Enhancement of the rate of pyrophosphate hydrolysis by nonenzymatic catalysts and by inorganic pyrophosphatase

To estimate the proficiency of inorganic pyrophosphatase as a catalyst, ³¹P NMR was used to determine rate constants and thermodynamics of activation for the spontaneous hydrolysis of inorganic pyrophosphate (PP_i) in the presence and absence of Mg²⁺ at elevated temperatures. These values were compared with rate constants and activation parameters determined for the reaction catalyzed by Escherichia coli inorganic pyrophosphatase using isothermal titration calorimetry. At 25 °C and pH 8.5, the hydrolysis of MgPP_i²⁻ proceeds with a rate constant of 2.8 × 10⁻¹⁰ s⁻¹, whereas E. coli pyrophosphatase was found to have a turnover number of 570 s⁻¹ under the same conditions. The resulting rate enhancement (2 × 10¹²-fold) is achieved entirely by reducing the enthalpy of activation (ΔΔH^‡ = −16.6 kcal/mol). The presence of Mg²⁺ ions or the transfer of the substrate from bulk water to dimethyl sulfoxide was found to increase the rate of pyrophosphate hydrolysis by as much as ∼10⁶-fold. Transfer to dimethyl sulfoxide accelerated PP_i hydrolysis by reducing the enthalpy of activation. Mg²⁺ increased the rate of PP_i hydrolysis by both increasing the entropy of activation and reducing the enthalpy of activation.     

A 2-dimensional geometry for biological time

This paper proposes an abstract mathematical frame for describing some features of biological time. The key point is that usual physical (linear) representation of time is insufficient, in our view, for the understanding key phenomena of life, such as rhythms, both physical (circadian, seasonal …) and properly biological (heart beating, respiration, metabolic …). In particular, the role of biological rhythms do not seem to have any counterpart in mathematical formalization of physical clocks, which are based on frequencies along the usual (possibly thermodynamical, thus oriented) time. We then suggest a functional representation of biological time by a 2-dimensional manifold as a mathematical frame for accommodating autonomous biological rhythms. The “visual” representation of rhythms so obtained, in particular heart beatings, will provide, by a few examples, hints towards possible applications of our approach to the understanding of interspecific differences or intraspecific pathologies. The 3-dimensional embedding space, needed for purely mathematical reasons, allows to introduce a suitable extra-dimension for “representation time”, with a cognitive significance.     

Homogeneity test of rate ratios in stratified matched-pair studies

This paper investigates homogeneity test of rate ratios in stratified matched-pair studies on the basis of asymptotic and bootstrap-resampling methods. Based on the efficient score approach, we develop a simple and computationally tractable score test statistic. Several other homogeneity test statistics are also proposed on the basis of the weighted least-squares estimate and logarithmic transformation. Sample size formulae are derived to guarantee a pre-specified power for the proposed tests at the pre-given significance level. Empirical results confirm that (i) the modified score statistic based on the bootstrap-resampling method performs better in the sense that its empirical type I error rate is much closer to the pre-specified nominal level than those of other tests and its power is greater than those of other tests, and is hence recommended, whilst the statistics based on the weighted least-squares estimate and logarithmic transformation are slightly conservative under some of the considered settings; (ii) the derived sample size formulae are rather accurate in the sense that their empirical powers obtained from the estimated sample sizes are very close to the pre-specified nominal powers. A real example is used to illustrate the proposed methodologies.     

Hydrolysis Rates of Different Small Interfering RNAs (siRNAs) by the RNA Silencing Promoter Complex,C3PO,Determines Their Regulation by Phospholipase Cβ

C3PO plays a key role in promoting RNA-induced gene silencing. C3PO consists of two subunits of the endonuclease translin-associated factor X (TRAX) and six subunits of the nucleotide-binding protein translin. We have found that TRAX binds strongly to phospholipase Cβ (PLCβ), which transmits G protein signals from many hormones and sensory inputs. The association between PLCβ and TRAX is thought to underlie the ability of PLCβ to reverse gene silencing by small interfering RNAs. However, this reversal only occurs for some genes (e.g. GAPDH and LDH) but not others (e.g. Hsp90 and cyclophilin A). To understand this specificity, we carried out studies using fluorescence-based methods. In cells, we find that PLCβ, TRAX, and their complexes are identically distributed through the cytosol suggesting that selectivity is not due to large scale sequestration of either the free or complexed proteins. Using purified proteins, we find that PLCβ binds ∼5-fold more weakly to translin than to TRAX but ∼2-fold more strongly to C3PO. PLCβ does not alter TRAX-translin assembly to C3PO, and brightness studies suggest one PLCβ binds to one C3PO octamer without a change in the number of TRAX/translin molecules suggesting that PLCβ binds to an external site. Functionally, we find that C3PO hydrolyzes siRNA(GAPDH) at a faster rate than siRNA(Hsp90). However, when PLCβ is bound to C3PO, the hydrolysis rate of siRNA(GAPDH) becomes comparable with siRNA(Hsp90). Our results show that the selectivity of PLCβ toward certain genes lies in the rate at which the RNA is hydrolyzed by C3PO.     

Long Term Ablation of Protein Kinase A (PKA)-mediated Cardiac Troponin I Phosphorylation Leads to Excitation-Contraction Uncoupling and Diastolic Dysfunction in a Knock-in Mouse Model of Hypertrophic Cardiomyopathy

The cardiac troponin I (cTnI) R21C (cTnI-R21C) mutation has been linked to hypertrophic cardiomyopathy and renders cTnI incapable of phosphorylation by PKA in vivo. Echocardiographic imaging of homozygous knock-in mice expressing the cTnI-R21C mutation shows that they develop hypertrophy after 12 months of age and have abnormal diastolic function that is characterized by longer filling times and impaired relaxation. Electrocardiographic analyses show that older R21C mice have elevated heart rates and reduced cardiovagal tone. Cardiac myocytes isolated from older R21C mice demonstrate that in the presence of isoproterenol, significant delays in Ca²⁺ decay and sarcomere relaxation occur that are not present at 6 months of age. Although isoproterenol and stepwise increases in stimulation frequency accelerate Ca²⁺-transient and sarcomere shortening kinetics in R21C myocytes from older mice, they are unable to attain the corresponding WT values. When R21C myocytes from older mice are treated with isoproterenol, evidence of excitation-contraction uncoupling is indicated by an elevation in diastolic calcium that is frequency-dissociated and not coupled to shorter diastolic sarcomere lengths. Myocytes from older mice have smaller Ca²⁺ transient amplitudes (2.3-fold) that are associated with reductions (2.9-fold) in sarcoplasmic reticulum Ca²⁺ content. This abnormal Ca²⁺ handling within the cell may be attributed to a reduction (2.4-fold) in calsequestrin expression in conjunction with an up-regulation (1.5-fold) of Na⁺-Ca²⁺ exchanger. Incubation of permeabilized cardiac fibers from R21C mice with PKA confirmed that the mutation prevents facilitation of mechanical relaxation. Altogether, these results indicate that the inability to enhance myofilament relaxation through cTnI phosphorylation predisposes the heart to abnormal diastolic function, reduced accessibility of cardiac reserves, dysautonomia, and hypertrophy.     

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.     

2000-2010年武汉市中心城区湖泊景观变化

武汉雅称"百湖之市",湖泊是武汉市的重要名片。以2000、2005和2010年三期Landsat TM数据为数据源,采用归一化差异水体指数(NDWI)提取湖泊水体信息,建立了各个时期武汉市中心城区湖泊矢量图层,计算了湖泊的面积萎缩率、斑块分维数和破碎度等景观指数,对2000—2010年武汉市中心城区湖泊变化特征进行了分析。研究表明,武汉市各个湖泊均有不同程度的萎缩,湖泊面积萎缩率大小和其所隶属的环线及政策因素有较大的关系;各湖泊斑块分维数在1-1.3之间,并越来越接近于1,表明在人类活动持续影响下,其形状变得越来越规则;同时斑块数目增加,湖泊在面积萎缩的同时,也变得越来越破碎,研究显示主要是道路修建所致导致的湖泊分割,湖泊被分割后,自净能力下降,会导致水体污染而最终被填埋。总之,10年武汉市湖泊景观环境朝着不良方向发展,应该制定更严格的政策进行水域管理。     

损伤神经自发放电节律分岔与频率变化的非线性特征

为了研究神经放电节律回周分岔与放电频率变化之间的关系,采用大鼠坐骨神经慢性结扎模型,记录损伤区的自发放电,观察放电节律转化的动力学规律,分析相应的放电频率的变化,并用理论模型进行数值模拟。结果表明,与放电节律加周分岔相对应,放电频率的变化呈现非线性的特征,数值模拟支持实验的发现。研究提示：神经放电的频率变化与刺激强度的改变并非呈简单的线性相关,可能具有更复杂的关系。     

小叶章种群高度的季节动态

探讨了三江平原以小叶章为优势种的典型草旬、沼泽化草甸、沼泽３个植被类型中小叶章种群高度的变化规律。结果表明,在３个类型中,种群高度的季节动态均呈单峰型,极大值均出现在７月末,分别为１１２．６７ｃｍ、１０１．６１ｃｍ、８３．８８ｃｍ。ＨＡＧＲ的季节动态在７月末之前〉０,之后〈０;典型草甸和沼泽化草甸的极大值出现在６月末至７月中旬,沼泽则出现在６月中旬至６月末;极小值典型草甸出现在８月末至９月中旬,而     

一类具有非单调增长率和功能反应的捕食者-食饵系统的定性分析

考虑一类食饵密度具有非单调增长率,捕食者具有功能性反应的捕食者一食饵系统。得到系统在第一象限内正平衡点的区域稳定的条件和极限环存在唯一的条件,完整地讨论了系统(1)的拓扑结构。