Interaction of the disintegrin and cysteine-rich domains of ADAM12 with integrin alpha7beta1

We describe a novel interaction between the disintegrin and cysteine-rich (DC) domains of ADAM12 and the integrin alpha7beta1. Integrin alpha7beta1 extracted from human embryonic kidney 293 cells transfected with alpha7 cDNA was retained on an affinity column containing immobilized DC domain of ADAM12. 293 cells stably transfected with alpha7 cDNA adhered to DC-coated wells, and this adhesion was partially inhibited by 6A11 integrin alpha7 function-blocking antibody. The X1 and the X2 extracellular splice variants of integrin alpha7 supported equally well adhesion to the DC protein. Integrin alpha7beta1-mediated cell adhesion to DC had different requirements for Mn2+ than adhesion to laminin. Furthermore, integrin alpha7beta1-mediated cell adhesion to laminin, but not to DC, resulted in efficient cell spreading and phosphorylation of focal adhesion kinase (FAK) at Tyr397. We also show that adhesion of L6 myoblasts to DC is mediated in part by the endogenous integrin alpha7beta1 expressed in these cells. Since integrin alpha7 plays an important role in muscle cell growth, stability, and survival, and since ADAM12 has been implicated in muscle development and regeneration, we postulate that the interaction between ADAM12 and integrin alpha7beta1 may be relevant to muscle development, function, and disease. We also conclude that laminin and the DC domain of ADAM12 represent two functional ligands for integrin alpha7beta1, and adhesion to each of these two ligands via integrin alpha7beta1 triggers different cellular responses.     

d-Ribose glycates β(2)-microglobulin to form aggregates with high cytotoxicity through a ROS-mediated pathway

β(2)-Microglobulin (β(2)M) modified with advanced glycation end products (AGEs) is a major component of the amyloid deposits in hemodialysis-associated amyloidosis (HAA). However, the effect of glycation on the misfolding and aggregation of β(2)M has not been studied so far. Here we examine the molecular mechanism of aggregate formation of HAA-related ribosylated β(2)M in vitro. We find that the glycating agent d-ribose interacts with human β(2)M to generate AGEs that form aggregates in a time-dependent manner. Ribosylated β(2)M molecules are highly oligomerized compared with unglycated β(2)M, and have granular morphology. Furthermore, such ribosylated β(2)M aggregates show significant cytotoxicity to both human SH-SY5Y neuroblastoma and human foreskin fibroblast FS2 cells and induce intracellular reactive oxygen species (ROS). Presence of the antioxidant N-acetylcysteine (1.0mM) attenuated intracellular ROS and prevented cell death induction in both SH-SY5Y and FS2 cells, indicating that the cytotoxicity of ribosylated β(2)M aggregates depends on a ROS-mediated pathway in both cell lines. In other words, d-ribose reacts with β(2)M and induces the ribosylated protein to form granular aggregates with high cytotoxicity through a ROS-mediated pathway. These findings suggest that ribosylated β(2)M aggregates could contribute to the dysfunction and death of cells and could play an important role in the pathogenesis of β(2)M-associated diseases such as HAA.     

Effect of a traditional Chinese medicine preparation Xindi soft capsule on rat model of acute blood stasis: a urinary metabonomics study based on liquid chromatography-mass spectrometry

Xindi soft capsule is a traditional Chinese medicine preparation which consists of sea buckthorn flavonoids and sea buckthorn berry oil. In this study, a urinary metabonomics method based on the ultra-performance liquid chromatography combined with quadrupole time-of-flight tandem mass spectrometry (UPLC Q-TOF MS) was used to evaluate the efficacy and study the mechanism of traditional Chinese medicine preparation to blood stasis. With pattern recognition analysis (principal component analysis and partial least squares-discriminate analysis) of urinary metabolites, a clear separation of acute blood stasis model group and healthy control group was achieved, the dose groups were located between acute blood stasis model group and healthy control group showing a tendency of recovering to healthy control group, high dose and middle dose were more effective than low dose. Some significantly changed metabolites like cholic acid, phenylalanine and kynurenic acid have been found and identified and used to explain the mechanism. The work shows that the metabonomics method is a valuable tool in the research mechanism of traditional Chinese medicine.     

大黄酚和大黄素甲醚的制备性分离

经系统分离得到大黄酚和大黄素甲醚的混合物后,取该混合物约1g,用少量的氯仿溶解,加入少量的层析用硅胶（100～200目）拌匀,减压使氯仿蒸干。将吸附有大黄酚和大黄素甲醚的硅胶装在已装好的硅胶层析柱的上端,然后进行洗脱。通过实验,得到了最佳分离大黄酚和大黄素甲醚的条件：硅胶与混合物的质量比=150：1;层析柱长与直径的比=23：1;洗脱剂比例：石油醚（60～90℃）：乙酸乙酯（V/V）=17：1;减压淋洗。     

Enzymatic esterification between n-alcohol homologs and n-caprylic acid in non-aqueous medium under microwave irradiation

The enzymatic esterification between n-alcohol homologs and n-caprylic acid catalyzed by lipozyme RM IM (LRI) in microwave field was investigated. Some interesting findings were obtained. The optimum reaction temperature slightly shifted from that in enzymatic esterification by conventional heating. n-Alcohol homologs used in this experiment showed substrate specificity in terms of the odd and even carbon numbers. THF expressed abnormal solvent effect. Whereas in the contrastive enzymatic esterification by conventional heating, the above mentioned substrate specificity and solvent effect were not observed. All the above phenomena could be explained by both thermal and non-thermal effect of microwave on enzyme and substrates. Further investigation revealed that microwave irradiation reduced the apparent activation energy of the enzymatic reaction according to Arrhenius equation, which is considered as one of the causes increasing initial reaction rate.     

Identification of genes necessary for jinggangmycin biosynthesis from Streptomyces hygroscopicus 10-22

A series of large chromosomal deletions in Streptomyces hygroscopicus 10-22 were aligned on the physical map of the wild-type strain and the mutants were assessed for their ability to produce the aminocyclitol antibiotic 5102-I (jinggangmycin). Twenty-eight mutants were blocked for jinggangmycin production and all of them were found to lack a 300 kb AseI-F fragment of the wild-type chromosome. An ordered cosmid library of the 300 kb AseI-F fragment was made and one of the cosmids conferred jinggangmycin productivity to Streptomyces lividans ZX1. Three of the overlapping cosmids (18G7, 5H3 and 9A2) also hybridized to the valA gene of the validamycin pathway from S. hygroscopicus 5008 as a probe. This gene resembles acbC from Actinoplanes sp. 50/110, which encodes a C7-cyclitol synthase that catalyses the transformation of sedoheptulose 7-phosphate into 2-5-epi-valiolone for acarbose biosynthesis. The valA/acbC-homolog (orf1) of S. hygroscopicus 10-22 was shown to be essential for jinggangmycin biosynthesis as an engineered mutant with a specific in-frame deletion removing a 609 bp sequence internal to orf1 completely abolished jinggangmycin production and the corresponding knock-out mutant (JXH4) could be complemented for jinggangmycin production by the introduction of an orf1-containing construct. Concurrently, the identities of the genes common to S. hygroscopicus strains 10-22 and 5008 prompted a comparison of the chemical structures of jinggangmycin and validamycin, which led to a clear demonstration that they are identical.The first two authors contributed equally to this study.     

几种细胞分裂素诱导石竹外植体直接再分化成花芽的机理

用ＭＳ＋２,４－Ｄ０．１ｍｇ／Ｌ为基本培养基,分别附加不同浓度的ＺＴ、ＢＡ和ＫＴ,对石竹的叶片、茎和花等外植体直接再分化成花芽的试验表明：叶片的外植体在有ＢＡ时可再分化出只有红色花瓣的不正常花芽;在含ＺＴ的培养基中再分化出具有１～２片叶或无叶的花芽,其花有两性花,雌花和仅单个雌蕊的不正常花,两性花可发育成结籽的果实;ＫＴ未能诱导花芽分化。茎外植体,３种ＣＴＫ都不能诱导出花芽。花等外植体仅在ＫＴ３ｍｇ／Ｌ的培养中再分化出无叶的不正常花芽,ＺＴ和ＢＡ的处理均不能诱导出花芽。