1,2-propanediol-trehalose mixture as a potent quantitative real-time PCR enhancer

Background  

Quantitative real-time PCR (qPCR) is becoming increasingly important for DNA genotyping and gene expression analysis. For continuous monitoring of the production of PCR amplicons DNA-intercalating dyes are widely used. Recently, we have introduced a new qPCR mix which showed improved amplification of medium-size genomic DNA fragments in the presence of DNA dye SYBR green I (SGI). In this study we tested whether the new PCR mix is also suitable for other DNA dyes used for qPCR and whether it can be applied for amplification of DNA fragments which are difficult to amplify.     

Identification of trehalose as a compatible solute in different species of acidophilic bacteria

The major industrial heap bioleaching processes are located in desert regions (mainly Chile and Australia) where fresh water is scarce and the use of resources with low water activity becomes an attractive alternative. However, in spite of the importance of the microbial populations involved in these processes, little is known about their response or adaptation to osmotic stress. In order to investigate the response to osmotic stress in these microorganisms, six species of acidophilic bacteria were grown at elevated osmotic strength in liquid media, and the compatible solutes synthesised were identified using ion chromatography and MALDI-TOF mass spectrometry. Trehalose was identified as one of, or the sole, compatible solute in all species and strains, apart from Acidithiobacillus thiooxidans where glucose and proline levels increased at elevated osmotic potentials. Several other potential compatible solutes were tentatively identified by MALDITOF analysis. The same compatible solutes were produced by these bacteria regardless of the salt used to produce the osmotic stress. The results correlate with data from sequenced genomes which confirm that many chemolithotrophic and heterotrophic acidophiles possess genes for trehalose synthesis. This is the first report to identify and quantify compatible solutes in acidophilic bacteria that have important roles in biomining technologies.     

Xerotolerance in fission yeasts and the role of glycerol as compatible solute

The effect on growth of reducing the water activity (a _w) of a medium with various solutes has been investigated for 27 strains of fission yeasts (Schizosaccharomyces). The minimum-tolerated a _w (MTA) was dependent on both the nature of the solute and the species. When the strains of each species were grouped together, the lowest mean MTA values were found with glucose, fructose or glycerol as stressing solutes, being in the range 0.89–0.90 for S. pombe, S. malidevorans, S. octosporus and S. slooffiae, but in the range 0.92–0.94 for S. japonicus. With the non-metabolizable sugars sorbose and xylose and the salts NH₄Cl, KCl, and NaCl, the mean MTA values were in the range 0.96–0.985, except for (1) the single strain of S. slooffiae, which was more tolerant of NH₄Cl and KCl with values of 0.95 and 0.94, respectively, and (2) the strains of S. pombe, S. malidevorans and S. japonicus, which were less tolerant of NaCl with mean values of about 0.99. One strain of each species was examined for intracellular solutes when actively growing in the presence of near-limiting concentrations of stressing solute. With glucose, fructose or glycerol, all five strains contained substantial amounts of glycerol but no other polyol; with the other solutes no glycerol or other polyol was found, except for small amounts of glycerol in strains of S. octosporus and S. slooffiae stressed with NH₄Cl, KCl, or NaCl.Abbreviations MTA Minimum-tolerated water activity - a _w water activity - YEPG yeast extract, phosphate, glucose medium     

以四氢嘧啶为主要相容性溶质的中度嗜盐菌I15的分离和特性研究

从草地土壤中分离到一株中度嗜盐菌I15,经过16S rDNA(GenBank登录号为DQ010162)序列分析、形态学和生理生化特征分析,该菌株初步鉴定为Virgibacillus marismortui。I15能在0%～25%NaCl的培养基中生长,最适生长NaCl浓度为10%,最适生长温度为30℃,最适pH为7.5～8.0。在高盐条件下,I15细胞内主要的相容性溶质为四氢嘧啶,在15%NaCl培养基中其含量达到1.608mmol/(g\5cdw),占到相容性溶质总摩尔含量的89.6%。渗透冲击试验表明I15细胞内四氢嘧啶在低渗冲击时能够快速分泌到细胞外,在高渗冲击冲剂时能够较快地重新合成。     

Carnitine: a novel compatible solute in Lactobacillus plantarum

The aim of this study was to unravel the identity of compatible solutes accumulated by Lactobacillus plantarum subjected to osmotic stress. Betaine was accumulated simulataneously with a novel compatible solute identified as carnitine, both present in the complex medium applied in this study. Beef extract provided the main source of carnitine in the medium. Both carnitine and betaine were accumulated to maximum concentrations of 248 and 231 mol.g dry weight^-1, respectively. A defined medium was devised devoid of carnitine. Addition of 0.5 mM carnitine to this medium increased the growth rate from 0.1 h^-1 to 0.2 h^-1 in media with 0.4 M sodium chloride. Also, carnitine made the organism more tolerant to sodium chloride. Growth occurred even when the sodium chloride concentration was raised from 0.5 M to 1.0 M. Quaternary compounds resembling the structure of carnitine and betaine enhanced the growth yield as well. -Butyrobetaine and succinylcholine restored the growth yield up to respectively 91 and 96% compared to non-stressed cells.Abbreviation MRS De Man, Rogosa and Sharpe (De Man et al. 1960)     

5-Hydroxytryptamine as a potent migration enhancer of human aortic endothelial cells

The purpose of the present study was to investigate whether 5-hydroxytryptamine (5-HT, serotonin) affects migration of vascular endothelial cells. 5-HT significantly enhanced migration of human aortic endothelial cells (HAECs), and this enhancement was completely inhibited by GR 55562, a 5-HT1 receptor antagonist, and fluoxetine, a 5-HT transporter inhibitor, but was not affected by ketanserin, a 5-HT2 receptor antagonist. 5-HT stimulation increased RhoA and ERK activity of HAECs, and inhibitors of RhoA (Y-27632 and H-1152) and inhibitors of MEK (U0126 and PD98059) abolished the 5-HT-induced increase in migration velocity. Inhibition of Rho kinase by Y-27632 blocked stress fiber formation and rear release of HAECs. Thus, 5-HT has a potent enhancing action on migration of HAECs through activating the RhoA and ERK pathways following 5-HT1 receptor stimulation.     

Glycinebetaine as an osmoregulant and compatible solute in the marine cyanobacterium Spirulina subsalsa

Glycinebetaine was found to be the major organic substrate accumulating under hypersaline growth conditions in the halotolerant cyanobacterium Spirulina subsalsa. In addition to its proposed role as osmolite, glycinebetaine is shown to specifically protect enzymatic activity. Glucose-6-phosphate dehydrogenase from S. subsalsa retained full activity in the presence of NaCl at concentrations as high as 1.5 M, provided that comparable concentrations of glycinebetaine were also present in the reaction mixture. A kinetic analysis indicated that glycinebetaine protected the enzyme against both NaCl-induced decrease in Vmax and reduction in affinity to glucose 6-phosphate. The alternative osmolites, glycerol and proline, protected the enzyme against the reduction in Vmax but not against the reduction in affinity to glucose 6-phosphate.     

Regulation of compatible solute accumulation in bacteria

In their natural habitats, microorganisms are often exposed to osmolality changes in the environment. The osmotic stress must be sensed and converted into an activity change of specific enzymes and transport proteins and/or it must trigger their synthesis such that the osmotic imbalance can be rapidly restored. On the basis of the available literature, we conclude that representative Gram-negative and Gram-positive bacteria use different strategies to respond to osmotic stress. The main focus of this paper is on the initial response of bacteria to hyper- and hypo-osmotic conditions, and in particular the osmosensing devices that allow the cell to rapidly activate and/or to synthesize the transport systems necessary for uptake and excretion of compatible solutes. The experimental data allow us to discriminate the transport systems by the physicochemical parameter that is sensed, which can be a change in external osmotic pressure, turgor pressure, membrane strain, internal osmolality and/or concentration of specific signal mmicule. We also evaluate the mmicular basis for osmosensing by reviewing the unique structural features of known osmoregulated transport systems.     

相容溶质四氢嘧啶的微生物合成研究进展北大核心CSCD

四氢嘧啶(ectoine)作为一种氨基酸的衍生物,是嗜盐微生物胞内重要的天然次级代谢物,具有保护细胞和稳定生物大分子的功能,可广泛应用于药物制备佐剂、器官移植与保存、皮肤创伤修复与新型化妆品研发等生物医学领域。由于四氢嘧啶的医用价值和商业市场需求,文中从野生菌株的筛选与诱变育种、构建基因工程菌株与系统代谢整合工程菌株、四氢嘧啶的优化发酵与生产工艺以及抽提纯化工艺等方面,系统论述了四氢嘧啶高效积聚和过量化生产研究策略,并展望多组学、计算生物学及“细胞工厂”等技术在后续四氢嘧啶高效生产上的应用前景。     

Function of leaf hamamelitol as a compatible solute during water stress treatment of Hedera helix L.

Hamamelitol is an unusual branched-chain sugar alcohol previously suggested to function as a leaf compatible solute. In this study, we have examined the leaf metabolism and intracelluiar compartmentalization of hamamelitol and other soluble sugars during long-term water stress treatment of Hedera helix (English ivy). Total leaf hamamelitol content was relatively low in greenhouse control plants, but increased 2-fold during water stress treatment to levels approaching those observed in field-grown plants (6–7 μmol g^?1 fresh weight). Using density gradient fractionation with non-aqueous solvents, we showed that hamamelitol occurs primarily in the cytoplasm and vacuoles of leaf mesophyll cells. During water stress treatment most of the increase in leaf hamamelitol occurred in the mesophyll cytoplasm, compensating osmotically for a decrease in cytoplasmic sucrose concentration. The maximum concentration of cytoplasmic hamamelitol was 155 mol m^?3 and occurred in field-grown plants. Labelling experiments showed that hamamelitol is slowly synthesized from ¹⁴CO₂ in leaves of H. helix, but is very long-lived (estimated t_1/2 of 4 years). Together, these data indicate that hamamelitol probably functions during long-term stress conditions as an osmotically active, compatible solute in plant leaves. We suggest that the signal for enhanced accumulation of hamamelitol during the water stress treatment was initiated by decreased plant growth and increased leaf sucrose hydrolysis.     

Betaine is the main compatible solute of halophilic eubacteria.

A number of moderately halophilic bacteria of diverse taxonomic groups have been studied to determine the intracellular concentrations of organic compounds at various salt concentrations. Betaine was accumulated in all of these organisms in proportion to the salinity of the medium, suggesting that this compound plays a major role in osmoregulation.     

In planta function of compatible solute transporters of the AtProT family

The three proline transporters of Arabidopsis thaliana (AtProTs) transport the compatible solutes proline and glycine betaine and the stress-induced compound γ-aminobutyric acid when expressed in heterologous systems. The aim of the present study was to show transport and physiological relevance of these three AtProTs in planta. Using single, double, and triple knockout mutants and AtProT-overexpressing lines, proline content, growth on proline, transport of radiolabelled betaine, and expression of AtProT genes and enzymes of proline metabolism were analysed. AtProT2 was shown to facilitate uptake of L- and D-proline as well as [(14)C]glycine betaine in planta, indicating a role in the import of compatible solutes into the root. Toxic concentrations of L- and D-proline resulted in a drastic growth retardation of AtProT-overexpressing plants, demonstrating the need for a precise regulation of proline uptake and/or distribution. Furthermore evidence is provided that AtProT genes are highly expressed in tissues with elevated proline content--that is, pollen and leaf epidermis.     

Glucosylglycerol,a compatible solute,sustains cell division under salt stress

The cyanobacterium Synechocystis sp. PCC 6803 accumulates the compatible solute glucosylglycerol (GG) and sucrose under salt stress. Although the molecular mechanisms for GG synthesis including regulation of the GG-phosphate synthase (ggpS) gene, which encodes GgpS, has been intensively investigated, the role of GG in protection against salt stress remains poorly understood. In our study of the role of GG in the tolerance to salt stress, we found that salt stress due to 450 mM NaCl inhibited cell division and significantly increased cell size in DeltaggpS mutant cells, whereas the inhibition of cell division and increase in cell size were observed in wild-type cells at high concentrations of NaCl, such as 800 mM. Electron microscopy revealed that, in DeltaggpS cells, separation of daughter cells was incomplete, and aborted division could be recognized by the presence of a structure that resembled a division ring. The addition of GG to the culture medium protected DeltaggpS cells against salt stress and reversed the adverse effects of NaCl on cell division and cell size. These observations suggest that GG is important for salt tolerance and thus for the proper division of cells under salt stress conditions.     

Isolation and characterisation of moderately halophilic bacteriumHalomonas ventosae DL7 synthesizing ectoine as compatible solute

A moderately halophilic Gram-negative bacterium, strain DL7, was isolated from saltern sediment of Dalian, China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that this isolate belongs to the genusHalomonas. Based on the taxonomic and DNA sequence in addition to high DNA-DNA homologies, we concluded that this strain was similar with the type strain ofHalomonas ventosae. Ectoine, one of the representative compatible solutes, was mainly detected in the cells as a result of¹H- and¹³C-NMR measurements when grown in the presence of wide concentration ranges of NaCl. The results showed that higher amount of ectoine was synthesized in a shorter incubation time compared with those of other strains reported earlier.     

Phylogenetic distribution of compatible solute synthesis genes support a freshwater origin for cyanobacteria

Previous work using ancestral state reconstruction of habitat salinity preference proposed that the early cyanobacteria likely lived in a freshwater environment. The aim of this study was to test that hypothesis by performing phylogenetic analyses of the genes underlying salinity preferences in the cyanobacteria. Phylogenetic analysis of compatible solute genes shows that sucrose synthesis genes were likely ancestral in the cyanobacteria, and were also likely inherited during the cyanobacterial endosymbiosis and into the photosynthetic algae and land plants. In addition, the genes for the synthesis of compatible solutes that are necessary for survival in marine and hypersaline environments (such as glucosylglycerol, glucosylglycerate, and glycine betaine) were likely acquired independently high up (i.e., more recently) in the cyanobacterial tree. Because sucrose synthesis is strongly associated with growth in a low salinity environment, this independently supports a freshwater origin for the cyanobacteria. It is also consistent with geologic evidence showing that the early oceans were much warmer and saltier than modern oceans—sucrose synthesis alone would have been insufficient for early cyanobacteria to have colonized early Precambrian oceans that had a higher ionic strength. Indeed, the acquisition of an expanded set of new compatible solute genes may have enabled the historical colonization of marine and hypersaline environments by cyanobacteria, midway through their evolutionary history.     

Continuous synthesis and excretion of the compatible solute ectoine by a transgenic, nonhalophilic bacterium

The compatible solute 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) acts in microorganisms as an osmotic counterweight against halostress and has attracted commercial attention as a protecting agent. Its production and application are restricted by the drawbacks of the discontinuous harvesting procedure involving salt shocks, which reduces volumetric yield, increases reactor corrosion, and complicates downstream processing. In order to synthesize ectoine continuously in less-aggressive media, we introduced the ectoine genes ectABC of the halophilic bacterium Chromohalobacter salexigens into an Escherichia coli strain using the expression vector pASK-IBA7. Under the control of a tet promoter, the transgenic E. coli synthesized 6 g liter-1 ectoine with a space-time yield of 40 mg liter-1 h-1, with the vast majority of the ectoine being excreted.     

Characterization of TAE684 as a potent LRRK2 kinase inhibitor

Leucine-rich repeat kinase 2 (LRRK2) is linked to Parkinson's disease and may represent an attractive therapeutic target. Here we report a 2,4-dianilino-5-chloro-pyrimidine, TAE684, a previously reported inhibitor of anaplastic lymphoma kinase (ALK), is also a potent inhibitor of LRRK2 kinase activity (IC(50) of 7.8nM against wild-type LRRK2, 6.1nM against the G2019S mutant). TAE684 substantially inhibits Ser910 and Ser935 phosphorylation of both wild-type LRRK2 and G2019S mutant at a concentration of 0.1-0.3μM in cells and in mouse spleen and kidney, but not in brain, following oral doses of 10mg/kg.     

Effect of the compatible solute ectoine on the stability of the membrane proteins

Mechanical single molecule techniques offer exciting possibilities for investigating protein folding and stability in native environments at sub-nanometer resolutions. Compatible solutes show osmotic activity which even at molar concentrations do not interfere with cell metabolism. They are known to protect proteins against external stress like temperature, high salt concentrations and dehydrating conditions. We studied the impact of the compatible solute ectoine (1M) on membrane proteins by analyzing the mechanical properties of Bacteriorhodopsin (BR) in its presence and absence by single molecule force spectroscopy. The unfolding experiments on BR revealed that ectoine decreases the persistence length of its polypeptide chain thereby increasing its tendency to coil up. In addition, we found higher unfolding forces indicating strengthening of those intra molecular interactions which are crucial for stability. This shows that force spectroscopy is well suited to study the effect of compatible solutes to stabilize membrane proteins against unfolding. In addition, it may lead to a better understanding of their detailed mechanism of action.