Expression of recombinant kringle 1-5 domains of human plasminogen by a prokaryote expression system

Kringle1-5 (K1-5), a proteolytic fragment containing five kringle domains of human plasminogen generated by plasmin-mediated proteolysis, has been already identified by Cao et al. with relation to anti-angiogenesis and proliferation of endothelial cells. To investigate anti-angiogenesis activity of recombinant human K1-5 (rhK1-5) expressed in Escherichia coli BL21, the cDNA of human K1-5 obtained from cloning vector pUC57-K1-5 by PCR, was inserted into an expression vector pET30(+) to construct a prokaryotic expression vector pET-K1-5. Recombinant K1-5 efficiently expressed in E. coli BL21 after IPTG induction was monitored by SDS-PAGE and Western blotting with an anti-angiostatin monoclonal antibody and an anti-hexahistidine tag antibody. The expressed K1-5 accounted for approximately 32% of the total bacterial proteins as estimated by densitometry, and existed mainly as inclusion bodies. The inclusion bodies were washed, lysed, purified, and refolded to a purity of 96% as estimated by capillary electrophoresis and the final purification yield of K1-5 in E. coli system was approximately 5.8 mg/L. Purified K1-5 protein was tested on chicken embryo chorioallantoic membranes (CAMs), and a large number of newly formed blood vessels were significantly regressed. In the present study, we demonstrated that bacterial-expressed K1-5 effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner through CAM assay. In addition, the rhK1-5 potently inhibited endothelial cell proliferation but not non-endothelial cells. For the first time, these findings demonstrate that the rhK1-5 produced by a prokaryote expression system effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner and specially suppressed in vitro the proliferation of human umbilical vein endothelial cells. This fact derived from the present study further suggests the rhK1-5 can be used for anti-angiogenesis therapy of cancer.     

Binding of Src to Na+/K+-ATPase forms a functional signaling complex

We have shown that ouabain activates Src, resulting in subsequent tyrosine phosphorylation of multiple effectors. Here, we tested if the Na+/K+-ATPase and Src can form a functional signaling complex. In LLC-PK1 cells the Na+/K+-ATPase and Src colocalized in the plasma membrane. Fluorescence resonance energy transfer analysis indicated that both proteins were in close proximity, suggesting a direct interaction. GST pulldown assay showed a direct, ouabain-regulated, and multifocal interaction between the 1 subunit of Na+/K+-ATPase and Src. Although the interaction between the Src kinase domain and the third cytosolic domain (CD3) of 1 is regulated by ouabain, the Src SH3SH2 domain binds to the second cytosolic domain constitutively. Functionally, binding of Src to either the Na+/K+-ATPase or GST-CD3 inhibited Src activity. Addition of ouabain, but not vanadate, to the purified Na+/K+-ATPase/Src complex freed the kinase domain and restored the Src activity. Consistently, exposure of intact cells to ouabain apparently increased the distance between the Na+/K+-ATPase and Src. Concomitantly, it also stimulated tyrosine phosphorylation of the proteins that are associated with the Na+/K+-ATPase. These new findings illustrate a novel molecular mechanism of signal transduction involving the interaction of a P-type ATPase and a nonreceptor tyrosine kinase.     

Involvement of lignocellulolytic enzymes in the decomposition of leaf litter in a subtropical forest

The involvement of ligninolytic and cellulolytic enzymes, such as laccase, lignin peroxidase, manganese peroxidase, carboxymethylcellulase (CMCase), and filter paper activity (FPA), in the decomposition process of leaf litter driven by 6 soil-inhabiting fungi imperfecti was studied under solid-state fermentations. All the tested fungi exhibited varied production profiles of lignocellulolytic enzymes and each caused different losses in total organic matter (TOM) during decomposition. Based on the results, the 6 fungi could be divided into 2 functional groups: Group 1 includes Alternaria sp., Penicillium sp., Acremonium sp., and Trichoderma sp., and Group 2 includes Pestalotiopsis sp. and Aspergillus fumigatus. Group 1, with higher CMCase and FPA activities, showed a higher decomposition rate than the fungi of Group 2 over the first 16 d, and thereafter the cellulolytic activities and decomposition rate slowed down. Group 2 showed the maximum and significantly higher CMCase and FPA activities than those of the Group 1 fungi during the later days. This, combined with the much higher laccase activity, produced a synergistic reaction that led to a much faster average mass loss rate. These results suggest that the fungi of Group 1 are efficient decomposers of cellulose and that the fungi of Group 2 are efficient decomposers of lignocellulose. During cultivation, Pestalotiopsis sp. produced an appreciable amount of laccase activity (0.56+/-0.09 U/ml) without the addition of inducers and caused a loss in TOM of 38.2%+/-3.0%, suggesting that it has high potential to be a new efficient laccase-producing fungus.     

Application of a sensitive collection heuristic for very large protein families: Evolutionary relationship between adipose triglyceride lipase (ATGL) and classic mammalian lipases

Background  

Manually finding subtle yet statistically significant links to distantly related homologues becomes practically impossible for very populated protein families due to the sheer number of similarity searches to be invoked and analyzed. The unclear evolutionary relationship between classical mammalian lipases and the recently discovered human adipose triglyceride lipase (ATGL; a patatin family member) is an exemplary case for such a problem.     

Magic-angle spinning NMR studies of cell wall bound aromatic-aliphatic biopolyesters associated with strengthening of intercellular adhesion in potato (Solanum tuberosum L.) tuber parenchyma

Intercellular adhesion strengthening, a phenomenon that compromises the texture and the edible quality of potatoes (Solanum tuberosum L.), has been induced reproducibly by exposure to low-pH acetic acid solutions under tissue culture conditions. The resulting parenchyma tissues have been examined by solid-state nuclear magnetic resonance (NMR) in order to characterize the biopolymer(s) thought to be associated with this syndrome. Cross polarization-magic angle spinning (CPMAS) (13)C NMR has been used to establish the presence of a polyphenol-suberin-like aromatic-aliphatic polyester within an abundant cell wall polysaccharide matrix in potato tubers that exhibit hardening due to strengthened intercellular adhesion. Dipolar dephasing and CP chemical shift anisotropy experiments suggest that the aromatic domain is composed primarily of guaiacyl and sinapyl groups. Two-dimensional wide-line separation experiments show that the biopolymer associated with parenchyma hardening contains rigid polysaccharide cell walls and mobile aliphatic long-chain fatty acids; (1)H spin diffusion experiments show that these flexible aliphatic chains are proximal to both the phenolics and a subpopulation of the cell wall polysaccharides. Finally, high-resolution MAS NMR of parenchyma samples swelled in DMSO in conjunction with two-dimensional through-bond and through-space NMR spectroscopy provides evidence for covalent linkages among the polysaccharide, phenolic, and aliphatic domains of the intercellular adhesion-strengthening biopolymer in potato parenchyma tissue.     

Development of new transformation-competent artificial chromosome vectors and rice genomic libraries for efficient gene cloning

The transformation-competent artificial chromosome vector (TAC) system has been shown to be very useful for efficient gene isolation in Arabidopsis thaliana (Proc. Natl. Acad. Sci. USA 96 (1998) 6535). To adapt the vector system for gene isolation in crops, two new TAC vectors and rice genomic libraries were developed. The new vectors pYLTAC17 and pYLTAC27 use the Bar gene and Hpt gene driven by the rice Act1 promoter as the plant selectable markers, respectively, and are suitable for transformation of rice and other grasses. Two representative genomic libraries (I and II) of an Indica rice variety Minghui63, a fertility restorer line for hybrid rice, were constructed with pYLTAC17 using different size classes of partially digested DNA fragments. Library I and library II consisted of 34,560 and 1.2 x 10(5) clones, with average insert sizes of approximately 77 and 39 kb, respectively. The genome coverage of the libraries I and II was estimated to be about 5 and 11 haploid genome equivalents, respectively. Clones of the library I were stored individually in ninety 384-well plates, and those of the library II were collected as bulked pools each containing 30-50 clones and stored in eight 384-well plates. A number of probes were used to hybridize high-density colony filters of the library I prepared by an improved replicating method and each detected 2-9 positive clones. A method for rapid screening of the library II by pooled colony hybridization was developed. A TAC clone having an 80 kb rice DNA insert was successfully transferred into rice genome via Agrobacterium-mediated transformation. The new vectors and the genomic libraries should be useful for gene cloning and genetic engineering in rice and other crops.     

祁连山红腹牧草蝗雌雄虫空间格局及关联性

应用地统计学的方法,研究了祁连山北坡石羊河上游,6—10月之间红腹牧草蝗Omocestus haemorrhoidalis(Charpentie)雌雄虫的空间分布格局、动态变化及空间相关性。结果表明:红腹牧草蝗雌雄虫的半变异函数为球状模型与高斯模型,二者空间分布类型相同,均呈聚集分布;随时间的变化均呈现"扩散-聚集-扩散-聚集"的动态特征,7—8月之间雌虫的变化强度明显强于雄虫;二者的模糊贴近度指数在0.53～0.71之间,且指数随红腹牧草蝗的生长呈逐渐增加到减少的变化特征,表明雌雄虫在数量与空间上有较强的跟随关系,且跟随关系随红腹牧草蝗的生长发生明显的阶段性变化。     

Interferon-β-armed oncolytic adenovirus induces both apoptosis and necroptosis in cancer cells

Interferon-β (IFN-β) has been widely used in cancer therapy, but the clinical trial results are generally disappointing. Our previous studies have shown that an oncolytic adenovirus carrying IFN-β (ZD55-IFN-β) exhibits significant anti-tumor activities. However, the underlying mechanisms are not clear. Here we showed that ZD55-IFN-β infection-induced S-phase cell cycle arrest in a p53-dependent manner by activating the ataxia telangiectasia mutated-dependent DNA damage pathway. In addition, ZD55-IFN-β infection could initiate both caspase-dependent apoptosis and necroptosis in cancer cells. More importantly, ZD55-IFN-β showed a synergistic effect on cancer cells when combined with doxorubicin. These results suggest that the combination of ZD55-IFN-β with doxorubicin may represent a promising clinical strategy in cancer therapy.     

Direct and indirect organogenesis of Clivia miniata and assessment of DNA methylation changes in various regenerated plantlets

Clivia miniata is an important indoor ornamental plant and has been reported to have medicinal value. We developed an efficient in vitro micropropagation protocol from young leaves (indirect organogenesis), young petals (indirect organogenesis) and shoot tips (direct organogenesis) of this plant. Using young leaves and shoot tips as explants, the regeneration frequencies were much higher than those in previous investigation and the regeneration was dependent upon less nutrition. We speculated that the leaf-derived callus can generate amino acids necessary for protein synthesis by itself. We employed the methylation-sensitive amplified polymorphism (MSAP) method to assess cytosine methylation variation in various regenerated plantlets and between organs. The MSAP profiles indicated that the frequency of somaclonal variation in the form of cytosine methylation was highest in petal-derived plantlets followed by secondary leaf-derived, primary leaf-derived and shoot tip-derived plantlets, but the methylation variation in petal-derived plantlets was lower than between petals and leaves of a single plant. The results indicated that the methylation variation in regenerated plantlets was related to the types of explants, regeneration pathways and number of regeneration generations. Two possible factors for the highest somaclonal variation rate in petal-derived plantlets are the callus phase and petal-specific set of epigenetic regulators. The property of meristem integrity can account for the lowest variation rate in shoot tip-derived plantlets. Moreover, the secondary plantlets underwent a longer total period of in vitro culture, which can explain why the methylation variation rate in the secondary plantlets is higher than in the primary ones. KEY MESSAGE: Methylation variation in regenerated plantlets of C. miniata was found to be related to the types of explants, regeneration pathways and number of regeneration generations.