Assembly of Plasmid DNA into Liposomes After Condensation by Cationic Lipid in Anionic Detergent Solution

A novel strategy to prepare negatively charged and small DNA-containing liposomes after condensation of plasmid DNA by a cationic lipid in deoxycholate micelle environment is described. The average diameter of resulting complexes was 62±8 nm. DNA-containing liposomes were then prepared by dialysis. The shape of the resulting liposomes was spherical. The average diameter and the surface charge of the liposomes were 86±6 nm and −24±3 mV, respectively. The plasmid DNA inside liposomes remained in a supercoiled form after incubation with DNase.     

Methylglyoxal induces apoptosis through activation of p38 MAPK in rat Schwann cells

The formation of glucose-derived methylglyoxal (MG), a highly reactive dicarbonyl compound, is accelerated under diabetic conditions. We examined whether MG was capable of inducing apoptosis in Schwann cells (SCs), since recent studies have suggested a potential involvement of apoptotic cell death in the development of diabetic neuropathy. MG induced apoptosis in SCs in a dose-dependent manner, accompanied by a reduction of intracellular glutathione content and activation of the p38 MAPK. Inhibiting the p38 MAPK activation by SB203580 successfully suppressed the MG-induced apoptosis in SCs. Aminoguanidine and N-acetyl-l-cysteine also inhibited the MG-induced p38 MAPK activation and apoptosis along with restoration of the intracellular glutathione content. These results suggest a potential role for MG in SC injury through oxidative stress-mediated p38 MAPK activation under diabetic conditions, and it may serve as a novel insight into therapeutic strategies for diabetic neuropathy.     

Genetic differentiation and relationships of populations in the Cycas balansae complex (Cycadaceae) and its conservation implications

BACKGROUND AND AIMS: The Cycas balansae complex is arguably a controversial group with regard to species delineation. Some taxonomists recognize a single polymorphic species while others distinguish five narrowly defined ones. The unresolved taxonomy has the potential to bring about significant problems for species conservation. Thus, an investigation to examine the genetic diversity and differentiation in the C. balansae complex was performed to determine the relationship of populations and to test whether the morphologically defined segregations represent genetically distinct units. METHODS: Inter-simple sequence repeat (ISSR) markers were employed to assess the genetic diversity in the C. balansae complex with a sample of 158 individuals from all extant populations in China. KEY RESULTS: ISSR markers revealed low genetic diversity in all populations studied (H(E) and H(O) averaged 0.0639 and 0.0798 at the population level, respectively). Phenetic analysis showed that the C. balansae complex grouped into five clusters closely corresponding to the narrowly defined C. balansae, C. parvula, C. shiwandashanica, C. tanqingii and C. simplicipinna. CONCLUSIONS: ISSR data suggest that the C. balansae complex has evolved into five genetically distinct units. These might be derived from a relatively widespread common ancestor through multiple vicariant events including geographical isolation resulting from the collision of the Indian plate with the Eurasian plate and from Pleistocene glaciations. In conservation, attention should be paid to each genetic unit.     

酵母纺锤体极体复制缺陷与染色体不稳定性的发生

纺锤体极体作为酵母细胞的微管组织中心,在功能上等同于高等真核细胞的中心体,它在细胞周期中的准确复制是两极纺锤体组装和染色体正确分离的前提。纺锤体极体复制缺陷会导致异倍体和多倍体的形成,造成染色体不稳定性的发生。以酿酒酵母细胞为模型,研究纺锤体极体复制过程相关蛋白质的突变,有助于揭示酵母细胞中染色体不稳定性发生的分子机制,并为动物细胞中心体复制的研究提供良好的借鉴。     

厚壁毛竹快速高生长期竹秆ATP酶超微细胞化学定位

采用电镜细胞化学技术对厚壁毛竹(Phyllostachys edulis ‘Pachyloen’)快速高生长期竹秆节间的伸长发育过程（包括：分生细胞期、伸长初期、快速伸长期和成熟期四个阶段）进行ATP酶超微细胞化学定位,以揭示竹秆节间快速伸长的细胞学基础。结果表明：分生细胞期,细胞质膜、核膜、细胞器膜系统上等均有很强的ATP酶活性。伸长初期,节间上部基本组织细胞质膜上ATP酶活性较强,且短细胞质膜上的ATP酶活性更强,节间基部各细胞均未观察到ATP酶活性。快速伸长期,节间基部基本组织ATP酶活性较节间上部高,细胞质膜、运输小泡膜、胞间隙及胞间连丝上均有ATP酶活性。成熟期,仅节间上部基本组织质膜上有较弱的ATP酶活性。ATP酶在节间伸长过程中主要参与新细胞壁物质的分泌和共质体运输,促进新细胞壁的形成,晶体和淀粉粒体外膜上ATP酶活性的存在表明其具有贮存物质的作用。节隔缺失节的节间基部未观察到ATP酶活性,节部韧皮结细胞ATP酶活性较高,节隔的缺失引起节部与节间与物质运输有关结构的变化,进而影响节间伸长生长。     

Analysis and comparison of a set of expressed sequence tags of the parthenogenetic water flea <Emphasis Type="Italic">Daphnia carinata</Emphasis>

The water flea Daphnia carinata (D. carinata) reproduces both sexually and parthenogenetically, yet little is known about the genes involved in these processes. To further clarify the reproductive biology of Daphnia and elucidate their unique mechanism of reproductive transformation, we have generated and characterized an expressed sequence tag (EST) data set from D. carinata. A set of 1,495 clusters were generated from sequencing 3,072 randomly chosen clones from a parthenogenetic, juvenile water flea cDNA library. The nucleic acid and deduced amino acid sequences were compared with known GenBank sequences. Functional annotation found that 959 clusters showed significant homology with known genes involved in a broad range of activities, including metabolism, translation, development and reproduction, as well as genes involved in sensing environmental factors. We speculate that genes involved in development and reproduction, along with genes that allow the organism to sense changes in the environment, play important roles in the process of parthenogenetic reproduction and could be markers of the early steps of sexual differentiation. Additionally, 86% of the D. Carinata unique sequences could be stringently mapped to the D. pulex genome, of which 125 mapped to intergenic and intronic regions on the current assembly. Our results provide practical insight into crustacean reproductive biology, in addition to establishing a new animal model for reproductive and developmental biology. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Xiaoqian Xu and Shuhui Song contributed equally to this work. Nucleotide sequence data reported are available in the GenBank databases under the accession numbers: GD269049−GD272045.     

Doxycycline enhances the Ras‐MAPK signaling and proliferation of mouse thymic epithelial cells

Depletion of T‐cell‐dependent immunity is a major consideration for patients suffering from infections of human immunodeficiency virus (HIV), those undergoing organ transplantation, and those receiving anti‐cancer chemotherapy and/or radiotherapy. In general, T‐cell regeneration occurs in the thymus through thymopoiesis. We have found that doxycycline (Dox), a tetracycline derivative, enhances the proliferation of mouse thymic epithelial cells, which are unique in their capacity to support positive selection and are essential throughout the development of thymocytes. Cell cycle analysis indicates that the increased cell proliferation is due to a shortened G₀/G₁ phase. To reveal the underlying mechanisms, we examined the expression of an array of molecules that regulate the cell cycle. The results show that in mouse thymic medullary‐type epithelial cell line 1 (MTEC1) Dox leads to elevated levels of H‐Ras, phosphorylated extracellular signal‐regulated kinase 1/2 (p‐ERK1/2), cyclin E, cyclin dependent kinase 4/2 (CDK4/CDK2), E2F3, and c‐myc. These data, and the observation that the proliferation‐enhancing effect is largely abolished following treatment with an ERK inhibitor support an active role of the Ras‐ERK/mitogen‐activated protein kinase (MAPK) signaling pathway. In conclusion, the present study reveals a new activity of an old family of antibiotics. The in vivo effect of Dox on immune reconstitution warrants further exploration. J. Cell. Biochem. 107: 494–503, 2009. © 2009 Wiley‐Liss, Inc.     

Exostosin glycosyltransferase 1 reduces porcine reproductive and respiratory syndrome virus infection through proteasomal degradation of nsp3 and nsp5

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a serious threat to the swine industry worldwide. Exostosin glycosyltransferase 1 (EXT1), an enzyme involved in the biosynthesis of heparin sulfate, has also been reported to be a host factor essential for a wide variety of pathogens. However, the role of EXT1 in PRRSV infection remains uncharted. Here, we identified that PRRSV infection caused an increase of EXT1 expression. EXT1 knockdown promoted virus infection, whereas its overexpression inhibited virus infection, suggesting an inhibitory function of EXT1 to PRRSV infection. We found that EXT1 had no effects on the attachment, internalization, or release of PRRSV but did restrict viral RNA replication. EXT1 was determined to interact with viral nonstructural protein 3 (nsp3) and nsp5 via its N-terminal cytoplasmic tail and to enhance K48-linked polyubiquitination of these two nsps to promote their degradation. Furthermore, the C-terminal glycosyltransferase activity domain of EXT1 was necessary for nsp3 and nsp5 degradation. We also found that EXT2, a EXT1 homolog, interacted with EXT1 and inhibited PRRSV infection. Similarly, EXT1 effectively restricted porcine epidemic diarrhea virus and porcine enteric alphacoronavirus infection in Vero cells. Taken together, this study reveals that EXT1 may serve as a broad-spectrum host restriction factor and suggests a molecular basis for the potential development of therapeutics against PRRSV infection.     

戊型肝炎病毒感染激活补体系统

为了探究补体系统与戊型肝炎病毒复制的相关性,分别在HEV感染的A549细胞和BALB/c小鼠中检测C3aR、CD55和CD59蛋白的表达.利用RT-qPCR定量检测细胞和组织中补体的表达,采用免疫组化法检测HEV感染BALB/c小鼠中补体CD59及C5b-9的表达,ELISA检测补体相关炎症因子的变化.HEV感染可以激活补体蛋白C3aR、C5b-9、CD55和CD59的表达,引起补体蛋白相关炎症因子IL-10表达水平下降,IL-12和TNF-α的表达水平的上升,从而导致机体的炎症反应,加剧组织损伤.HEV感染激活补体系统并参与早期的抗病毒反应,HEV感染对补体的持续激活导致炎症因子过度表达,加重机体损伤.