Effects of somatic mutations on CDR loop flexibility during affinity maturation

Prior studies suggest that antibody affinity maturation is achieved, in part, via prearranging the CDRs for binding. The implication is that the entropy cost of binding is reduced and that this rigidification occurs as a consequence of somatic mutations during maturation. However, how these mutations modulate CDR flexibility is unclear. Here, molecular dynamics simulations captured CDR flexibility differences between four mature antibodies (7G12, AZ28, 28B4, and 48G7) and their germline predecessors. Analysis of their trajectories: (1) rationalized how mutations during affinity maturation restrict CDR motility, (2) captured the equilibrium between bound and unbound conformations for the H3 loop of unliganded 7G12, and (3) predicted a set of new mutations that, according to our simulations, should diminish binding by increasing flexibility.     

Nox2 B-loop peptide, Nox2ds, specifically inhibits the NADPH oxidase Nox2

In recent years, reactive oxygen species (ROS) derived from the vascular isoforms of NADPH oxidase, Nox1, Nox2, and Nox4, have been implicated in many cardiovascular pathologies. As a result, the selective inhibition of these isoforms is an area of intense current investigation. In this study, we postulated that Nox2ds, a peptidic inhibitor that mimics a sequence in the cytosolic B-loop of Nox2, would inhibit ROS production by the Nox2-, but not the Nox1- and Nox4-oxidase systems. To test our hypothesis, the inhibitory activity of Nox2ds was assessed in cell-free assays using reconstituted systems expressing the Nox2-, canonical or hybrid Nox1-, or Nox4-oxidase. Our findings demonstrate that Nox2ds, but not its scrambled control, potently inhibited superoxide (O₂^•−) production in the Nox2 cell-free system, as assessed by the cytochrome c assay. Electron paramagnetic resonance confirmed that Nox2ds inhibits O₂^•− production by Nox2 oxidase. In contrast, Nox2ds did not inhibit ROS production by either Nox1- or Nox4-oxidase. These findings demonstrate that Nox2ds is a selective inhibitor of Nox2-oxidase and support its utility to elucidate the role of Nox2 in organ pathophysiology and its potential as a therapeutic agent.     

Stimulation of IFN-γ production by garlic lectin in mouse spleen cells: Involvement of IL-12 via activation of p38 MAPK and ERK in macrophages

Several lectins, present in beans and edible plant products, have immuno-potentiating and anti-tumor activities. We here report the effects of garlic lectin purified from garlic bulbs on the production of cytokines such as interleukin-12 (IL-12) and interferon-γ (IFN-γ) in the mouse. Garlic lectin induced IFN-γ production in spleen cells in a bell-shaped time (24-60 h)- and concentration (0.25-2.0 mg/ml)-dependent manner. The maximal enhancement was observed at 36 h with 0.5 mg/ml of garlic lectin. The stimulatory effect of garlic lectin on IFN-γ production was completely inhibited by both actinomycin D and cycloheximide, an inhibitor of ribosomal protein synthesis and DNA-dependent RNA polymerase, respectively, and was associated with an increase in IFN-γ mRNA level. Garlic lectin also induced IL-12 production in mouse peritoneal macrophages in a concentration (0.25-1.0 mg/ml)- and bell-shaped time (3-24 h)-dependent manner. The lectin increased the phosphorylation of extracellular signal-regulated kinases (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) in macrophages. Furthermore, specific pharmacological inhibitors of ERK kinase (U0126) and p38 MAPK (SB203580) also suppressed the production of IL-12 induced by garlic lectin. The present findings suggest that garlic lectin induces IL-12 production via activation of p38 MAPK and ERK in mouse macrophages, which, in turn, stimulates IFN-γ production through an increase in IFN-γ mRNA in the spleen cells.     

Identification of novel allosteric modulators for the G-protein coupled US28 receptor of human cytomegalovirus

The highly constitutively active G-protein coupled receptor US28 of human cytomegalovirus (HCMV) is an interesting pharmacological target because of its implication on viral dissemination, cardiovascular diseases and tumorigenesis. We found that dihydroisoquinolinone and tetrahydroisoquinoline scaffolds may be promising lead structures for novel US28 allosteric inverse agonists. These scaffolds were rapidly synthesized by radical carboamination reactions followed by non-radical transformations. Our novel US28 allosteric modulators provide valuable scaffolds for further ligand optimization and may be helpful chemical tools to investigate molecular mechanisms of US28 constitutive signaling and its role in pathogenesis.     

肿瘤能量代谢机制研究进展

细胞的能量主要来自糖酵解和线粒体的有氧氧化.细胞活性和能量状态密切相关,恶性肿瘤由于其生长迅速,常常出现葡萄糖摄取量增高、糖酵解增加和乳酸堆积现象.通过介绍肿瘤能量代谢机制研究进展,结合代谢成像技术,将会有助于深入揭示肿瘤能量代谢改变与肿瘤进展的因果关联,为靶向能量代谢的肿瘤治疗策略提供新的视野和契机.     

黄颡单尾虫(粘体门,双壳目)的重描述及基于28S rDNA和ITS-5.8S序列的系统地位分析

采用形态分类学方法与以28S rDNA和ITS-5.8S序列为基础的分子系统学研究方法,对采自嘉陵江重庆市磁器口江段的黄颡单尾虫Unicauda pelteobagrusMa,1998进行了形态学和分子生物学的研究。基于28S rDNA数据探讨了黄颡单尾虫以及单尾虫属与相邻种属粘孢子虫间的系统地位;基于5.8S rDNA数据比较分析了粘孢子虫的系统地位。补充了黄颡单尾虫重庆种群形态学信息和28S rDNA、ITS-5.8S rDNA序列的分子信息。     

直突摇蚊亚科部分属28S rDNA D1序列的分子系统发育关系(双翅目,摇蚊科)

利用28S rDNA D1部分基因序列对直突摇蚊亚科代表性属级阶元进行了分子系统学研究。测定了12个内群属和2种外群的28SrDNAD1片段,并结合GenBank中3个同亚科种类该基因的同源序列进行了分析。采用2种建树方法(距离邻近法NJ和最大俭约法MP)分析了直突摇蚊亚科内属级分类单元的分子系统发育关系。结果表明,滨海摇蚊属Clunio位于系统发育树的基部,与该属营海洋生活的特殊性一致。心突摇蚊属和真开氏摇蚊属互为姐妹群,流环足摇蚊属和刀突摇蚊属互为姐妹群,此结果与基于形态学的系统发育研究相结果一致。其它属间的系统发育关系因尚无前人研究而有待做进一步研究。本研究同时证明28S rDNA D1基因片段在分析摇蚊科昆虫属级及属内阶元关系上具有一定的指导意义。     

Comparative modeling and genomics for galactokinase (Gal1p) enzyme

The Gal1p (Galactokinase) protein is known for regulation of D-galactose metabolism. It catalyzes the formation of galactose -1-phosphate from alpha - D-galactose, which is an important step in galactose catabolism. The knowledge of Gal1p protein structure, its protein interacting partners and enumeration of functional site residues will provide great insight in understanding the functional role of Gal1p. These studies are lacking in case of the Gal11p kinase enzyme. Structure of this enzyme has already been determined in S. cerevisiae, however, no structural information for this protein is available for K. lactis and E. coli. We used the homology modeling based approach to model the structures of Gal1p for K. lactis and E. coli. Furthermore, functional residues were predicted for these Gal1 proteins and the strength of interaction between Gal1p and other Gal proteins was determined by protein-protein interaction studies via patchdock software. The interaction studies revealed that the affinity for Gal1p for other Gal proteins varies in different organisms. Sequence and structural based comparison of Gal1p kinase enzyme showed that the orthologs in K.lactis and S. cervisiae are more similar to each other as compared to the ortholog in E. coli. These studies carried out by us will help in better understanding of the galactose metabolism. Our sequence and structure comparison studies revealed that Human Gal1p shows more homology for Gal1p protein of E. coli. The above studies may be applied to Human Gal1p, where it can help in gaining useful insight into Galactosemia disease.     

研究报告：骨形态发生蛋白9通过p38激酶途径调控间充质干细胞C3H10T1/2成骨分化

前期研究发现骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)具有较强的诱导间充质干细胞成骨分化的能力.为进一步揭示其诱导和调控间充质干细胞成骨分化的机理,利用BMP9重组腺病毒感染间充质干细胞C3H10T1/2,通过体外细胞实验和体内动物实验,初步分析BMP9是否可通过p38激酶途径调控间充质干细胞成骨分化.结果发现,BMP9可以通过促进p38激酶磷酸化而导致其活化,p38抑制剂SB203580可抑制由BMP9诱导的C3H10T1/2细胞的碱性磷酸酶(alkalinephosphatase,ALP)活性、骨桥蛋白(osteopontin,OPN)表达和钙盐沉积,而且利用抑制剂SB203580抑制p38激酶活性后,BMP9诱导的Smad经典途径的激活也相应受到抑制,RNA干扰导致p38基因沉默同样也可抑制BMP9诱导的ALP活性、OPN表达、钙盐沉积以及裸鼠皮下异位成骨.因此,BMP9可通过活化p38激酶途径调控间充质干细胞C3H10T1/2成骨分化.