Genomic fluidity and pathogenic bacteria: applications in diagnostics, epidemiology and intervention

The increasing availability of DNA-sequence information for multiple pathogenic and non-pathogenic variants of individual bacterial species has indicated that both DNA acquisition and genome reduction have important roles in genome evolution. Such genomic fluidity, which is found in human pathogens such as Escherichia coli, Helicobacter pylori and Mycobacterium tuberculosis, has important consequences for the clinical management of the diseases that are caused by these pathogens and for the development of diagnostics and new molecular epidemiological methods.     

Identification of transposable element-mediated deletions in 27 Korean individuals based on whole genome sequencing data

The human genome has various genomic structural variations such as insertion/deletions between human individuals. These structural variations have led to genomic fluidity and rearrangements in individuals and populations. To investigate Korean-specific structural genomic variations, we performed next generation sequencing with 30× mean coverage from 27 Korean individuals using illumina-HiSeq 2000 platform. We collected a total of 119 deletion loci as transposable element-mediated Korean-specific deletion (KSD) candidates. Of the 119 loci, 35 were filtered out due to computational overlapping regions. A total of 78 loci were validated by PCR amplification with 27 Korean individuals and 80 human individuals from four different populations. We confirmed deletion breakpoints of the 78 loci using Sanger sequencing. We also investigated different deletion mechanisms based on sequencing alignment analysis. We found at least one KSD locus in 80 human individual panel. It has not been previously reported in human genomes. Here, for the first time, we report transposable element-mediated KSD study based on whole genome sequencing data of 27 Korean.     

Age specificity of lipid fluidity in the sarcoplasmic reticulum of the rat heart

The lipid fluidity in heart sarcoplasmic reticulum membranes prepared from adult (12 mo.) and old (24 mo.) rats has been measured by the fluorescence probe (DPPH) and spin probe (5NS) methods at 22 and 37 degrees C. The lipid fluidity in the old rat membranes is higher than that in the adult rat ones. It has been suggested that this difference is caused by age lowering in reliability of membrane fluidity stabilization systems.     

Lipid mobility in the assembly and expression of the activity of the prothrombinase complex

A phospholipid or membrane surface is a required component of the prothrombinase complex, yet little is known about the influence of the lipid on the assembly and expression of this complex. Vesicles composed of synthetic phospholipids were used to investigate the effects of membrane "fluidity" on the prothrombinase complex. All vesicle types studied were capable of supporting the prothrombinase reaction which in each case was characterized by a similar apparent Km. The binding constants for the interaction of Factor Va and prothrombin with synthetic phospholipid vesicles were not significantly affected by temperature. The rate of thrombin production, however, increased with increasing temperature. The fluidity of the vesicles was assessed by measuring the fluorescence lifetimes, steady state anisotropies, and differential phase fluorometry of diphenylhexatriene embedded in the vesicles. No correlation was observed between the fluidity of the vesicles and the steady-state rate of thrombin production, even when the enzymatic activity was monitored below and above the phase transition temperature of the lipid vesicles. A distinct correlation, however, was found between the fluidity of the vesicle and the time required to reach the maximum rate of thrombin production (pre-steady-state interval). We believe that this "lag" time corresponds to the time required for the assembly of the prothrombinase complex. Thus, although lipid fluidity does affect the assembly of the prothrombinase complex, after the complex is assembled, this property has little effect on the catalytic process itself.     

Lipid fluidity directly modulates the overall protein rotational mobility of the Ca-ATPase in sarcoplasmic reticulum

We have developed a quantitative and relatively model-independent measure of lipid fluidity using EPR and have applied this method to compare the temperature dependence of lipid hydrocarbon chain fluidity, overall protein rotational mobility, and the calcium-dependent enzymatic activity of the Ca-ATPase in sarcoplasmic reticulum. We define membrane lipid fluidity to be T/eta, where eta is the viscosity of a long chain hydrocarbon reference solvent in which a fatty acid spin label gives the same EPR spectrum (quantitated by the order parameter S) as observed for the same probe in the membrane. This measure is independent of the reference solvent used as long as the spectral line shapes in the membrane and the solvent match precisely, indicating that the same type of anisotropic probe motion occurs in the two systems. We argue that this empirical measurement of fluidity, defined in analogy to the macroscopic fluidity (T/eta) of a bulk solvent, should be more directly related to protein rotational mobility (and thus to protein function) than are more conventional measures of fluidity, such as the rate or amplitude of rotational motion of the lipid hydrocarbon chains themselves. This new definition thus offers a fluidity measure that is more directly relevant to the protein's behavior. The direct relationship between this measure of membrane fluidity and protein rotational mobility is supported by measurements in sarcoplasmic reticulum. The overall rotational motion of the spin-labeled Ca-ATPase protein was measured by saturation-transfer EPR. The Arrhenius activation energy for protein rotational mobility (11-12 kcal/mol/degree) agrees well with the activation energy for lipid fluidity, if defined as in this study, but not if more conventional definitions of lipid fluidity are used. This agreement, which extends over the entire temperature range from 0 to 40 degrees C, suggests that protein mobility depends directly on lipid fluidity in this system, as predicted from hydrodynamic theory. The same activation energy is observed for the calcium-dependent ATPase activity under physiological conditions, suggesting that protein rotational mobility (dependent on lipid fluidity) is involved in the rate-limiting step of active calcium transport.     

Control of centrosome reproduction: the right number at the right time.

It is of great importance for the cell to precisely coordinate the doubling of the interphase centrosome with nuclear events during the cell cycle and limit the number of centrosomes it contains at the onset of mitosis to two and only two. The penalties for mistakes are abnormal spindle assembly, inappropriate chromosome distribution, and consequently, genomic instability. We review the functional properties of the mechanisms that control when the centrosome duplicates in the cell cycle and the controls for centrosome copy number. We look to limits that are intrinsic to the centrosome itself and controls imposed by cell cycle linked changes in cytoplasmic conditions. Control of centrosome reproduction is exercised at both levels.     

Membrane fluidity and its roles in the perception of environmental signals

Poikilothermic organisms are exposed to frequent changes in environmental conditions and their survival depends on their ability to acclimate to such changes. Changes in ambient temperature and osmolarity cause fluctuations in the fluidity of cell membranes. Such fluctuations are considered to be critical to the initiation of the regulatory reactions that ultimately lead to acclimation. The mechanisms responsible for the perception of changes in membrane fluidity have not been fully characterized. However, the analysis of genome-wide gene expression using DNA microarrays has provided a powerful new approach to studies of the contribution of membrane fluidity to gene expression and to the identification of environmental sensors. In this review, we focus on the mechanisms that regulate membrane fluidity, on putative sensors that perceive changes in membrane fluidity, and on the subsequent expression of genes that ensures acclimation to a new set of environmental conditions.     

Controlling the membrane fluidity of yeasts during coupled thermal and osmotic treatments

Yeasts are often exposed to variations in osmotic pressure in their natural environments or in their substrates when used in fermentation industries. Such changes may lead to cell death or activity loss. Previous work by our team has allowed us to relate the mortality of cells exposed to a combination of thermal and osmotic treatments to leakage of cellular components through an unstable membrane when lipid phase transition occurs. In this study, yeast viability was measured after numerous osmotic and thermal treatments. In addition, the fluidity of yeast membranes was assessed according to a(w) and temperature by means of 1,6-diphenyl-1,3,5-hexatriene (DPH) anisotropy measurement. The results show that there is a negative correlation between the overall fluidity variation undergone by membranes during treatments and yeast survival. Using a diagram of membrane fluidity according to a(w) and temperature, we defined dehydration and rehydration methods that minimize fluidity fluctuations, permitting significantly increased yeast survival. Thus, such membrane fluidity diagram should be a potential tool for controlling membrane state during dehydration and rehydration and improve yeast survival. Overall fluidity measurements should now be completed by accurate structural analysis of membranes to better understand the plasma membrane changes occurring during dehydration and rehydration.     

Genetic instability and hypervariability in Streptomyces ambofaciens: towards an understanding of a mechanism of genome plasticity

Many Streptomyces species exhibit a very high degree of genetic instability which is usually manifested as genomic rearrangements such as large deletions. In Streptomyces ambofaciens DSM40697, two levels of genetic instability were previously described: (i) a basic genetic instability similar to that reported for other strains, and (ii) hypervariability, a phenomenon that we believe to be a new aspect of instability closely associated with DNA amplification. A large DNA region undergoes deletions, amplifications and large genomic changes strictly associated with both aspects of genetic instability. The genetic and molecular analyses of the different aspects of genetic instability allow us to propose that they result from a cascade of molecular events and to investigate the relationships between genetic instability phenomena and genome fluidity.     

基因组分析方法在微生物分类学中的应用

细菌分类学始于19世纪后半叶,当时主要是以表型标记和生理生化特性为基础的简单分类,之后DNA-DNA分子杂交、16S rRNA基因序列分析方法的出现给微生物分类带来了极大的便利。尽管如此,这些分类学方法仍然存在一些局限性,而基因组时代的到来,为微生物分类带来了新思路。本文主要介绍了5种基于全基因组数据的微生物分类方法,包括平均核苷酸同源性分析、核心基因组分析、最大唯一匹配指数分析、K串组分矢量法和基因流动性分析,并论述了这些方法在微生物分类学中的应用。     

Concanavalin A-induced increase in the membrane fluidity of chicken erythrocytes.

To determine the fluidity of the membrane lipid phase, chicken erythrocytes were labeled with a stearic acid derivative spin label. When chicken erythrocytes were treated with concanavalin A (Con A), ESR spectra showed a change in the peaks of the labels in membrane lipids, indicating an increase of membrane fluidity. The degree of the increase in fluidity of the membrane lipid phase depended on the valency of the lectin used. Tetravalent Con A induced an increase of membrane fluidity at a concentration as low as 30 micrograms/ml, while a monovalent derivative of Con A did not affect membrane fluidity. This increase in membrane fluidity was observed within 10 min after the addition of Con A. If bound Con A was removed with methyl alpha-D-mannoside later than 60 min after its addition, a complete return of the fluidity to the normal level could not be observed. However, no change was found in the composition of phospholipids or in the fatty acid compositions of phosphatidylcholine and phosphatidylethanolamine of chicken erythrocytes after the addition of Con A, indicating that this increase in membrane fluidity is not caused by a change of lipid composition. The clustering of membrane receptors of chicken erythrocytes for Con A was demonstrated when the two-dimensional distribution of ferritin-conjugated Con A on the membranes was assayed by transmission electron microscopy. Furthermore, it was shown that major receptors for Con A of chicken erythrocytes were transmembrane glycoproteins having apparent molecular weights of 100K, 45, and 33K.     

Disorders in blood rheological properties in the post-resuscitation period after the agonal state

It has been discovered in experiments on anesthetized dogs that the early postresuscitation period following agony after a two-hour hypovolemic hypotension was characterized by pronounced disturbances of blood rheological properties: blood viscosity was significantly increased as compared with initial values under the shift rate 1.34 s-1; the limit of blood fluidity was twice as increased. Insignificantly changed was the magnitude of hematocrit following the recovery of the blood volume. Infusion of polyglucin (20-25 ml/kg) promptly after the blood volume recovery and on the first day after it (10 ml/kg) decreased the hematocrit magnitude to 0.26 l/l (days 5-7) but did not make blood fluidity return to normal. A significant increase of blood viscosity and fluidity could be observed for 1-3 months after resuscitation, the hematocrit magnitude being equal to 0.55 l/l instead of 0.47 l/l in an initial state (P less than 0.005).     

老年学习记忆减退大鼠脑突触体膜流动性改变

选用Morris水迷宫将老年大鼠分为学习记忆正常和学习记忆减退两部分,采用荧光偏振技术,对青年、老年记忆正常和老年记忆减退鼠脑分离实触体膜流动性进行测定,并检测神经节苷脂GM1对膜流动性的影响．结果表明老年记忆减退鼠新皮质、海马结构突触体膜荧光各向异性明显增加,即膜流动性显著降低,GM1对膜流动性有明显改善作用．相关分析表明新皮质、海马结构实触膜流动性与老年学习记忆减退密切相关,GM1的积极作用为临床治疗提供实验依据．     

Oxidative and drug-induced alterations in brush border membrane hemileaflet fluidity, functional consequences for glucose transport

Oxidation of biological membranes has been suggested as a major pathological process in a variety of disease states including intestinal ischemia and inflammatory bowel disease. Previous studies on the small intestinal brush border membrane have shown that part of the decrease in the activity of the Na(+)-dependent glucose transporter (SGLT1) observed after oxidation could be secondary to the derangement in membrane fluidity that accompanied oxidative damage. The present study examined the relationship between oxidative-induced hemileaflet fluidity alterations and the resultant change in Na(+)-dependent glucose transport activity. To address this issue, in vitro oxidation of guinea pig brush border membrane vesicles was induced by incubation of the vesicles with ferrous sulfate and ascorbate. We found that oxidation decreased the fluidity of both the outer and inner hemileaflets, the decrease being greater in the outer leaflet. Moreover, the preferential alteration in hemileaflet fluidity was accompanied by a decrease in glucose transport. However, when membrane perturbing agents such as hexanol and A(2)C were used to restore membrane fluidity to levels comparable to controls, rates of glucose transport could not be interpreted in terms of variation of bulk membrane fluidity or variation in fluidity of any specific membrane leaflet. On the basis of these experiments, we propose that previous studies that reported coincidental alteration in membrane fluidity and glucose transport cannot be interpreted on the basis of bulk fluidity or hemileaflet fluidity.     

The effect of membrane composition and alcohols on the insulin-sensitive reconstituted monosaccharide-transport system of rat adipocyte plasma membranes.

The monosaccharide transporter from the plasma membranes of rat adipocytes and insulin-stimulated adipocytes has been reconstituted in sonicated liposomes. The stereospecific D-glucose uptake by liposomes made from a range of phospholipids and incorporating fatty acids has been investigated. D-Glucose uptake is correlated with an increase in lipid fluidity as a consequence of the addition of fluidizing fatty acids, changes in phospholipid acyl chain length and temperature. Benzyl alcohol and ethyl alcohol, which are generally considered to increase bilayer fluidity, decrease stereo-specific D-glucose uptake in both whole adipocytes and reconstituted liposomes. It is suggested that, although these alcohols may affect D-glucose transport by lipid-mediated fluidity changes, they also interact directly with the transporter resulting in inhibition of transport.     

Correlations between the thermal stability of chloroplast (thylakoid) membranes and the composition and fluidity of their polar lipids upon acclimation of the higher plant,Nerium oleander,to growth temperature

The thermal stability of excitation transfer from pigment proteins to the Photosystem II reaction center of Nerium oleander adjusts by 10 Celsius degrees when cloned plants grown at 20°C/15°C, day/night growth temperatures are shifted to 45°C/32°C growth temperature or vice versa. Concomitant with this adjustment is a decrease in the fluidity of thylakoid membrane polar lipids as determined by spin labeling. The results are consistent with the hypothesis that there is a limiting maximum fluidity compatible with maintenance of native membrane structure and function. This limiting fluidity was about the same as for a number of other species which exhibit a range of thermal stabilities. Inversely correlated shifts in lipid fluidity and thermal stability occurred during the time course of acclimation of N. oleander to new growth temperatures. Thus, the temperature at which the limiting fluidity was reached changed during acclimation while the limiting fluidity remained constant. Although the relative proportion of the major classes of membrane polar lipids remained constant during adjustments in fluidity, large changes occured in the abundance of specific fatty acids. These changes were different for the phospho- and galacto-lipids suggesting that the fatty acid composition of these two lipid classes is regulated by different mechanisms. Comparisons between membrane lipid fluidity and fatty acid composition indicate that fluidity is not a simple linear function of fatty acid composition.     

Age and sex related changes of plasma membrane fluidity in isolated rat hepatocytes

The influence of sex and age on membrane fluidity, has been investigated in 6, 12, 18 weeks old Sprague-Dawley rats. Fluorescence polarization (P) was determined at 37 degrees C with a Perkin Elmer MPF 44A fluorescence spectrophotometer. The fluorescent probe TMA-DPH was added to isolated hepatocytes prepared by collagenase method. The membrane fluidity was constantly lower in males than in females, but the difference was statistically significant only in the 12 weeks old group. Major differences appeared related to aging with a significant age-related decrease in fluidity in all animals.     

Escherichia coli membrane fluidity as detected by excimerization of dipyrenylpropane: sensitivity to the bacterial fatty acid profile.

A coordinated study of membrane fluidity and fatty acid composition has been carried out in Escherichia coli W3110. The lipid acyl chain profile of the bacteria, altered by growing cells in steady state at 30, 37, 42, or 45 degrees C, was determined by gas chromatography of the fatty acid methyl esters. In parallel experiments, total membranes obtained from cells of the above-mentioned cultures were labeled with dipyrenylpropane and their relative fluidity was measured on the basis of the excimer to monomer fluorescence intensity ratio of the fluorophore. It has been found that, at constant assay temperature, fluidity determined with dipyrenylpropane decreases gradually with the growth temperature increment, from 30 to 45 degrees C. Interestingly, when fatty acid composition is taken into account, fluidity increases linearly in the range under study, with the proportion of unsaturated fatty acyl chains, both variables being highly correlated (0.924 相似文献