Involvement of PCH family proteins in cytokinesis and actin distribution

Pombe Cdc15 homology (PCH) proteins constitute an extensive protein family whose members have been found in diverse eukaryotic organisms. These proteins are characterized by the presence of several conserved sequence and structural motifs. Recent studies in yeast and mammalian cultured cells have implicated these proteins in actin-based processes, in particular, cytokinesis. Here we review the recent findings on the in vivo localization, function, and binding partners of PCH family members. We also provide new microscopy data regarding the in vivo dynamics of a budding yeast PCH protein involved in cytokinesis.     

大规模MCM基板互连探针测试和路径优化

已有的路径优化算法在MCM基板互连测试中已经发挥了一定的作用，但由于MCM的高密互连特性，使得测试变得更加复杂和困难，因此人们希望能引入新的方法与思路，以解决MCM基板互连测试的路径优化问题。将蚁群算法应用到互连测试探针路径优化问题当中，根据MCM基板互连测试的特点，建立探针路径优化的模型。提出一种针对大规模MCM基板互连探针测试的方法，首先将MCM基板进行分片，然后对每片进行优化，最后将优化结果连接在一起，成为一条完整的路径。实验结果表明，蚁群算法能在较短的时间内得到更优的路径。     

协同过程建模方法MCM的图形化构建环境

针对协同过程新的建模方法协同关联图MCM（Multi-Context Map）构建困难的问题,采用图形化工具Microsoft Visio,结合MCM的特点,开发了一个MCM图形化构建环境,并给出了应用实例。     

Monitoring of S100 homodimerization and heterodimeric interactions by the yeast two-hybrid system

The S100 family consists of 19 members, which function as transducers of calcium signals in a tissue-specific manner. Upon calcium binding, the conformation of many S100 proteins changes dramatically. Several hydrophobic residues are exposed, allowing the S100 proteins to interact with their target proteins, and thereby to transduce calcium signals into specific biological responses. To further elucidate the exact contribution of the S100 calciproteins in the calcium signalling pathways, several groups have applied the yeast two-hybrid technology to identify putative target proteins for the various S100 calciproteins. Two-hybrid large screens using S100 proteins as baits have confirmed the biochemical and structural feature of S100, which enable them to form homodimers and the ability of some members to form specific heterodimers in vivo. Yeast two-hybrid investigations have allowed the identification of conserved hydrophobic residues and domains that are crucial for the stabilization of S100 homo- and heterodimers. Furthermore, this method clearly underlines that the homo- and heterodimerization mechanisms differ among the members of the S100 family. However, several lines of evidence strongly suggest that two-hybrid methodology is limited to the analysis of interactions that are calcium-independent, since no target proteins other than S100 family members themselves have been detected with this methodology.     

Molecular mechanisms of S100-target protein interactions

S100 proteins have no known enzymatic activity and exert their intracellular effects via interaction with and regulation of the activity of other proteins, termed target proteins, in both a Ca(2+)-dependent and Ca(2+)-independent manner. Structural studies have identified the linker region between the two EF-hand Ca(2+) binding domains and the C-terminus as Ca(2+)-dependent target protein binding sites in several S100 family members. In fact, C-terminal aromatic residues are obligatory for interaction of S100A1 with several of its Ca(2+)-dependent target proteins. Pharmacological studies suggest the presence of additional Ca(2+)-dependent binding motifs on some family members. A minimum of seven family members interact with and regulate the activity of aldolase A in a Ca(2+)-independent manner. In the case of S100A1, Ca(2+)-independent target protein interactions utilize a binding motif distinct from the C-terminal Ca(2+)-dependent target protein binding site. Several studies suggest that ionic interactions participate in the interaction of S100 family members with Ca(2+)-independent target proteins. While some target proteins are activated by multiple family members, other target proteins exhibit family member-specific activation, i.e., they are activated by a single family member. As predicted, family member specific interactions appear to be mediated by regions that exhibit the most divergence in amino acid sequence among family members, the linker or "hinge" region and the C terminus. Further specificity in S100-target protein interactions may arise from the different biochemical/biophysical properties of the individual family members, including affinity for metal ions (Ca(2+), Zn(2+), and Cu(2+)), oligomerization properties, heterodimerization, post-translational modifications, and lipid-binding. Delineation of the structural motifs that mediate S100-target protein interactions and determination of the in vivo relevance of these interactions are needed to fully understand the role of S100 proteins in normal and diseased cells.     

内容可寻址存储器MCM69C432及其在防火墙中的应用

介绍了MCM69CA32的结构、特点及使用方法,针对其特点提出了一种基于它的嵌入式防火墙方案,通过构建CAM规则匹配单元对数据包头和数据载荷进行并行过滤,对过滤的结果根据规则响应要求集中进行处理,提高了过滤效率,使得系统处理能力强,简单可靠,相对与纯硬件的基于ASIC的防火墙使用更灵活,适用性更强,具有良好的应用前景.     

Intracellular and extracellular roles of S100 proteins

S100, a multigenic family of non-ubiquitous Ca(2+)-modulated proteins of the EF-hand type expressed in vertebrates exclusively, has been implicated in intracellular and extracellular regulatory activities. Members of this protein family have been shown to interact with several effector proteins within cells thereby regulating enzyme activities, the dynamics of cytoskeleton constituents, cell growth and differentiation, and Ca(2+) homeostasis. Structural information indicates that most of S100 proteins exist in the form of antiparallelly packed homodimers (in some cases heterodimers), capable of functionally crossbridging two homologous or heterologous target proteins in a Ca(2+)-dependent (and, in some instances, Ca(2+)-independent) manner. In addition, extracellular roles have been described for several S100 members, although secretion (via an unknown mechanism) has been documented for a few of them. Extracellular S100 proteins have been shown to exert regulatory effects on inflammatory cells, neurons, astrocytes, microglia, and endothelial and epithelial cells, and a cell surface receptor, RAGE, has been identified as a potential S100A12 and S100B receptor transducing the effects of these two proteins on inflammatory cells and neurons. Other cell surface molecules with ability to interact with S100 members have been identified, suggesting that RAGE might not be a universal S100 protein receptor and/or that a single S100 protein might interact with more than one receptor. Collectively, these data indicate that members of the S100 protein family are multifunctional proteins implicated in the regulation of a variety of cellular activities.     

Apoptosis in normal and neoplastic mammary gland development

Apoptosis plays important roles in mammary development from early embryonic formation of the mammary gland to the regression that follows cessation of cycling. The most dramatic occurrence of apoptosis is found during mammary involution. Most of the secretory epithelium in the lactating breast undergoes apoptosis as the mammary gland regresses and is reorganized for another cycle of lactation. We used the morphology, biochemical changes, and gene expression detected in apoptotic mammary epithelium during involution as a model for studying cell death during other stages of mammary development and for approaching the failure of apoptosis found in mammary hyperplasia. Morphological studies and gene expression have suggested that apoptosis during involution is comprised of two phases: an early limited apoptosis in response to hormone ablation and later protease promoted widespread apoptosis in response to altered cell-matrix interactions and loss of anchorage. We examined protein expression during involution for changes associated with loss of hormone stimulation and altered cell-matrix interactions. One of the proteins whose expression is able to inhibit apoptosis, and is altered during mammary epithelial cell was the serine-threonine protein kinase, Akt 1. Akt 1 activation is common to hormone, growth factor, and anchorage-mediated survival of epithelial cells. We found regulated expression of activated Akt 1 in the mammary gland during involution. Akt 1 activation peaked in pregnancy and lactation, and decreased significantly during apoptosis in mammary involution. Mechanisms of Akt 1 action include modulation of the ratio bcl-2 family members implicated in control of apoptosis. Bcl-2 family proteins were also expressed in pattern consistent with Akt 1 regulation. These observations led us to examine expression of activated Akt 1 and bcl-2 family proteins in premalignant hyperplasias. Akt 1 activation was increased; expression of anti-apoptotic proteins bcl-2 and bcl-x was strongly increased while pro-apoptotic bax was greatly diminished in three different lines of transplantable premalignant mammary hyperplasia. This data suggest that activation of Akt 1 by hormone- or anchorage-mediated pathways regulates survival of mammary epithelium and can contribute to initiation of neoplasia. These data suggest that perturbation of normal cell turnover can contribute to initiation of neoplasia.     

Meta-analysis of the cell cycle related <i>C12orf48</i>

The cell cycle is a conserved process from yeast to mammals and focuses on mechanisms that regulate the timing and frequency of DNA replication and cell division. The temporal and spatial expression of the genes is tightly regulated to ensure accurate replication and transmission of DNA to daughter cells during the cycle. Although the genes involved in interphase are well studied, most of the genes which are involved in mitotic events still remain unidentified. Since, the discovery of mitosis related genes is still incomplete, we performed a co-expression and gene ontology analysis for revealing novel mitosis regulated genes. In this study, we showed that C12orf48 is co-expressed with well-known mitotic genes. Moreover, it is also co-expressed with the genes that have roles in interphase such as DNA replication. Furthermore, our results showed that C12orf48 is also differentially expressed in various cancers. Therefore, the results presented in this study suggest that C12orf48 may be an important molecule for both interphase and mitosis. Since, the molecules involved in these mechanisms are crucial for proliferation as well as in carcinogenesis, C12orf48 should be considered as a novel cell cycle and carcinogenesis related gene.     

Rab7 and actin cytoskeleton participate during mobilization of β1EHFNR in fibronectin-stimulated Entamoeba histolyticatrophozoites

In vitro interaction of Entamoeba histolytica trophozoites with fibronectin (FN) induces redistribution of the amoebic fibronectin receptor (β1EhFNR). Trafficking of beta1 integrins is important for cell adhesion and migration in higher eukaryotes and requires the participation of Rab proteins. In E. histolytica, the machinery involved in integrin trafficking is not completely known. EhRab7 is a 24.5-kDa protein whose alignment with other Rab7 proteins demonstrated that it shared significant homology with Rab7 proteins from other organisms, including humans. Using different types of microscopy (fluorescence and confocal microscopy), it was established that Rab7 and the actin cytoskeleton participated in the mobilization of β1EhFNR in FN-stimulated trophozoites. β1EhFNR and Rab7 co-localized only in vesicular structures at 5 min, and at longer time (1 h), both co-localized in both plasma membrane and in vesicular structures; at the same time, Rab7 co-localized with specific actin structures (phagocytic vacuoles). At 5 h the β1EhFNR, Rab7, and actin co-localized at the plasma membrane, and only β1EhFNR and Rab7 decorated vesicles of different sizes. Actin and Rab7 co-localized in a cap-like structure at one end of the cell. Fluorescence resonance energy transfer and electron microscopy confirmed the close interaction between β1EhFNR and Rab7. Moreover, the use of a lysosome-specific marker (LysoTracker) and a Golgi-specific marker (NBD C(6)-ceramide) allowed us to establish that, at some point within the endocytic route, β1EhFNR and Rab7 co-localized within a lysosome-type organelle, but not a Golgi-like organelle, which indicated that this integrin-like molecule was returned to the plasma membrane via exocytic or secretory vesicles.     

应用多模式组合加工技术修正大口径非球面环带误差

为了满足大口径非球面光学元件加工的需求,提出了用多模式组合加工(MCM)技术修正大口径非球面反射镜环带误差的方法。本文讨论的MCM技术以经典加工工艺为基础,采用抛光盘的多工位加工和抛光模式的组合完成光学元件的抛光,实现对光学表面中低频段误差的有效控制。介绍了MCM技术的重要组成部分JP-01抛光机械手的工作原理,分析了MCM的工作模式。采用MCM技术对Φ1230mm的非球面反射镜进行环带误差的修正,给出了镜面面形检测结果。实验结果表明,MCM技术可以有效地控制光学表面的中低频误差,使光学表面误差得到有效收敛,从而显著提高抛光效率。目前,采用MCM技术加工1～2m口径的同轴非球面,其精度可以达到30nm(RMS)。     

Construction and application of a yeast expression system for thymosin alpha1.

We want to construct a yeast expression system for thymosin alpha1 (Talpha1) to make the orally administered Talpha1 preparation possible. The whole Talpha1 DNA fragment was obtained by PCR. After being digested with restriction enzymes, it was cloned into pYES2 vector. Sequencing was performed to identify the recombinant. The sequence of Talpha1 in recombinant coincided with the original one reported in Genbank. When pYES2-Talpha1 plasmid was transformed into yeast, galactose instead of glucose was used to induce Talpha1 expression. Western blot was performed to identify the quality of the expressed Talpha1. Dried yeast containing pYEST2-Talpha1 was fed to Balb/c mice whose immunities were inhibited by cyclophosphamide in advance. Synthesized Talpha1 peptide was used as positive control and empty yeast was used as negative control. Compared with the negative control group, both dried yeast containing pYEST2-Talpha1 and synthesized Talpha1 peptide can significantly increase the CD8+ level (22.74 +/- 1.09 and 18.77 +/- 4.72 vs 7.49 +/- 2.14, p < 0.01), while both of them had little effect on the CD4+ lymphocytes (61.86 +/- 6.94 and 65.91 +/- 4.78 vs 57.93 +/- 10.40,p > 0.05). We concluded that a high effective yeast expression system for Talpha1 was constructed successfully and the Talpha1 protein expressed by this system can improve CD8+ level in immune inhibited mice.     

Effect of sugars on the association between cowpea vicilin (7S storage proteins) and fungal cells

Vicilins (7S storage proteins) found in various legume seeds have been previously shown to interfere with the germination of spores or conidia of phytopathogenic fungi and inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells. In the present work vicilins from cowpea (Vigna unguiculata) seeds were added to the growth medium of Saccharomyces cerevisiae cells and Fusarium oxysporum conidia. Helix pomatia lectin, wheat germ agglutinin and Ulex europaeus lectin were used to identify differences in the binding of the vicilins to the surface of cells of S. cerevisiae and F. oxysporum treated with this protein. After the growth period, the material in suspension (yeast cells) was centrifuged and the final pellet was also treated with different sugar (glucose, sucrose, glucosamine, N-acetyl-glucosamine) concentrations and 0.1 M HCl for extraction of vicilins associated to chitinous structures present in yeast cells. Our results showed that vicilin sub-units were present in the different sugar extracts of yeast cells pretreated with the vicilins and these proteins were eluted by 0.5 M solutions of sugars in the following order of efficiency of elution: N-acetyl-glucosamine, sucrose/glucose and glucosamine.     

Mitochondrial apoptotic pathways.

Apoptosis or programmed cell death (PCD) is a physiological process characteristic of pluricellular organisms leading to self-destruction of the cell. It is therefore involved in development, homeostasis and host defense. However, a significant difference has been shown between mammalian cell apoptosis and non-mammalian cell apoptosis: mitochondria are implicated only in the former. Execution of PCD includes the release of several proapoptotic proteins from the intermembrane space of mitochondria. They could exert their actions through a caspase dependent as well as a caspase independent way. On the other hand, regulation of PCD is mainly given by the Bcl-2 family members, which are in turn essentially regulated by activation of death receptors and/or DNA damage. Nowadays, execution of apoptosis is better known than its regulation. Nevertheless, we are still far of a complete understanding of the apoptotic process.     

Probing DNA structure and function with a multiwavelength fluorescence confocal laser microscope

Three levels of organization in DNA structure in the interphase cell nucleus are assessed by confocal laser scanning microscopy: (i) the conformational state of the double helix; (ii) the distribution of eu- and heterochromatin; and (iii) the localization of replication complexes throughout S phase. Multi-parameter measurements were carried out in each optical section using two laser sources and combined stereoscopic reconstructions were used to assess the co-localization of nuclear components. DNA is highly polymorphic and can adopt a variety of different helical conformations as well as unusual structures (curved, cruciform, multi-stranded). We have assessed by laser scanning microscopy the presence of left-handed Z-DNA in polytene chromosomes of Diptera as well as the spatio-temporal distribution of Z-DNA binding proteins in whole-mount Drosophila embryos and ovaries. We have determined the 3-D distribution of replication sites relative to heterochromatin regions, nucleoli and nuclear membrane by using short pulses of BrdU incorporation in synchronized mouse and human fibroblasts. Replication sites were visualized with a monoclonal anti-BrdU antibody combined with DNA fluorescent staining and antibody labelling of nuclear lamin. The implications of dynamic DNA movement and structural rearrangement to the organization of the nucleus in domains are discussed.     

Association between preterm birth risk and polymorphism and expression of the DNA repair genes <i>OGG1</i> and <i>APE1</i> in Saudi women

Genomic instability and mutations caused by increases in oxidative stress during pregnancy can damage the fetoplacental unit and can upshot preterm birth. Oxidative damage to DNA may possibly be involved in etiology of preterm birth (PTB) which can be repaired by DNA repair gene. In the present study, we assessed the association of base excision repair gene family by analyzing the association of single nucleotide polymorphisms and genes expression in 8-oxoguanine glycosylase-1 (OGG1) and apurinic-apyrimidinic endonuclease 1 (APE1) genes with risk of preterm birth in Saudi women. We analyzed genotypes of four single nucleotide polymorphisms (SNPs) (rs1052133, rs293795, rs2072668 and rs2075747) in OGG1 gene and three SNPs (rs1130409, rs3136814, and rs3136817) in APE1 gene using TaqMan Genotyping assay kits in 50 pairs of preterm cases and individually matched controls. Also, gene expression level was explored by RT-PCR in 10 pairs of preterm placental tissues and individually matched normal placental tissues. Two OGG1 SNP, rs1052133 (OR=0.497; c2=1.11; p=0.292) and rs2072668 (OR=0.408; c2=1.90; p=0.167) and one APE1 SNP rs3136817 (OR=0.458; c2=0.40; p=0.527) showed nonsignificant protective effect against PTB development. The expression of both genes under study was found lower in the PTB patients. Genotype and allele frequencies of both gene SNPs did not show any association with the risk of preterm delivery in Saudi women (P˃0.05). However, synthesis and release of OGG1 and APE1 proteins decreased in preterm placental tissues compared to term delivery reflects the probability of being one of the mechanisms leading to preterm birth.     

腐蚀对新型高强度铝合金疲劳裂纹萌生机制及扩展行为的作用

根据编制的某机场环境加速试验谱,通过腐蚀试验模拟飞机服役过程中遭受的潮湿空气、盐雾、二氧化硫、酸雨和干/湿交变等严酷条件的侵蚀作用,采用飞机主承力结构新型高强度铝合金7B04-T74,制备单边缺口拉伸(Single edge notch tension, SENT)试样进行预腐蚀和疲劳试验,分析不同程度腐蚀损伤对疲劳裂纹萌生、裂纹扩展行为和疲劳寿命的影响,揭示腐蚀对裂纹萌生及扩展行为的作用机理。结果表明,在腐蚀初期,疲劳裂纹萌生源主要为单个蚀孔,裂纹扩展路径较为平直;随着腐蚀程度的加重,在多个蚀孔处同时萌生多条小疲劳裂纹,萌生疲劳裂纹的蚀孔具有隧道效应,扩展路径不规则,形成“之”字形裂纹;疲劳裂纹萌生机制是材料第二相与腐蚀损伤之间相互竞争的结果;腐蚀导致疲劳寿命显著降低,尤其是裂纹萌生寿命,腐蚀12年试验件裂纹萌生寿命仅为未腐蚀试验件裂纹萌生寿命的2.2%。     

III. Chemokines and other mediators, 8. Chemokines and their receptors in cell-mediated immune responses in the lung

Chemokines constitute a large family of chemotactic cytokines that belong to a super-gene family of 8-10 kDa proteins. The chemokines are considered to be primarily beneficial in host defense against invading pathogens. However, the reactions induced by chemokines can be occasionally excessive, resulting in a harmful response to the host. Recent studies in chemokine biology have elucidated that chemokines are involved in the initiation, development, and maintenance of numbers of diseases including lung diseases. In addition to its chemotactic activity, evidence suggests that chemokines can modify the outcome of the cell-mediated immune responses by altering the Th1/Th2 cytokine profile. Chemokines are also capable of dictating the direction of specific immune responses. Chemokine action is mediated by a large super-family of G-protein coupled receptors, and the receptors are preferentially expressed on Th1/Th2 cells. Certain chemokine receptors are constitutively expressed in immune surveying cells such as dendritic cells and naive T cells. The corresponding chemokines are present in normal lymphoid tissues, suggesting a role of chemokines/receptors in cell homing and cell-cell communication in lymphoid tissue that can be an initial step for immune recognition. Thus, comprehension of the chemokine biology in immune responses appears to be fundamental for understanding the pathogenesis of T cell-mediated immune responses. The following review will highlight the current insight into the role of chemokines and their receptors in the cell-mediated immune response, with a special focus on lung diseases.