编码流感病毒血凝素基因和CD40L的核酸疫苗抗小鼠流感研究

为了检测表达CD40L的质粒能台作为核酸疫苗佐剂提高A／PR8／34流感病毒血凝素(HA)DNA疫苗的免疫应答,构建了表达鼠CD40L的质粒,行将它与A型流感病毒的HADNA疫苗用电击的方法共同免疫BALB／C小鼠(各30μg),免疫两次,间隔三周,第二次免疫后1周,用致死量流感病毒A／PR8／34攻击。发现与单独免疫30μgHA相比,血清中抗HA的IgG抗体节明显提高,且以IgG2a抗体提高为主,小鼠体重减轻非常少且体重恢复加快。实验结果显示,CD40L能作为流感病毒核酸疫苗佐剂,提高小鼠抗流感病毒攻击的能力。     

The role of YKL-40 in the pathogenesis of autoimmune diseases: a comprehensive review

YKL-40, a chitinase-3-like protein 1 (CHI3L1) or human cartilage glycoprotein 39 (HC gp-39), is expressed and secreted by various cell-types including macrophages, chondrocytes, fibroblast-like synovial cells and vascular smooth muscle cells. Its biological function is not well elucidated, but it is speculated to have some connection with inflammatory reactions and autoimmune diseases. Although having important biological roles in autoimmunity, there were only attempts to elucidate relationships of YKL-40 with a single or couple of diseases in the literature. Therefore, in order to analyze the relationship between YKL-40 and the overall diseases, we reviewed 51 articles that discussed the association of YKL-40 with rheumatoid arthritis, psoriasis, systemic lupus erythematosus, Behçet disease and inflammatory bowel disease. Several studies showed that YKL-40 could be assumed as a marker for disease diagnosis, prognosis, disease activity and severity. It is also shown to be involved in response to disease treatment. However, other studies showed controversial results particularly in the case of Behçet disease activity. Therefore, further studies are needed to elucidate the exact role of YKL-40 in autoimmunity and to investigate its potential in therapeutics.     

A loop of coagulation factor VIIa influencing macromolecular substrate specificity

Coagulation factor VIIa (FVIIa) belongs to a family of proteases being part of the stepwise, self-amplifying blood coagulation cascade. To investigate the impact of the mutation Met(298{156})Lys in FVIIa, we replaced the Gly(283{140})-Met(298{156}) loop with the corresponding loop of factor Xa. The resulting variant exhibited increased intrinsic activity, concurrent with maturation of the active site, a less accessible N-terminus, and, interestingly, an altered macromolecular substrate specificity reflected in an increased ability to cleave factor IX (FIX) and a decreased rate of FX activation compared to that of wild-type FVIIa. In complex with tissue factor, activation of FIX, but not of FX, returned to normal. Deconvolution of the loop graft in order to identify important side chain substitutions resulted in the mutant Val(158{21})Asp/Leu(287{144})Thr/Ala(294{152})Ser/Glu(296{154}) Ile/Met(298{156})Lys-FVIIa with almost the same activity and specificity profile. We conclude that a lysine residue in position 298{156} of FVIIa requires a hydrophilic environment to be fully accommodated. This position appears critical for substrate specificity among the proteases of the blood coagulation cascade due to its prominent position in the macromolecular exosite and possibly via its interaction with the corresponding position in the substrate (i.e. FIX or FX).     

Syntheses and antitumor activities of N′1,N′3-dialkyl-N′1,N′3-di-(alkylcarbonothioyl) malonohydrazide: The discovery of elesclomol

A series of N′¹,N′³-dialkyl-N′¹,N′³-di(alkylcarbonothioyl) malonohydrazides have been designed and synthesized as anticancer agents by targeting oxidative stress and Hsp70 induction. Structure–activity relationship (SAR) studies lead to the discovery of STA-4783 (elesclomol), a novel small molecule that has been evaluated in a number of clinical trials as an anticancer agent in combination with Taxol.     

冠心病患者介入治疗前后血清IL-18、sCD40L和hs-CRP水平的变化及其意义

目的：探讨冠心病患者血清白介素18（Interleukin-18,IL-18）、白细胞分化抗原40配体（CD40L）、及高敏C反应蛋白（hs-CRP）水平在经皮冠状动脉介入术治疗前后的变化和意义。方法：选择经冠状动脉造影确诊的冠心病患者85例,根据病变程度分为单支病变组（n=32）、双支病变组（n=28）和多支病变组（n=25）,采用双抗体夹心ELISA法测定PCI术前术后血清IL-18、CD40L和hs-CRP水平。结果：血清IL-18水平测定结果：多支病变组高于双支病变组,双支病变组高于单支病变组;支架置入术后显著高于术前,差异均有统计学意义（P〈0.01）。血清CD40L水平测定结果：多支病变组高于双支病变组和单支病变组,差异均有统计学意义（P〈0.01）,双支病变组与单支病变组间差异无统计学意义（P〉0.05）;支架置入术后较术前显著升高,差异有统计学意义（P〈0.01）。血清hs-CRP水平测定结果：多支病变组高于双支病变组,双支病变组高于单支病变组;支架置入术后显著高于术前,差异均有统计学意义（P〈0.01）。结论：冠心病患者血清IL-18、CD40L和hs-CRP与冠脉病变程度密切相关,介入治疗可使冠心病患者血清IL-18、CD40L和hs-CRP水平升高,监测血清中IL-18、CD40L和hs-CRP水平变化可了解治疗效果和炎症程度。     

A natural extracellular factor that induces Hsp72, inhibits apoptosis, and restores stress resistance in aged human cells

Experiments with cultured cells showed that most cellular stress resistance components are specialized for certain types of damage. For example, superoxide dismutase protects from oxidative damage; DNA repair enzymes guard against mutagens and other DNA-damaging agents. On the other hand, the major inducible heat shock protein Hsp72 protects cells from a large variety of stresses and thus represents a generalized repair/stress resistance component. Hsp72 not only refolds damaged proteins but also interferes with programmed cell death signaling pathways, thus providing cells with time to repair the damage, hence its universality as a stress protector. In the present study we demonstrate the occurrence in murine and human ascites fluids (AF) of a natural nontoxic extracellular factor (ascites Hsp72-inducing factor, AHIF) capable of activating Hsp72 expression in different types of cells via a pathway distinct from the heat shock response pathway. AHIF is unique in that it is the first physiological factor capable of inducing synthesis of Hsp72 not only in young cells but, remarkably, also in aged human cells that largely have lost the ability to express Hsp72 in response to stresses, a manifestation at the cellular level of a progressive impairment in the ability to adapt to environmental changes which characterizes aging. Pretreatment of aged human cells with AF triggers Hsp72 expression at levels seen in young stressed cells and protects cells from a variety of otherwise lethal stressful treatments such as heat shock, TNF, UV irradiation, etoposide, and menadione. Activation of Hsp72 expression is essential for antiapoptotic action of AHIF because specific inhibition of Hsp72 expression by antisense RNA abolishes the cytoprotective effect of AF. In view of an important link between stress resistance and longevity in different organisms, the abilities of AHIF make it a unique candidate for the role of a systemic regulator of the aging process. While a cell-autonomous stress response diminishes with aging, aged cells retain the ability to respond to an extracellular factor which induces the expression of Hsp72. This finding opens up exciting possibilities for using AF factor to restore stress resistance to old cells and organisms and the possibility of interfering with the aging process. The ability to induce stress resistance in young cells and to restore it in aged cells could serve as a basis for developing effective antiapoptotic therapies.     

Culture of large vessel endothelial cells on floating collagen gels promotes a phenotype characteristic of endothelium in vivo

The vascular endothelium in vivo is a remarkably quiescent cell layer that displays a highly differentiated and tissue-specific phenotype. Once established in culture, endothelial cells (EC) are phenotypically different from their in situ counterparts, displaying altered gene expression, increased mitotic index, and decreased cell density. To determine whether manipulating the microenvironment of cells in vitro would lead to a more differentiated phenotype, we cultured bovine aortic EC on floating collagen gels. EC cultured to confluence on floating gels for 24 or 48 hr display mitotic indices nearly identical to those of quiescent endothelium in vivo, nearly two log orders lower than that of EC cultured to confluence on plastic, and cell density on floating gels also resembles that observed for endothelium in vivo. Culture of EC on floating gels leads to decreased expression of platelet-derived growth factor-B, fibronectin, and fibronectin isoform ED-B, and increased levels of connexin40, relative to cells cultured on plastic. We conclude that culture of bovine aortic EC under standard culture conditions results in a phenotype reminiscent of development and/or wound healing, and that culturing them on a floating collagen gel leads to a more differentiated phenotype, reminiscent of that observed for large vessel EC in vivo.     

A novel mammalian display system for the selection of protein-protein interactions by decoy receptor engagement

The emerging field of proteomics has created a need for new high-throughput methodologies for the analysis of gene products. An attractive approach is to develop systems that allow for clonal selection of interacting protein pairs from large molecular libraries. In this study, we have characterized a novel approach for identification and selection of protein-protein interactions, denoted SPIRE (selection of protein interactions by receptor engagement), which is based on a mammalian expression system. We have demonstrated proof of concept by creating a general plasma membrane bound decoy receptor, by displaying a protein or a peptide genetically fused to a trunctated version of the CD40 molecule. When this decoy receptor is engaged by a ligand to the displayed protein/peptide, the receptor expressing cell is rescued from apoptosis. To design a high-throughput system with a highly parallel capacity, we utilized the B cell line WEHI-231, as carrier of the decoy receptor. One specific peptide-displaying cell could be identified and amplified, based on a specific receptor engagement, in a background of 12 500 wild-type cells after four selections. This demonstrates that the approach may serve as a tool in post-genomic research for identifying protein-protein interactions, without prior knowledge of either component.     

Crystallography and mutagenesis point to an essential role for the N-terminus of human mitochondrial ClpP

We have determined a 2.1 A crystal structure for human mitochondrial ClpP (hClpP), the proteolytic component of the ATP-dependent ClpXP protease. HClpP has a structure similar to that of the bacterial enzyme, with the proteolytic active sites sequestered within an aqueous chamber formed by face-to-face assembly of the two heptameric rings. The hydrophobic N-terminal peptides of the subunits are bound within the narrow (12 A) axial channel, positioned to interact with unfolded substrates translocated there by the associated ClpX chaperone. Mutation or deletion of these residues causes a drastic decrease in ClpX-mediated protein and peptide degradation. Residues 8-16 form a mobile loop that extends above the ring surface and is also required for activity. The 28 amino acid C-terminal domain, a unique feature of mammalian ClpP proteins, lies on the periphery of the ring, with its proximal portion forming a loop that extends out from the ring surface. Residues at the start of the C-terminal domain impinge on subunit interfaces within the ring and affect heptamer assembly and stability. We propose that the N-terminal peptide of ClpP is a structural component of the substrate translocation channel and may play an important functional role as well.