A role for the granzyme B inhibitor serine protease inhibitor 6 in CD8+ memory cell homeostasis

Generation and maintenance of protective immunological memory is the goal of vaccination programs. It has recently become clear that CD8+ memory T cells are derived directly from CTLs. The mechanisms underlying this transformation and the subsequent survival of memory cells are not completely understood. However, some effector molecules required by CTLs to eliminate infected cells have also been shown to control the number of Ag-specific cells. We report that memory cells express high levels of serine protease inhibitor (Spi) 6, an inhibitor of the effector molecule granzyme B, and that Spi6 can protect T cells from granzyme B-mediated apoptosis. In mouse models, both elevated expression of Spi6 and the complete absence of granzyme B in CD8+ T cells led to an increase in memory cells after infection with lymphocytic choriomeningitis virus. This was not the result of increased levels of antilymphocytic choriomeningitis virus CD8+ T cells during the expansion or contraction phases, but rather transgenic Spi6 directly influenced the survival of CD8+ memory T cells. We propose that expression of protective molecules, like Spi6, serves to shield metabolically active CD8+ memory T cells from their own effector molecules.     

Determination of spectinomycin hydrochloride and its related substances by HPLC-ELSD and HPLC-MSn

A new and simple high-performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) method for the determination of spectinomycin hydrochloride and its related substances was developed. The column was Agilent SB-C(18) (250 mm x 4.6 mm, 5 microm).The mobile phase was 25 mM trifluoroacetic acid. The drift tube temperature was 40 degrees C. The pressure of nebulizing gas was 3.5 bar. Good separation of spectinomycin from main related substances could be achieved. The standard curve was rectilinear in the range of 0.07-3.8 mg/ml (r = 0.9997). Precision was 1.0% (R.S.D.). The limit of detection was 6 microg/ml. The method is simple and rapid, and the results are accurate and reproducible. The HPLC-MS(n) method was used to characterize the structures of impurities contained in the spectinomycin. In positive mode, impurities were elucidated by use of electrospray ion trap mass spectrometry in the multi-stage MS full scan mode. The possible structures of impurities C and D in spectinomycin were deduced based on the HPLC-MS(n) data.     

EDNRB基因编码区点突变及多态性在浙江地区散发性先天性巨结肠症中的检测与分析

本实验应用聚合酶链反应-单链构象多态性(single strand confbrmation polymorphism analysis of polymerase chain reaction products,PCR-SSCP)和DNA直接测序技术,对75例浙江地区散发性先天性巨结肠病例EDNRB基因编码区的全部7个外显子,进行了点突变与单核苷酸多态性的检测与分析,探讨浙江地区先天性巨结肠患者EDNRB基因的突变特征,阐明EDNRB基因与散发性先天性巨结肠症发病之间可能存在的关系。结果有6例患者在第4外显子上检测到密码子277位点 CTG→CTA的置换,导致亮氨酸的同义突变(L277L),属于单核苷酸多态性,发生率为8％(6/75)。有 2例患者在第2外显子上检测到密码子185位点GTG→ATG的置换,导致缬氨酸到蛋氨酸的错义突变(V185M),此突变型未在国内外文献中报道过,认为是新的基因突变型,突变率2．7％(2/75)。研究结果表明,浙江地区先天性巨结肠群体可发生EDNRB基因的杂合性突变,提示EDNRB基因与先天性巨结肠症的发病存在一定程度的关联。     

Self-assembly of monoglycerides in beta-lactoglobulin adsorbed films at the air-water interface. Structural, topographical, and rheological consequences

In this work, we have analyzed the structural, topographical, and surface dilatational characteristics of pure beta-lactoglobulin adsorbed films and the effect of the self-assembly of monoglycerides (monopalmitin or monoolein) in beta-lactoglobulin films at the air-water interface. Measurements were performed in a single device that incorporates a Wilhelmy-type film balance, Brewster angle microscopy, and interfacial dilatational rheology. The structural and topographical characteristics of beta-lactoglobulin adsorbed and spread films are similar. However, the surface dilatational modulus of beta-lactoglobulin films shows a complex behavior depending on film formation. The self-assembly of monoglyceride in a beta-lactoglobulin adsorbed film has an effect on the structural, topographical, and dilatational properties of the mixed films, depending on the interfacial composition and the surface pressure (pi). At low pi, a mixed film of monoglyceride and beta-lactoglobulin may exist. At high pi (after the collapse of beta-lactoglobulin), the mixed films are dominated by monoglyceride molecules. However, the small amounts of collapsed beta-lactoglobulin have a significant effect on the surface dilatational properties of the mixed films. Protein displacement by monoglyceride is higher for monopalmitin than for monoolein. However, some degree of interaction exists between proteins and monoglycerides, and these interactions are more evident in adsorbed films than in spread films.     

小叶榕叶中具有抗HSV活性的黄烷成分(英文)

从小叶榕(Ficus microcarpa L.)叶的乙醇提取物中首次分离鉴定了(+)(2R,3S)阿夫儿茶素(1)、(-)(2R,3R)表阿夫儿茶素(2)、(-)(2R,3S)阿夫儿茶素-(4α-8)(2R,3S)阿夫儿茶素(3)、(-)(2R,3S)阿夫儿茶素-(4α-8)(2R,3R)表阿夫儿茶素(4)4个黄烷化合物,其中化合物1和2对兔肾细胞中培养的2型单纯疱疹病毒(HSV-2)具有抑制作用,半数抑制浓度(IC50)分别为0.49和0.55mgmL-1,治疗指数(TI)分别为4.31和3.19,可用作小叶榕叶药材质量控制的指标成分。从该提取物中还分离鉴定了另外7个已知成分:儿茶素(5)、phaseic acid(6)、苯甲酸(7)、4-羟基苯甲酸(8)、间苯三酚(9)、胡萝卜苷(10)和β-谷甾醇(11)。     

宫腔镜下甲氨蝶呤注入输卵管保守治疗非破裂型输卵管妊娠的疗效观察

目的探讨宫腔镜下甲氨蝶呤注入输卵管保守治疗非破裂型输卵管妊娠的临床疗效。方法对34例确诊为非破裂型输卵管妊娠,且包块直径≤4 cm,血β-HCG〈2 000 IU,无腹腔积液的病例,行宫腔镜下将MTX 40 mg注入输卵管;动态观察患者血HCG变化,定期复查彩超,观察包块变化情况。结果32例治愈,治愈率94.12%。其中有1例疗程结束后1周复查血β-HCG〉1000 u/L,追加用药后成功;失败2例,均因治疗期间腹痛加剧出现内出血征象而转行手术治疗,内出血分别为700 ml、480 ml,转腹腔镜手术,恢复良好。结论宫腔镜下甲氨蝶呤注入输卵管保守治疗非破裂型输卵管妊娠具有损伤小、避免开腹手术等优点,值得临床推广。     

猪肝酯酶催化苯乙二醇环碳酸酯水解最佳反应条件的研究

猪肝酯酶是手性合成中重要的水解酶,在猪肝酯酶的催化下,苯乙二醇环碳酸酯发生水解,生成苯乙二醇。实验围绕影响猪肝酯酶催化反应活性的4个主要因素进行了系统研究,得到了最优的酶浓度（15g/L）、pH值（8.0）、温度（25~30℃）及有机溶剂种类和浓度（二氧六环,65%v/v）,为猪肝酯酶催化苯乙二醇环碳酸酯反应的进一步研究奠定了基础。     

Activation of the N-Terminally Truncated Form of the Stk Receptor Tyrosine Kinase Sf-Stk by Friend Virus-Encoded gp55 Is Mediated by Cysteine Residues in the Ecotropic Domain of gp55 and the Extracellular Domain of Sf-Stk

Friend virus induces an erythroleukemia in susceptible mice that is initiated by the interaction of the Friend virus-encoded glycoprotein gp55 with the erythropoietin (Epo) receptor and the product of the host Fv2 gene, a naturally occurring truncated form of the Stk receptor tyrosine kinase (Sf-Stk). We have previously demonstrated that the activation of Sf-Stk, recruitment of a Grb2/Gab2/Stat3 signaling complex, and induction of Pu.1 expression by Stat3 are required for the development of the early stage of Friend disease both in vitro and in vivo. Here we demonstrate that the interaction of gp55 with Sf-Stk is dependent on cysteine residues in the ecotropic domain of gp55 and the extracellular domain of Sf-Stk. Point mutation of these cysteine residues or deletion of these domains inhibits the ability of gp55 to interact with Sf-Stk, resulting in the inability of these proteins to promote the Epo-independent growth of erythroid progenitor cells. We also demonstrate that the interaction of gp55 with Sf-Stk does not promote dimerization of Sf-Stk but results in enhanced phosphorylation of Sf-Stk and the relocalization of Sf-Stk from the cytosol to the plasma membrane. Finally, we demonstrate that a constitutively active form of Sf-Stk (Sf-StkM330T), as well as its human counterpart, Sf-Ron, promotes Epo-independent colony formation in the absence of gp55 and that this response is also dependent on the cysteines in the extracellular domains of Sf-StkM330T and Sf-Ron. These data suggest that the cysteines in the extracellular domains of Sf-Stk and Sf-Ron may also mediate the interaction of these truncated receptors with other cellular factors that regulate their ability to promote cytokine-independent growth.Since Friend disease was first reported in 1957 (19), the acute erythroleukemia induced by the various strains of Friend virus have provided an excellent model to study multistage carcinogenesis (5). In the first stage, the virus infects erythroid progenitor cells and a viral glycoprotein, gp55, interacts with both the erythropoietin receptor (EpoR) and a naturally occurring truncated form of the stem cell-derived tyrosine kinase (Stk), Sf-Stk, resulting in the Epo-independent (Epo^ind) expansion of erythroid progenitor cells. The late stage of erythroleukemia in Friend disease is marked by inactivation of the p53 locus (6, 28, 38, 39, 51) and proviral integration into the Spi-1 locus (36, 43, 44), resulting in enhanced expression of Pu.1, which causes a block in erythroid differentiation and promoting the onset of acute erythroleukemia.Friend virus is a complex of two viruses, the spleen focus-forming virus (SFFV), which is a replication-defective C-type retrovirus, and the ecotropic Friend murine leukemia virus (F-MuLV). SFFV is responsible for the rapid splenomegaly and acute erythroleukemia induced by Friend virus infection (7, 64, 65, 67), while F-MuLV provides helper function and can be substituted for by other murine leukemia viruses (35). Specifically, the glycoprotein gp55, encoded by the SFFV env gene, acts as the transforming viral oncoprotein (2, 65).Several loci in the mouse genome that control Friend virus susceptibility have been identified. Fv1, Fv3, and Fv4 affect the ability of Friend virus to infect early erythroid progenitor cells. The Fv1 gene product inhibits Friend virus infection by interacting with the viral capsid protein (60). The Fv3 gene encodes cytidine deaminase Apobec3, which broadly inhibits retrovirus infection (42, 53, 57). The Fv4 gene product affects viral binding by competing for receptors on the cell membrane (59). Another set of genes, W, Sl, f, and Fv2, are required for the development or expansion of infected progenitor cells. Our previous work demonstrated that W, Sl, and f, which encode the kit receptor, its ligand SCF, and Smad5, respectively, also play key roles in the BMP4-dependent stress erythropoiesis pathway(46, 47, 55). Analysis of those mutants showed that Friend virus activates this pathway, leading to acute amplification of stress progenitors, which are targets of Friend virus in the spleen, and resulting in rapid onset of disease.The Friend virus susceptibility gene Fv2 encodes the stem cell-derived tyrosine kinase (Stk) receptor (48). A naturally occurring N-terminally truncated form of Stk, short-form Stk (Sf-Stk), is required for Friend virus susceptibility. Fv2^r/r mice, including C57BL/6, lack expression of Sf-Stk and are resistant to Friend virus infection, while full-length Stk expression is unaffected in these mice. An internal promoter within the Stk locus drives Sf-Stk expression, and Fv2^r/r mice harbor mutations in the internal promoter. Sf-Stk lacks the N-terminal ligand binding domain of full-length Stk but retains the transmembrane and tyrosine kinase domains. In vitro and in vivo expression of Sf-Stk in C57BL/6 bone marrow cells has been shown to confer Friend virus susceptibility to Fv2^r/r mice (18).Sf-Stk covalently interacts with gp55, resulting in constitutive activation of Sf-Stk (41). However, the mechanism by which this occurs is currently unknown. Here, we identify cysteines in the extracellular domains of Sf-Stk and gp55 that mediate this interaction. Furthermore, we demonstrate that while the association with gp55 is not required for the dimerization of Sf-Stk, the interaction of gp55 with Sf-Stk promotes tyrosine phosphorylation of Sf-Stk. In addition, while the extracellular cysteines in Sf-Stk promote retention of Sf-Stk in the cytoplasm in the absence of gp55, the interaction of Sf-Stk with gp55 through these cysteines results in enhanced cell surface localization of Sf-Stk. These changes in receptor activation and subcellular localization mediate the ability of Sf-Stk to induce gene expression and promote the Epo^ind growth of primary erythroblasts.