2+ is dispensable under normal growth conditions. Myo2 is hence the major contractile protein of the CAR whilst Myp2 plays a more subtle and, as yet, incompletely documented role. Using a fission yeast strain in which the chromosomal copy of the myo2+ gene is fused to the gene encoding green fluorescent protein (GFP), we analysed CAR formation and function in the presence and absence of Myp2. No change in the rate of CAR contraction was observed when Myp2 was absent although the CAR persisted longer in the contracted state and was occasionally observed to split into two discrete rings. This was also observed in myp2, cells following actin depolymerisation with latrunculin. CAR contraction in the absence of Myp2 was completely abolished in the presence of elevated levels of chloride ions. Thus, Myp2 appears to contribute to the stability of the CAR, in particular at a late stage of CAR contraction, and to be a component of the signalling pathway that regulates cytokinesis in response to elevated levels of chloride. To determine whether the presence of two type II myosins was a feature of cytokinesis in other fungi that divide by septation, we searched the genomes of two filamentous fungi, Aspergillus fumigatus and Neurospora crassa, for myosin genes. As in fission yeast, both A. fumigatus and N. crassa contained myosins of classes I, II, and V. Unlike fission yeast, both contained a single type II myosin gene that, on the basis of its tail structure, was more reminiscent of Myp2 than Myo2. The significance of these observations to our understanding of septum to formation and cleavage is discussed. Cell Motil. Cytoskeleton 54:208,216, 2003. © 2003 Wiley-Liss, Inc. [source] Cloning and characterization of genes encoding trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2) from Zygosaccharomyces rouxiiFEMS YEAST RESEARCH, Issue 4 2003Hawk-Bin Kwon Abstract In many organisms, trehalose protects against several environmental stresses, such as heat, desiccation, and salt, probably by stabilizing protein structures and lipid membranes. Trehalose synthesis in yeast is mediated by a complex of trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2). In this study, genes encoding TPS1 and TPS2 were isolated from Zygosaccharomyces rouxii (designated ZrTPS1 and ZrTPS2, respectively). They were functionally identified by their complementation of the tps1 and tps2 yeast deletion mutants, which are unable to grow on glucose medium and with heat, respectively. Full-length ZrTPS1 cDNA is composed of 1476 nucleotides encoding a protein of 492 amino acids with a molecular mass of 56 kDa. ZrTPS2 cDNA consists of 2843 nucleotides with an open reading frame of 2700 bp, which encodes a polypeptide of 900 amino acids with a molecular mass of 104 kDa. The amino acid sequence encoded by ZrTPS1 has relatively high homology with TPS1 of Saccharomyces cerevisiae and Schizosaccharomyces pombe, compared with TPS2. Western blot analysis showed that the antibody against S. cerevisiae TPS1 recognizes ZrTPS1. Under normal growth conditions, ZrTPS1 and ZrTPS2 were highly and constitutively expressed, unlike S. cerevisiae TPS1 and TPS2. Salt stress and heat stress reduced the expression of the ZrTPS1 and ZrTPS2 genes, respectively. [source] Escherichia coli tRNase Z can shut down growth probably by removing amino acids from aminoacyl-tRNAsGENES TO CELLS, Issue 11 2008Hiroaki Takaku In most organisms, tRNase Z is considered to be essential for 3, processing of tRNA molecules. The Escherichia coli tRNase Z gene, however, appears to be dispensable under normal growth conditions, and its existence remained an enigma. Here we intensively examined various (pre-)tRNAs for good substrates of E. coli tRNase Z in vitro, and found that the enzyme can remove the 3, terminal CCA residues from mature tRNAs regardless of their nucleotide modifications. Furthermore, we discovered that E. coli tRNase Z, when sufficiently expressed in the cell, can shut down growth probably by removing amino acids from aminoacyl-tRNAs. We confirmed in vitro that E. coli tRNase Z exceptionally possesses the activity that cleaves off the 3, terminal residues charging an amino acid from an aminoacyl-tRNA molecule. The current data suggest that tRNase Z might help modulate a cell growth rate by repressing translation under some stressful conditions. [source] Expression of a High Mobility Group Protein Isolated from Cucumis sativus Affects the Germination of Arabidopsis thaliana under Abiotic Stress ConditionsJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 5 2008Ji Young Jang Abstract Although high mobility group B (HMGB) proteins have been identified from a variety of plant species, their importance and functional roles in plant responses to changing environmental conditions are largely unknown. Here, we investigated the functional roles of a CsHMGB isolated from cucumber (Cucumis sativus L.) in plant responses to environmental stimuli. Under normal growth conditions or when subjected to cold stress, no differences in plant growth were found between the wild-type and transgenic Arabidopsis thaliana overexpressing CsHMGB. By contrast, the transgenic Arabidopsis plants displayed retarded germination compared with the wild-type plants when grown under high salt or dehydration stress conditions. Germination of the transgenic plants was delayed by the addition of abscisic acid (ABA), implying that CsHMGB affects germination through an ABA-dependent way. The expression of CsHMGB had affected only the germination stage, and CsHMGB did not affect the seedling growth of the transgenic plants under the stress conditions. The transcript levels of several germination-responsive genes were modulated by the expression of CsHMGB in Arabidopsis. Taken together, these results suggest that ectopic expression of a CsHMGB in Arabidopsis modulates the expression of several germination-responsive genes, and thereby affects the germination of Arabidopsis plants under different stress conditions. [source] RNA interference-mediated knockdown of ,-synuclein protects human dopaminergic neuroblastoma cells from MPP+ toxicity and reduces dopamine transportJOURNAL OF NEUROSCIENCE RESEARCH, Issue 2 2007Timothy M. Fountaine Abstract The critical observation in the pathology of Parkinson's disease (PD) is that neurodegeneration is largely restricted to dopaminergic neurons that develop cytoplasmic inclusions called Lewy bodies. These aggregations contain the protein ,-synuclein. Furthermore, it is becoming apparent that ,-synuclein expression levels are a major factor in PD pathogenesis. Patients with additional copies of the ,-synuclein gene develop PD with a severity proportional to levels of ,-synuclein overexpression. Similarly, overexpression of ,-synuclein in in vitro and in vivo models has been shown to be toxic. However, little is known about the effects of reducing ,-synuclein expression in human neurons. To investigate this, we have developed a system in which levels of ,-synuclein can be acutely suppressed by using RNA interference (RNAi) in a physiologically relevant human dopaminergic cellular model. By using small interfering RNA (siRNA) molecules targeted to endogenous ,-synuclein, we achieved 80% protein knockdown. We show that ,-synuclein knockdown has no effect on cellular survival either under normal growth conditions over 5 days or in the presence of the mitochondrial inhibitor rotenone. Knockdown does, however, confer resistance to the dopamine transporter (DAT)-dependent neurotoxin N-methyl-4-phenylpyridinium (MPP+). We then demonstrate for the first time that ,-synuclein suppression decreases dopamine transport in human cells, reducing the maximal uptake velocity (Vmax) of dopamine and the surface density of its transporter by up to 50%. These results show that RNAi-mediated ,-synuclein knockdown alters cellular dopamine homeostasis in human cells and may suggest a mechanism for the increased survival in the presence of MPP+, a toxin used extensively to model Parkinson's disease. © 2006 Wiley-Liss, Inc. [source] Burkholderia pseudomallei RpoS regulates OxyR and the katG-dpsA operon under conditions of oxidative stressMICROBIOLOGY AND IMMUNOLOGY, Issue 7 2010Witawat Jangiam ABSTRACT Burkholderia pseudomallei, the causative agent of the potentially fatal tropical disease melioidosis, is known to be highly resistant to oxidative stress although the mechanism of this resistance remains to be fully elucidated. Previous studies have shown that an OxyR is involved in the regulation of oxidative stress via the katG and dpsA genes encoding KatG and DpsA and that the alternative sigma factor, RpoS, plays a critical role in resistance to oxidative stress by regulating katG and katE genes. Here it is shown that RpoS is essential for expression of the oxidative stress regulator OxyR, since a mutant strain lacking RpoS failed to induce oxyR expression both during normal growth and under conditions of oxidative stress. It is further demonstrated that the RpoS acts as a positive transcriptional regulator of oxyR and dpsA expression, while OxyR acts as a negative transcriptional regulator of the katG-dpsA operon via OxyR repressor under normal growth conditions, and as a positive transcriptional regulator via OxyR under conditions of oxidative stress. Therefore both RpoS and OxyR are required to promote expression of both the katG-dpsA operon and dpsA gene. [source] Heat shock protein 101 effects in A. thaliana: genetic variation, fitness and pleiotropy in controlled temperature conditionsMOLECULAR ECOLOGY, Issue 6 2008S. J. TONSOR Abstract The Hsp100/ClpB heat shock protein family is ancient and required for high temperature survival, but natural variation in expression and its phenotypic effects is unexplored in plants. In controlled environment experiments, we examined the effects of variation in the Arabidopsis cytosolic AtHsp101 (hereafter Hsp101). Ten wild-collected ecotypes differed in Hsp101 expression responses across a 22 to 40 °C gradient. Genotypes from low latitudes expressed the least Hsp101. We tested fitness and pleiotropic consequences of varying Hsp101 expression in ,control' vs. mild thermal stress treatments (15/25 °C D/N vs. 15/25° D/N plus 3 h at 35 °C 3 days/week). Comparing wild type and null mutants, wt Columbia (Col) produced ~33% more fruits compared to its Hsp101 homozygous null mutant. There was no difference between Landsberg erecta null mutant NIL (Ler) and wt Ler; wt Ler showed very low Hsp101 expression. In an assay of six genotypes, fecundity was a saturating function of Hsp101 content, in both experimental treatments. Thus, in addition to its essential role in acquired thermal tolerance, Hsp101 provides a substantial fitness benefit under normal growth conditions. Knocking out Hsp101 decreased fruit production, days to germination and days to bolting, total dry mass, and number of inflorescences; it increased transpiration rate and allocation to root mass. Root : total mass ratio decayed exponentially with Hsp101 content. This study shows that Hsp101 expression is evolvable in natural populations. Our results further suggest that Hsp101 is primarily an emergency high-temperature tolerance mechanism, since expression levels are lower in low-latitude populations from warmer climates. Hsp101 expression appears to carry an important trade-off in reduced root growth. This trade-off may select for suppressed expression under chronically high temperatures. [source] Response of superoxide dismutase isoenzymes in tomato plants (Lycopersicon esculentum) during thermo-acclimation of the photosynthetic apparatusPHYSIOLOGIA PLANTARUM, Issue 3 2007Daymi Camejo Seedlings of Lycopersicon esculentum Mill. var. Amalia were grown in a growth chamber under a photoperiod of 16 h light at 25°C and 8 h dark at 20°C. Five different treatments were applied to 30-day-old plants: Control treatment (plants maintained in the normal growth conditions throughout the experimental time), heat acclimation (plants exposed to 35°C for 4 h in dark for 3 days), dark treatment (plants exposed to 25°C for 4 h in dark for 3 days), heat acclimation plus heat shock (plants that previously received the heat acclimation treatment were exposed to 45°C air temperature for 3 h in the light) and dark treatment plus heat shock (plants that previously received the dark treatment were exposed to 45°C air temperature for 3 h in the light). Only the heat acclimation treatment increased the thermotolerance of the photosynthesis apparatus when the heat shock (45°C) was imposed. In these plants, the CO2 assimilation rate was not affected by heat shock and there was a slight and non-significant reduction in maximum carboxylation velocity of Rubisco (Vcmax) and maximum electron transport rate contributing to Rubisco regeneration (Jmax). However, the plants exposed to dark treatment plus heat shock showed a significant reduction in the CO2 assimilation rate and also in the values of Vcmax and Jmax. Chlorophyll fluorescence measurements showed increased thermotolerance in heat-acclimated plants. The values of maximum chlorophyll fluorescence (Fm) were not modified by heat shock in these plants, while in the dark-treated plants that received the heat shock, the Fm values were reduced, which provoked a significant reduction in the efficiency of photosystem II. A slight rise in the total superoxide dismutase (SOD) activity was found in the plants that had been subjected to both heat acclimation and heat shock, and this SOD activity was significantly higher than that found in the plants subjected to dark treatment plus heat shock. The activity of Fe-SOD isoenzymes was most enhanced in heat-acclimated plants but was unaltered in the plants that received the dark treatment. Total CuZn-SOD activity was reduced in all treatments. Darkness had an inhibitory effect on the Mn-SOD isoenzyme activity, which was compensated by the effect of a rise in air temperature to 35°C. These results show that the heat tolerance of tomatoplants may be increased by the previous imposition of a moderately high temperature and could be related with the thermal stability in the photochemical reactions and a readjustment of Vcmax and Jmax. Some isoenzymes, such as the Fe-SODs, may also play a role in the development of heat-shock tolerance through heat acclimation. In fact, the pattern found for these isoenzymes in heat-acclimated Amalia plants was similar to that previously described in other heat-tolerant tomato genotypes. [source] Understanding the relationship between the primary structure of proteins and its propensity to be soluble on overexpression in Escherichia coliPROTEIN SCIENCE, Issue 3 2005Susan Idicula-Thomas Abstract Solubility of proteins on overexpression in Escherichia coli is a manifestation of the net effect of several sequence-dependent and sequence-independent factors. This study aims to delineate the relationship between the primary structure and solubility on overexpression. The amino acid sequences of proteins reported to be soluble or to form inclusion bodies on overexpression in E. coli under normal growth conditions were analyzed. The results show a positive correlation between thermostability and solubility of proteins, and an inverse correlation between the in vivo half-life of proteins and solubility. The amino acid (Asn, Thr, Tyr) composition and the tripeptide frequency of the protein were also found to influence its solubility on overexpression. The amino acids that were seen to be present in a comparatively higher frequency in inclusion body-forming proteins have a higher sheet propensity, whereas those that are seen more in soluble proteins have a higher helix propensity; this is indicative of a possible correlation between sheet propensity and inclusion body formation. Thus, the present analysis shows that thermostability, in vivo half-life, Asn, Thr, and Tyr content, and tripeptide composition of a protein are correlated to the propensity of a protein to be soluble on overexpression in E. coli. The precise mechanism by which these properties affect the solubility status of the overexpressed protein remains to be understood. [source] The chloroplastic lipocalin AtCHL prevents lipid peroxidation and protects Arabidopsis against oxidative stressTHE PLANT JOURNAL, Issue 4 2009Gabriel Levesque-Tremblay Summary Lipocalins are small ligand-binding proteins with a simple tertiary structure that gives them the ability to bind small, generally hydrophobic, molecules. Recent studies have shown that animal lipocalins play important roles in the regulation of developmental processes and are involved in tolerance to oxidative stress. Plants also possess various types of lipocalins, and bioinformatics analyses have predicted that some lipocalin members may be present in the chloroplast. Here we report the functional characterization of the Arabidopsis thaliana chloroplastic lipocalin AtCHL. Cellular fractionation showed that AtCHL is a thylakoid lumenal protein. Drought, high light, paraquat and abscisic acid treatments induce AtCHL transcript and protein accumulation. Under normal growth conditions, knockout (KO) and over-expressing (OEX) lines do not differ from wild-type plants in terms of phenotype and photosynthetic performance. However, KO plants, which do not accumulate AtCHL, show more damage upon photo-oxidative stress induced by drought, high light or paraquat. In contrast, a high level of AtCHL allows OEX plants to cope better with these stress conditions. When exposed to excess light, KO plants display a rapid accumulation of hydroxy fatty acids relative to the wild-type, whereas the lipid peroxidation level remains very low in OEX plants. The increased lipid peroxidation in KO plants is mediated by singlet oxygen and is not correlated with photo-inhibition of the photosystems. This work provides evidence suggesting that AtCHL is involved in the protection of thylakoidal membrane lipids against reactive oxygen species, especially singlet oxygen, produced in excess light. [source] Arabidopsis Bax inhibitor-1 functions as an attenuator of biotic and abiotic types of cell deathTHE PLANT JOURNAL, Issue 6 2006Naohide Watanabe Summary Programmed cell death (PCD) is a common process in eukaryotes during development and in response to pathogens and stress signals. Bax inihibitor-1 (BI-1) is proposed to be a cell death suppressor that is conserved in both animals and plants, but the physiological importance of BI-1 and the impact of its loss of function in plants are still unclear. In this study, we identified and characterized two independent Arabidopsis mutants with a T-DNA insertion in the AtBI1 gene. The phenotype of atbi1-1 and atbi1-2, with a C-terminal missense mutation and a gene knockout, respectively, was indistinguishable from wild-type plants under normal growth conditions. However, these two mutants exhibit accelerated progression of cell death upon infiltration of leaf tissues with a PCD-inducing fungal toxin fumonisin B1 (FB1) and increased sensitivity to heat shock-induced cell death. Under these conditions, expression of AtBI1 mRNA was up-regulated in wild-type leaves prior to the activation of cell death, suggesting that increase of AtBI1 expression is important for basal suppression of cell death progression. Over-expression of AtBI1 transgene in the two homozygous mutant backgrounds rescued the accelerated cell death phenotypes. Together, our results provide direct genetic evidence for a role of BI-1 as an attenuator for cell death progression triggered by both biotic and abiotic types of cell death signals in Arabidopsis. [source] BAK and BAX deletion using zinc-finger nucleases yields apoptosis-resistant CHO cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Gregory J. Cost Abstract Anoxic and metabolic stresses in large-scale cell culture during biopharmaceutical production can induce apoptosis. Strategies designed to ameliorate the problem of apoptosis in cell culture have focused on mRNA knockdown of pro-apoptotic proteins and over-expression of anti-apoptotic ones. Apoptosis in cell culture involves mitochondrial permeabilization by the pro-apoptotic Bak and Bax proteins; activity of either protein is sufficient to permit apoptosis. We demonstrate here the complete and permanent elimination of both the Bak and Bax proteins in combination in Chinese hamster ovary (CHO) cells using zinc-finger nuclease-mediated gene disruption. Zinc-finger nuclease cleavage of BAX and BAK followed by inaccurate DNA repair resulted in knockout of both genes. Cells lacking Bax and Bak grow normally but fail to activate caspases in response to apoptotic stimuli. When grown using scale-down systems under conditions that mimic growth in large-scale bioreactors they are significantly more resistant to apoptosis induced by starvation, staurosporine, and sodium butyrate. When grown under starvation conditions, BAX - and BAK -deleted cells produce two- to fivefold more IgG than wild-type CHO cells. Under normal growth conditions in suspension culture in shake flasks, double-knockout cultures achieve equal or higher cell densities than unmodified wild-type cultures and reach viable cell densities relevant for large-scale industrial protein production. Biotechnol. Bioeng. 2010; 105: 330,340. © 2009 Wiley Periodicals, Inc. [source] | ||||||||||||||||||||||