染色体易位重组位点的自动识别方法研究

染色体易位重组位点的识别对很多染色体遗传性疾病的诊断有着重要的意义.本文基于实际诊断中采集到的24类染色体数据和9号正常与异常染色体数据,构建了一套自动识别染色体易位重组位点的模型和方法.首先,对染色体图像进行预处理,得到了方向梯度直方图特征(HOG)和局部二值模式特征(LBP),构建了基于纹理特征的染色体24分类多通...     

格特隐球菌HOG1基因的敲除及重建

目的 构建格特隐球菌HOG1基因缺陷株和HOG1基因重建株.方法 从格特隐球菌基因组扩增HOG1基因,通过部分基因缺失方法,获得缺陷基因dHOG1.将获得的HOG1基因及其缺陷基因dHOG1分别亚克隆到真核表达载体pGAPzα-A,构建pGAPzα-HOG1及pGAPzcα-dHOG1质粒.将pGAPzα-dHOG1质粒转染隐球菌原始株,通过筛选获得HOG1基因缺陷菌株;同样方法将pGAPzα-HOG1质粒转染格特隐球菌HOGI基因敲除菌株,获得HOGI基因重建株.结果 RTPCR结果示:格特隐球菌HOG1基因缺陷株不转录表达完整的HOG1基因,而HOG1基因重建株可以转录表达完整的HOG1基因片段.结论 成功获得格特隐球菌HOG1缺陷株和HOG1基因重建株,为后续格特隐球菌毒力和致病机制的研究奠定了基础.     

Simulations in Evolution. III. Randomness as a Generator of Opportunities

In Neo‐Darwinism, variation and natural selection are the two evolutionary mechanisms which propel biological evolution. Our previous reports presented a histogram model to simulate the evolution of populations of individuals classified into bins according to an unspecified, quantifiable phenotypic character, and whose number in each bin changed generation after generation under the influence of fitness, while the total population was maintained constant. The histogram model also allowed Shannon entropy (SE) to be monitored continuously as the information content of the total population decreased or increased. Here, a simple Perl (Practical Extraction and Reporting Language) application was developed to carry out these computations, with the critical feature of an added random factor in the percent of individuals whose offspring moved to a vicinal bin. The results of the simulations demonstrate that the random factor mimicking variation increased considerably the range of values covered by Shannon entropy, especially when the percentage of changed offspring was high. This increase in information content is interpreted as facilitated adaptability of the population.     

Analysis of mitogen-activated protein kinase signaling specificity in response to hyperosmotic stress: use of an analog-sensitive HOG1 allele

When confronted with a marked increase in external osmolarity, budding yeast (Saccharomyces cerevisiae) cells utilize a conserved mitogen-activated protein kinase (MAPK) signaling cascade (the high-osmolarity glycerol or HOG pathway) to elicit cellular responses necessary to permit continued growth. One input that stimulates the HOG pathway requires the integral membrane protein and putative osmosensor Sho1, which recruits and enables activation of the MAPK kinase kinase Ste11. In mutants that lack the downstream MAPK kinase (pbs2Delta) or the MAPK (hog1Delta) of the HOG pathway, Ste11 activated by hyperosmotic stress is able to inappropriately stimulate the pheromone response pathway. This loss of signaling specificity is known as cross talk. To determine whether it is the Hog1 polypeptide per se or its kinase activity that is necessary to prevent cross talk, we constructed a fully functional analog-sensitive allele of HOG1 to permit acute inhibition of this enzyme without other detectable perturbations of the cell. We found that the catalytic activity of Hog1 is required continuously to prevent cross talk between the HOG pathway and both the pheromone response and invasive growth pathways. Moreover, contrary to previous reports, we found that the kinase activity of Hog1 is necessary for its stress-induced nuclear import. Finally, our results demonstrate a role for active Hog1 in maintaining signaling specificity under conditions of persistently high external osmolarity.     

使用图像特征构建快速有效的蛋白质折叠识别方法

蛋白质结构自动分类是探索蛋白质结构- 功能关系的一种重要研究手段。首先将蛋白质折叠子三维空间结构映射成为二维距离矩阵,并将距离矩阵视作灰度图像。然后基于灰度直方图和灰度共生矩阵提出了一种计算简单的折叠子结构特征提取方法,得到了低维且能够反映折叠结构特点的特征,并进一步阐明了直方图中零灰度孤峰形成原因,深入分析了共生矩阵特征中灰度分布、不同角度和像素距离对应的结构意义。最后应用于27类折叠子分类,对独立集测试的精度达到了71.95 %,对所有数据进行10 交叉验证的精度为78.94 %。与多个基于序列和结构的折叠识别方法的对比结果表明,此方法不仅具有低维和简洁的特征,而且无需复杂的分类系统,能够有效和高效地实现多类折叠子识别。     

Activation of the HOG pathway upon cold stress in Saccharomyces cerevisiae

When Saccharomyces cerevisiae cells are exposed to hyper-osmotic stress, the high-osmolarity glycerol response (HOG) pathway is activated to induce osmotic responses. The HOG pathway consists of two upstream osmosensing branches, the SLN1 and SHO1 branches, and a downstream MAP kinase cascade. Although the mechanisms by which these upstream branches transmit signals to the MAP kinase cascade are well understood, the mechanisms by which they sense and respond to osmotic changes are elusive. Here we show that the HOG pathway is activated in an SLN1 branch-dependent manner when cells are exposed to cold stress (0 degrees C treatment). Dimethyl sulfoxide (DMSO) treatment, which rigidifies the cell membrane, also activates the HOG pathway in both SLN1 branch- and SHO1 branch-dependent manners. Moreover, cold stress, as well as hyper-osmotic stress, exhibits a synergistic effect with DMSO treatment on HOG pathway activation. On the other hand, ethanol treatment, which fluidizes the cell membrane, partially represses the cold stress-induced HOG pathway activation. Our results suggest that both osmosensing branches respond to the rigidification of the cell membrane to activate the HOG pathway.     

Functional characterization of ARAKIN (ATMEKK1): a possible mediator in an osmotic stress response pathway in higher plants.

The Arabidopsis thaliana ARAKIN (ATMEKK1) gene shows strong homology to members of the (MAP) mitogen-activated protein kinase family, and was previously shown to functionally complement a mating defect in Saccharomyces cerevisiae at the level of the MEKK kinase ste11. The yeast STE11 is an integral component of two MAP kinase cascades: the mating pheromone pathway and the HOG (high osmolarity glycerol response) pathway. The HOG signal transduction pathway is activated by osmotic stress and causes increased glycerol synthesis. Here, we first demonstrate that ATMEKK1 encodes a protein with kinase activity, examine its properties in yeast MAP kinase cascades, then examine its expression under stress in A. thaliana. Yeast cells expressing the A. thaliana ATMEKK1 survive and grow under high salt (NaCl) stress, conditions that kill wild-type cells. Enhanced glycerol production, observed in non-stressed cells expressing ATMEKK1 is the probable cause of yeast survival. Downstream components of the HOG response pathway, HOG1 and PBS2, are required for ATMEKK1-mediated yeast survival. Because ATMEKK1 functionally complements the sho1/ssk2/ssk22 triple mutant, it appears to function at the level of the MEKK kinase step of the HOG response pathway. In A. thaliana, ATMEKK1 expression is rapidly (within 5 min) induced by osmotic (NaCl) stress. This is the same time frame for osmoticum-induced effects on the electrical properties of A. thaliana cells, both an immediate response and adaptation. Therefore, we propose that the A. thaliana ATMEKK1 may be a part of the signal transduction pathway involved in osmotic stress.     

Stimulation of the yeast high osmolarity glycerol (HOG) pathway: evidence for a signal generated by a change in turgor rather than by water stress

The Saccharomyces cerevisiae HOG pathway controls responses to osmotic shock such as production of the osmolyte glycerol. Here we show that the HOG pathway can be stimulated by addition of glycerol. This stimulation was strongly diminished in cells expressing an unregulated Fps1p glycerol channel, presumably because glycerol rapidly equilibrated across the plasma membrane. Ethanol, which passes the plasma membrane readily and causes water stress by disturbing the hydration of biomolecules, did not activate the HOG pathway. These observations suggest that stimulation of the HOG pathway is mediated by a turgor change and not by water stress per se.     

Hyperosmotic stress response and regulation of cell wall integrity in Saccharomyces cerevisiae share common functional aspects

The osmosensitive phenotype of the hog1 strain is suppressed at elevated temperature. Here, we show that the same holds true for the other commonly used HOG pathway mutant strains pbs2 and sho1ssk2ssk22, but not for ste11ssk2ssk22. Instead, the ste11ssk2ssk2 strain displayed a hyperosmosensitive phenotype at 37 degrees C. This phenotype is suppressed by overexpression of LRE1, HLR1 and WSC3, all genes known to influence cell wall composition. The suppression of the temperature-induced hyperosmosensitivity by these genes prompted us to investigate the role of STE11 and other HOG pathway components in cellular integrity and, indeed, we were able show that HOG pathway mutants display sensitivity to cell wall-degrading enzymes. LRE1 and HLR1 were also shown to suppress the cell wall phenotypes associated with the HOG pathway mutants. In addition, the isolated multicopy suppressor genes suppress temperature-induced cell lysis phenotypes of PKC pathway mutants that could be an indication for shared targets of the PKC pathway and high-osmolarity response routes.     

Estimating the ventilation-perfusion distribution: an ill-posed integral equation problem.

The distribution of ventilation-perfusion ratio over the lung is a useful indicator of the efficiency of lung function. Information about this distribution can be obtained by observing the retention in blood of inert gases passed through the lung. These retentions are related to the ventilation-perfusion distribution through an ill-posed integral equation. An unusual feature of this problem of estimating the ventilation-perfusion distribution is the small amount of data available; typically there are just six data points, as only six gases are used in the experiment. A nonparametric smoothing method is compared to a simpler method that models the distribution as a histogram with five classes. Results from the smoothing method are found to be very unstable. In contrast, the simpler method gives stable solutions with parameters that are physiologically meaningful. It is concluded that while such smoothing methods may be useful for solving some ill-posed integral equation problems, the simpler method is preferable when data are scarce.     

Requirement of STE50 for Osmostress-Induced Activation of the STE11 Mitogen-Activated Protein Kinase Kinase Kinase in the High-Osmolarity Glycerol Response Pathway

Exposure of yeast cells to increases in extracellular osmolarity activates the HOG1 mitogen-activated protein (MAP) kinase cascade, which is composed of three tiers of protein kinases: (i) the SSK2, SSK22, and STE11 MAP kinase kinase kinases (MAPKKKs), (ii) the PBS2 MAPKK, and (iii) the HOG1 MAP kinase. Activation of the MAP kinase cascade is mediated by two upstream mechanisms. The SLN1-YPD1-SSK1 two-component osmosensor activates the SSK2 and SSK22 MAPKKKs by direct interaction of the SSK1 response regulator with these MAPKKKs. The second mechanism of HOG1 MAP kinase activation is independent of the two-component osmosensor and involves the SHO1 transmembrane protein and the STE11 MAPKKK. Only PBS2 and HOG1 are common to the two mechanisms. We conducted an exhaustive mutant screening to identify additional elements required for activation of STE11 by osmotic stress. We found that strains with mutations in the STE50 gene, in combination with ssk2Δ ssk22Δ mutations, were unable to induce HOG1 phosphorylation after osmotic stress. Both two-hybrid analyses and coprecipitation assays demonstrated that the N-terminal domain of STE50 binds strongly to the N-terminal domain of STE11. The binding of STE50 to STE11 is constitutive and is not affected by osmotic stress. Furthermore, the two proteins relocalize similarly after osmotic shock. It was concluded that STE50 fulfills an essential role in the activation of the high-osmolarity glycerol response pathway by acting as an integral subunit of the STE11 MAPKKK.     

Predicting the Valence of a Scene from Observers’ Eye Movements

Multimedia analysis benefits from understanding the emotional content of a scene in a variety of tasks such as video genre classification and content-based image retrieval. Recently, there has been an increasing interest in applying human bio-signals, particularly eye movements, to recognize the emotional gist of a scene such as its valence. In order to determine the emotional category of images using eye movements, the existing methods often learn a classifier using several features that are extracted from eye movements. Although it has been shown that eye movement is potentially useful for recognition of scene valence, the contribution of each feature is not well-studied. To address the issue, we study the contribution of features extracted from eye movements in the classification of images into pleasant, neutral, and unpleasant categories. We assess ten features and their fusion. The features are histogram of saccade orientation, histogram of saccade slope, histogram of saccade length, histogram of saccade duration, histogram of saccade velocity, histogram of fixation duration, fixation histogram, top-ten salient coordinates, and saliency map. We utilize machine learning approach to analyze the performance of features by learning a support vector machine and exploiting various feature fusion schemes. The experiments reveal that ‘saliency map’, ‘fixation histogram’, ‘histogram of fixation duration’, and ‘histogram of saccade slope’ are the most contributing features. The selected features signify the influence of fixation information and angular behavior of eye movements in the recognition of the valence of images.     

Regulated nucleo/cytoplasmic exchange of HOG1 MAPK requires the importin beta homologs NMD5 and XPO1.

MAP kinase signaling modules serve to transduce extracellular signals to the nucleus of eukaryotic cells, but little is known about how signals cross the nuclear envelope. Exposure of yeast cells to increases in extracellular osmolarity activates the HOG1 MAP kinase cascade, which is composed of three tiers of protein kinases, namely the SSK2, SSK22 and STE11 MAPKKKs, the PBS2 MAPKK, and the HOG1 MAPK. Using green fluorescent protein (GFP) fusions of these kinases, we found that HOG1, PBS2 and STE11 localize to the cytoplasm of unstressed cells. Following osmotic stress, HOG1, but neither PBS2 nor STE11, translocates into the nucleus. HOG1 translocation occurs very rapidly, is transient, and correlates with the phosphorylation and activation of the MAP kinase by its MAPKK. HOG1 phosphorylation is necessary and sufficient for nuclear translocation, because a catalytically inactive kinase when phosphorylated is translocated to the nucleus as efficiently as the wild-type. Nuclear import of the MAPK under stress conditions requires the activity of the small GTP binding protein Ran-GSP1, but not the NLS-binding importin alpha/beta heterodimer. Rather, HOG1 import requires the activity of a gene, NMD5, that encodes a novel importin beta homolog. Similarly, export of dephosphorylated HOG1 from the nucleus requires the activity of the NES receptor XPO1/CRM1. Our findings define the requirements for the regulated nuclear transport of a stress-activated MAP kinase.     

Calcofluor antifungal action depends on chitin and a functional high-osmolarity glycerol response (HOG) pathway: evidence for a physiological role of the Saccharomyces cerevisiae HOG pathway under noninducing conditions

We have isolated several Saccharomyces cerevisiae mutants resistant to calcofluor that contain mutations in the PBS2 or HOG1 genes, which encode the mitogen-activated protein kinase (MAPK) and MAP kinases, respectively, of the high-osmolarity glycerol response (HOG) pathway. We report that blockage of either of the two activation branches of the pathway, namely, SHO1 and SLN1, leads to partial resistance to calcofluor, while simultaneous disruption significantly increases resistance. However, chitin biosynthesis is independent of the HOG pathway. Calcofluor treatment also induces an increase in salt tolerance and glycerol accumulation, although no activation of the HOG pathway is detected. Our results indicate that the antifungal effect of calcofluor depends on its binding to cell wall chitin but also on the presence of a functional HOG pathway. Characterization of one of the mutants isolated, pbs2-14, revealed that resistance to calcofluor and HOG-dependent osmoadaptation are two different physiological processes. Sensitivity to calcofluor depends on the constitutive functionality of the HOG pathway; when this is altered, the cells become calcofluor resistant but also show very low levels of basal salt tolerance. Characterization of some multicopy suppressors of the calcofluor resistance phenotype indicated that constitutive HOG functionality participates in the maintenance of cell wall architecture, a conclusion supported by the antagonism observed between the protein kinase and HOG signal transduction pathways.     

Role of Proteolipid Protein in HSV-1 Entry in Oligodendrocytic Cells

Herpes simplex virus type 1 (HSV-1) has the ability to enter many different hosts and cell types by several strategies. This highly prevalent alphaherpesvirus can enter target cells using different receptors and different pathways: fusion at a neutral pH, low-pH-dependent and low-pH-independent endocytosis. Several cell receptors for viral entry have been described, but several observations suggest that more receptors for HSV-1 might exist. In this work, we propose a novel role for the proteolipid protein (PLP) in HSV-1 entry into the human oligodendrocytic cell line HOG. Cells transfected with PLP-EGFP showed an increase in susceptibility to HSV-1. Furthermore, the infection of HOG and HOG-PLP transfected cells with the R120vGF virus–unable to replicate in ICP4-defficient cells- showed an increase in viral signal in HOG-PLP, suggesting a PLP involvement in viral entry. In addition, a mouse monoclonal antibody against PLP drastically inhibited HSV-1 entry into HOG cells. PLP and virions colocalized in confocal immunofluorescence images, and in electron microscopy images, which suggest that PLP acts at the site of entry into HOG cells. Taken together these results suggest that PLP may be involved in HSV-1 entry in human oligodendrocytic cells.     

The alpha isoform of diacylglycerol kinase exhibits arachidonoyl specificity with alkylacylglycerol

We compared the diacylglycerol kinase (DGK) catalyzed phosphorylation of 1-O-hexanoyl-2-oleoylglycerol (HOG) with 1-O-hexanoyl-2-arachidonoylglycerol (HAG). We assayed the activity of DGKalpha and DGKzeta using a liposomal-based assay system. Liposomal assays show that the DGKalpha and, to a lesser extent, DGKzeta preferentially act on substrates containing an arachidonoyl group when this group is incorporated into alkylacylglycerols. The activity of DGKalpha was 82 times greater with HAG compared to HOG. DGKzeta is 10 times more active in catalyzing the phosphorylation of HAG compared to HOG. Although diacylglycerols were better substrates for both DGKalpha and DGKzeta than the alkylacylglycerols, no specificity was exhibited for arachidonoyl-containing diacylglycerols. However, this specificity for HAG over HOG is modulated by the phospholipid composition of the liposome. Addition of cholesterol and/or phosphatidylethanolamine partially reduces the substrate selectivity. We also analyzed the kinetic constants for the phosphorylation of both diacylglycerol and 1-alkyl-2-acylglycerol catalyzed by the alpha, epsilon, or zeta isoforms using a soluble Triton mixed micelle system. We found that all three isoforms of DGK can phosphorylate 1-alkyl-2-acylglycerols but generally at a lower rate than for the corresponding diacylglycerol. The specificity of DGKepsilon for diacylglycerols containing an arachidonoyl group was retained when the ester group in the C-1 position is replaced with an ether linkage. In contrast, DGKalpha and, to a lesser extent, DGKzeta had greater specificity for arachidonoyl-containing 1-alkyl-2-acylglycerols than for arachidonoyl-containing diacylglycerols. This demonstrates that the acyl chain specificity is affected by the structure of the lipid headgroup.