地噻咪松诱导乳鼠胸腺细胞凋亡的研究

本文用透射电镜的方法,研究了地噻咪松诱导乳鼠胸腺细胞的凋亡,并确立了乳腺胸腺细胞中凋亡细胞学形态的特征;含溴化乙锭的琼脂多糖电泳检测乳鼠胸腺细胞中凋亡细胞的断裂ＤＮＡ;末端标记法直接,持异的标记乳鼠胸腺细胞中凋亡细胞断裂ＤＮＡ。     

Exploring the conformational space of protein side chains using dead-end elimination and the A* algorithm

We describe an algorithm which enables us to search the conformational space of the side chains of a protein to identify the global minimum energy combination of side chain conformations as well as all other conformations within a specified energy cutoff of the global energy minimum. The program is used to explore the side chain conformational energy surface of a number of proteins, to investigate how this surface varies with the energy model used to describe the interactions within the system and the rotamer library. Enumeration of the rotamer combinations enables us to directly evaluate the partition function, and thus calculate the side chain contribution to the conformational entropy of the folded protein. An investigation of these conformations and the relationships between them shows that most of the conformations near to the global energy minimum arise from changes in side chain conformations that are essentially independent; very few result from a concerted change in conformation of two or more residues. Some of the limitations of the approach are discussed. Proteins 33:227–239, 1998. © 1998 Wiley-Liss, Inc.     

Peptide-binding motif of HLA-A*6603

The peptide motif of HLA-A*6603 was determined and compared with the available data on the peptide motifs of A*6601 and A*6602. A*6601 differs from A*6602 by two amino acids at positions 90 (Asp90Ala; outer loop) and 163 (Arg163Glu; pocket A). A*6603 differs from A*6601 and A*6602 by a single amino-acid exchange at position 70 (His70Gln; pockets A, B and C). No significant differences were found between the A*6602 and A*6603 peptide motifs suggesting that the Gln70His variation is of minor importance. However, the auxiliary anchors at position P1 of peptides bound by A*6601 (polar/acidic: Asp, Glu) and A*6602/6603 (polar/neutral: Ser) had striking differences. This finding may be best explained by the Arg163Glu substitution that results in a shift towards higher acidity in pocket A of A*6602/6603, apparently leading to the loss of preference for acidic auxiliary anchors. The similarity of A*6602 and A*6603 peptide motifs suggests low allogenicity when mismatched in stem cell transplantation. Inversely, the differences in A*6601 versus A*6602/6603 peptide motifs suggest that mismatches will have a higher allogenicity. These data will contribute to both assessing permissive mismatches in the A*66 group and weighting the impact of this individual amino-acid variation for matching and peptide binding algorithms.     

Assessment of promoter elements of the germ cell-specific proacrosin gene

Presence of the simian virus 40 (SV40) has recently been demonstrated in a relatively high percentage of human mesotheliomas and it is associated with the development of these malignancies in pleural cavities. Therefore, we have initiated a study to identify candidate peptides presented by the human HLA-A*0201 molecule for vaccination approaches against SV40 and monitoring of SV40 directed human immune responses. Initial screening of SV40 large T (Tag) domains required for transformation of cells for HLA-A*0201 binding motifs revealed ten possible binding peptides. Screening of these candidate peptides showed that seven of the ten peptides could bind and stabilize HLA-A*0201 molecules. In an in vitro immunization assay the two peptides with the highest binding affinity for HLA-A*0201, Tag aa 396-405 and aa 577-585, were tested for their ability to induce peptide specific cytotoxic T cells in two healthy donors. One donor developed cytotoxic T cells against Tag aa 396-405 and in T cell cultures of both donors Tag aa 577-585 specific T cells were initiated. The T cells against Tag aa 577-585 not only recognized and killed peptide pulsed cells, but, most importantly, SV40 transformed human mesothelial cells. This is the first demonstration of the induction of SV40 specific human cytotoxic T lymphocytes that recognize endogenously processed peptides from SV40. This peptide identification study opens the possibility to investigate immune responses against SV40 in mesothelioma patients and in individuals exposed to SV40.     

Colony expansion model for describing the spatial distribution of populations

Two equations have been used frequently to describe the relation between the sample variance (s ²) and sample mean (m) of the number of individuals per quadrat: Taylor's power law, s ² = am ^b , and Iwao's m ^*−m regression, s ² = cm + dm ², where a, b, c, and d are constants. We can obtain biological information such as colony size and the degree of aggregation of colonies from parameters c and d of Iwao's m ^*−m regression. However, we cannot obtain such biological information from parameters a and b of Taylor's power law because these parameters have not been described by simple functions. To mitigate such in-convenience, I propose a mechanistic model that produces Taylor's power law; this model is called the colony expansion model. This model has the following two assumptions: (1) a population consists of a fixed number of colonies that lie across several quadrats, and (2) the number of individuals per unit occupied area of colony becomes v times larger in an allometric manner when the occupied area of colony becomes h times larger (v≥ 1, h≥ 1). The parameter h indicates the dispersal rate of organisms. We then obtain Taylor's power law with b = {ln[E(h)] + ln[E(v ²)]}/{ln[E(h)] + ln[E(v)]}, where E indicates the expectation. We can use the inverse of the exponent, 1/b, as an index of dispersal of individuals because it increases with increasing E(h). This model also yields a relation, known as the Kono–Sugino relation, between the proportion of occupied quadrats and the mean density per quadrat: −ln(1 −p) = fm ^g , where p is the proportion of occupied quadrats, f is a constant, and g = ln[E(h)]/{ln[E(h)] + ln[E(v)]}. We can use g as an index of dispersal as it increases with increasing E(h). The problem at low densities where Taylor's power law is not applicable is also discussed. Received: January 27, 2000 / Accepted: June 20, 2000     

石蒜属植物分支系统学分析

基于37个形态学、解剖学、孢粉学和细胞学性状及解剖学性状之外的28个形态学、孢粉学和细胞学性状,分别对石蒜属进行分支系统学分析,试图建立石蒜属种间的系统发育关系。利用PAUP^*软件分别构建了最大简约树(MP),所得树的拓扑结构是一致的。同时,基于解剖学9个性状,对石蒜、换锦花、忽地笑、江苏石蒜、长筒石蒜、乳白石蒜、夏水仙、红兰石蒜、安徽石蒜、短蕊石蒜、中国石蒜11个种进行系统发育树构建,其结果也是支持上述系统发育树的。系统发育树结构结果表明,石蒜属16种明显聚为两大类:石蒜、玫瑰石蒜、稻草石蒜和江苏石蒜;广西石蒜、红兰石蒜、换锦花、香石蒜、夏水仙、长筒石蒜、安徽石蒜、中国石蒜、忽地笑、乳白石蒜、短蕊石蒜和陕西石蒜。除换锦花、红兰石蒜及江苏石蒜系统发育位置不同之外,大类群的划分与RAPD指纹图谱基本一致。类群一均属于石蒜亚属(Lycoris亚属)。类群二又可以聚为两小类:广西石蒜、红兰石蒜、换锦花、香石蒜、夏水仙归为一类;长筒石蒜、安徽石蒜、中国石蒜、忽地笑、乳白石蒜、短蕊石蒜和陕西石蒜归为一类。前一子类群除广西石蒜外,都属于整齐花亚属(Symman thus亚属)。后一子类群除长筒石蒜与安徽石蒜外,均属于石蒜亚属(Lycoris亚属)。因此,花冠整齐与否是一个重要的分类特征,但作为石蒜属植物亚属的划分依据,没有得到本研究支持。而在本文中,雄蕊与花被片的位置关系可以作为大分类群划分依据,能否依此来对石蒜属植物亚属进行划分,仍需探讨。另外研究表明叶微形态特征在研究种间亲缘关系时,具有一定的作用。而在种间亲缘关系鉴定时,出叶期不应成为重要的依据。同时研究还表明中国石蒜与忽地笑具有非常近的亲缘关系,与形态学研究一致。     

In and out of refugia: historical patterns of diversity and demography in the North American Caesar's mushroom species complex

Some of the effects of past climate dynamics on plant and animal diversity make‐up have been relatively well studied, but to less extent in fungi. Pleistocene refugia are thought to harbour high biological diversity (i.e. phylogenetic lineages and genetic diversity), mainly as a product of increased reproductive isolation and allele conservation. In addition, high extinction rates and genetic erosion are expected in previously glaciated regions. Some of the consequences of past climate dynamics might involve changes in range and population size that can result in divergence and incipient or cryptic speciation. Many of these dynamic processes and patterns can be inferred through phylogenetic and coalescent methods. In this study, we first delimit species within a group of closely related edible ectomycorrhizal Amanita from North America (the American Caesar's mushrooms species complex) using multilocus coalescent‐based approaches; and then address questions related to effects of Pleistocene climate change on the diversity and genetics of the group. Our study includes extensive geographical sampling throughout the distribution range, and DNA sequences from three nuclear protein‐coding genes. Results reveal cryptic diversity and high speciation rates in refugia. Population sizes and expansions seem to be larger at midrange latitudes (Mexican highlands and SE USA). Range shifts are proportional to population size expansions, which were overall more common during the Pleistocene. This study documents responses to past climate change in fungi and also highlights the applicability of the multispecies coalescent in comparative phylogeographical analyses and diversity assessments that include ancestral species.     

通路克隆入门载体pEN-L4~*-PrbcS-~*T-gfp-L3~*的构建及其应用

为了利用通路克隆(Gateway)技术构建一个含有两个目的基因表达盒的植物表达载体,并把目的基因编码的蛋白质定位到转基因植物的叶绿体中,通过定点突变技术,在含有attL4和attL3重组位点的Gateway入门载体pEN-L4-2-L3中产生HindⅢ和XhoⅠ的酶切位点,然后在这两个酶切位点之间插入一个含有1,5-二磷酸核酮糖羧化酶小亚基的光诱导型启动子(PrbcS)及其叶绿体基质定位序列(*T)和绿色荧光蛋白(GFP)报告基因(gfp)的DNA片段,获得pEN-L4*-PrbcS-*T-gfp-L3*入门载体.用该载体和另一个含有attL1和attL2重组位点的入门载体(pENTR*-PrbcS-*T-gus)与Gateway的目的载体pK7 m34GW2-8 m21GW3进行LR重组反应可以构建一个能串联gfp和gus两个报告基因表达盒的植物表达载体pKm-35S-PrbcS-*T-gfp-PROLD-PrbcS-*T-gus,所构建的植物表达载体转化烟草后,gfp和gus基因能插入到转基因烟草的基因组中并正常表达,所表达的GFP蛋白可正确定位到转基因植物的叶绿体中,而GUS蛋白也可以在叶片中表达.利用此表达载体通过一次转化事件不仅可以完成两个目的基因的转化操作,而且还可以利用叶绿体基质定位序列(*T)把PrbcS控制表达的目的蛋白直接定位到转基因植物的叶绿体中.因此pEN-L4*-PrbcS-*T-gfp-L3*入门载体的应用进一步扩大了Gateway技术及植物表达载体的应用范围,为叶绿体基因工程操作提供了一个更方便的技术平台.     

Allele frequency distribution of CYP2C9*2 and CYP2C9*3 polymorphisms in six Mexican populations

Allele frequency differences of functional CYP2C9 polymorphisms are responsible for some of the variation in drug response observed in human populations. The most relevant CYP2C9 functional variants are CYP2C9*2 (rs1799853) and CYP2C9*3 (rs1057910). These polymorphisms show variation in allele frequencies among different population groups. The present study aimed to analyze these polymorphisms in 947 Mexican-Mestizo from Mexico City and 483 individuals from five indigenous Mexican populations: Nahua, Teenek, Tarahumara, Purepecha and Huichol. The CYP2C9*2 allele frequencies in the Mestizo, Nahua and Teenek populations were 0.051, 0.007 and 0.005, respectively. As for CYP2C9*3, the allelic frequencies in the Mestizo, Nahua and Teenek populations were 0.04, 0.005 and 0.005, respectively. The CYP2C9*2 and CYP2C9*3 alleles were not observed in the Tarahumara, Purepecha and Huichol populations. These findings are in agreement with previous studies reporting very low allele frequencies for these polymorphisms in American Indigenous populations.