Microtubule-associated protein 1B: a neuronal binding partner for gigaxonin

Giant axonal neuropathy (GAN), an autosomal recessive disorder caused by mutations in GAN, is characterized cytopathologically by cytoskeletal abnormality. Based on its sequence, gigaxonin contains an NH2-terminal BTB domain followed by six kelch repeats, which are believed to be important for protein-protein interactions (Adams, J., R. Kelso, and L. Cooley. 2000. Trends Cell Biol. 10:17-24.). Here, we report the identification of a neuronal binding partner of gigaxonin. Results obtained from yeast two-hybrid screening, cotransfections, and coimmunoprecipitations demonstrate that gigaxonin binds directly to microtubule-associated protein (MAP)1B light chain (LC; MAP1B-LC), a protein involved in maintaining the integrity of cytoskeletal structures and promoting neuronal stability. Studies using double immunofluorescent microscopy and ultrastructural analysis revealed physiological colocalization of gigaxonin with MAP1B in neurons. Furthermore, in transfected cells the specific interaction of gigaxonin with MAP1B is shown to enhance the microtubule stability required for axonal transport over long distance. At least two different mutations identified in GAN patients (Bomont, P., L. Cavalier, F. Blondeau, C. Ben Hamida, S. Belal, M. Tazir, E. Demir, H. Topaloglu, R. Korinthenberg, B. Tuysuz, et al. 2000. Nat. Genet. 26:370-374.) lead to loss of gigaxonin-MAP1B-LC interaction. The devastating axonal degeneration and neuronal death found in GAN patients point to the importance of gigaxonin for neuronal survival. Our findings may provide important insights into the pathogenesis of neurodegenerative disorders related to cytoskeletal abnormalities.     

水稻OsRacD蛋白G15V、T20N点突变对其细胞定位的影响及其与靶蛋白的相互作用

OsRacD是水稻小GTP结合蛋白Rho家族成员,功能之一是作为“分子开关",通过控制花粉管的延伸生长,参与光敏核不育水稻光周期育性转换过程. 为研究该蛋白的作用机制,采用重叠延伸PCR方法,分别在其GTPase结构域中引入G15V、T20N点突变,模拟GTP和GDP结合形式的OsRacD. 进一步构建了受控于CaMV35S的与绿色荧光蛋白融合表达的双元植物表达载体;采用农杆菌介导法转化洋葱表皮细胞,在荧光显微镜下观察蛋白在活细胞内定位的特点. 结果显示,野生型蛋白在细胞质和细胞膜都有分布,组成型激活的蛋白主要分布在细胞膜上,而失活型蛋白则大都集中到细胞核周围. 蛋白相互作用的酵母双杂交体系分析显示,OsRacD及其2个突变体具有不同的靶蛋白结合特性. 研究证实,水稻OsRacD蛋白G15V和T20N点突变不仅影响其在活细胞内的定位,而且也影响了与靶蛋白的相互作用.说明处于不同鸟苷酸结合状态的OsRacD具有不同的胞内定位方式,可能通过结合不同的靶蛋白,引发不同的细胞应答事件.     

T4-Like genome organization of the Escherichia coli O157:H7 lytic phage AR1

We report the genome organization and analysis of the first completely sequenced T4-like phage, AR1, of Escherichia coli O157:H7. Unlike most of the other sequenced phages of O157:H7, which belong to the temperate Podoviridae and Siphoviridae families, AR1 is a T4-like phage known to efficiently infect this pathogenic bacterial strain. The 167,435-bp AR1 genome is currently the largest among all the sequenced E. coli O157:H7 phages. It carries a total of 281 potential open reading frames (ORFs) and 10 putative tRNA genes. Of these, 126 predicted proteins could be classified into six viral orthologous group categories, with at least 18 proteins of the structural protein category having been detected by tandem mass spectrometry. Comparative genomic analysis of AR1 and four other completely sequenced T4-like genomes (RB32, RB69, T4, and JS98) indicated that they share a well-organized and highly conserved core genome, particularly in the regions encoding DNA replication and virion structural proteins. The major diverse features between these phages include the modules of distal tail fibers and the types and numbers of internal proteins, tRNA genes, and mobile elements. Codon usage analysis suggested that the presence of AR1-encoded tRNAs may be relevant to the codon usage of structural proteins. Furthermore, protein sequence analysis of AR1 gp37, a potential receptor binding protein, indicated that eight residues in the C terminus are unique to O157:H7 T4-like phages AR1 and PP01. These residues are known to be located in the T4 receptor recognition domain, and they may contribute to specificity for adsorption to the O157:H7 strain.     

Factors affecting lipid accumulation by <Emphasis Type="Italic">Nannochloropsis oculata</Emphasis> in a two-stage cultivation process

Reserve lipids of microalgae are promising for biodiesel production. However, optimization of cultivation conditions for both biomass yield and lipid production of microalgae is a contradictory problem because required conditions for both targets are different. In this study, a two-stage cultivation strategy is proposed to enhance lipid production of the microalga Nannochloropsis oculata. Biomass growth and lipid production were carried out in two separate and non-interacting stages. In first-stage cultivation, microalgae were cultivated in optimal conditions for cell growth. Then, microalgae were harvested and transferred into a growth-limited environment, thus enhancing lipid production of microalgae. Here, optimization of the lipid production stage (second stage) with respect to different levels of inoculum concentration, salinity of culture broth, and intensity of irradiance was performed. The results show that irradiance exhibits a significant influence on lipid production. The highest lipid productivity of 0.324 g L⁻¹ day⁻¹ was obtained with an inoculum concentration of 2.3 g L⁻¹, a salinity of 35 g L⁻¹, and an irradiance of 500 μmol photons m⁻² s⁻¹. The final yield of lipid obtained from the two-stage process was 2.82-times higher than that from traditional single-stage batch cultivation systems.     

Regulation of Secretion of Alzheimer Amyloid Precursor Protein by the Mitogen-Activated Protein Kinase Cascade

Abstract: Activation of protein kinase C (PKC) regulates the processing of Alzheimer amyloid precursor protein (APP) into its soluble form (sAPP) and amyloid β-peptide (Aβ). However, little is known about the intermediate steps between PKC activation and modulation of APP metabolism. Using a specific inhibitor of mitogen-activated protein (MAP) kinase kinase activation (PD 98059), as well as a dominant negative mutant of MAP kinase kinase, we show in various cell lines that stimulation of PKC by phorbol ester rapidly induces sAPP secretion through a mechanism involving activation of the MAP kinase cascade. In PC12-M1 cells, activation of MAP kinase by nerve growth factor was associated with stimulation of sAPP release. Conversely, M1 muscarinic receptor stimulation, which is known to act in part through a PKC-independent pathway, increased sAPP secretion mainly through a MAP kinase-independent pathway. Aβ secretion and its regulation by PKC were not affected by PD 98059, supporting the concept of distinct secretory pathways for Aβ and sAPP formation.     

Improved execution efficiency of model-based roentgen stereophotogrammetric analysis: simplification and segmentation of model meshes

Recently, the model-based roentgen stereophotogrammetric analysis (RSA) method has been developed as an in vivo tool to estimate static pose and dynamic motion of the instrumented prostheses. The two essential inputs for the RSA method are prosthetic models and roentgen images. During RSA calculation, the implants are often reversely scanned and input in the form of meshes to estimate the outline error between prosthetic projection and roentgen images. However, the execution efficiency of the RSA iterative calculation may limit its clinical practicability, and one reason for inefficiency may be very large number of meshes in the model. This study uses two methods of mesh manipulation to improve the execution efficiency of RSA calculation. The first is to simplify the model meshes and the other is to segment and delete the meshes of insignificant regions. An index (i.e. critical percentage) of an optimal element number is defined as the trade-off between execution efficiency and result accuracy. The predicted results are numerically validated by total knee prosthetic system. The outcome shows that the optimal strategy of the mesh manipulation is simplification and followed by segmentation. On average, the element number can even be reduced to 1% of the original models. After the mesh manipulation, the execution efficiency can be increased about 75% without compromising the accuracy of the predicted RSA results (the increment of rotation and translation error: 0.06° and 0.02 mm). In conclusion, prosthetic models should be manipulated by simplification and segmentation methods prior to the RSA calculation to increase the execution efficiency and then to improve clinical applicability of the RSA method.     

Proteins differentially expressed during limonene biotransformation by <Emphasis Type="Italic">Penicillium digitatum</Emphasis> DSM 62840 were examined using iTRAQ labeling coupled with 2D-LC–MS/MS

This study focused on the differences in protein expression at various periods during limonene biotransformation by Penicillium digitatum DSM 62840. A total of 3644 protein-species were quantified by iTRAQ during limonene biotransformation (0 and 12 h). A total of 643 proteins were differentially expressed, 316 proteins were significantly up-regulated and 327 proteins were markedly down-regulated. GO, COG, and pathway enrichment analysis showed that the differentially expressed proteins possessed catalytic and binding functions and were involved in a variety of cellular and metabolic process. Furthermore, the enzymes involved in limonene transformation might be related to cytochrome P-450. This study provided a powerful platform for further exploration of biotransformation, and the identified proteins provided insight into the mechanism of limonene transformation.     

凝胶条带内DNA回收测序评估DGGE可靠性

为了评估DGGE的可靠性,对DGGE条带中回收的DNA片段进行了测序比较分析,并引入了DGGE可靠性指数的概念评价其可靠性。结果显示同一条DGGE条带回收的DNA来自同一属的概率为64.7%,相同位置的DGGE条带可以被认为是同一OTU;不同的DGGE条带回收到类似的DNA序列（16S rDNA V3区差异小于4 bp）的概率为10.5%;DGGE可靠性指数为74.8%。以上结果表明尽管DGGE技术与理论预期存在一定的差距,但是DGGE技术基本能够反映微生物群落的多样性。       

Wogonin alleviates liver injury in sepsis through Nrf2-mediated NF-κB signalling suppression

Sepsis is a life-threatening organ dysfunction syndrome, and liver is a susceptible target organ in sepsis, because the activation of inflammatory pathways contributes to septic liver injury. Oxidative stress has been documented to participate in septic liver injury, because it not only directly induces oxidative genotoxicity, but also exacerbates inflammatory pathways to potentiate damage of liver. Therefore, to ameliorate oxidative stress is promising for protecting liver in sepsis. Wogonin is the compound extracted from the medicinal plant Scutellaria baicalensis Geogi and was found to exert therapeutic effects in multiple inflammatory diseases via alleviation of oxidative stress. However, whether wogonin is able to mitigate septic liver injury remains unknown. Herein, we firstly proved that wogonin treatment could improve survival of mice with lipopolysaccharide (LPS)- or caecal ligation and puncture (CLP)-induced sepsis, together with restoration of reduced body temperature and respiratory rate, and suppression of several pro-inflammatory cytokines in circulation. Then, we found that wogonin effectively alleviated liver injury via potentiation of the anti-oxidative capacity. To be specific, wogonin activated Nrf2 thereby promoting expressions of anti-oxidative enzymes including NQO-1, GST, HO-1, SOD1 and SOD2 in hepatocytes. Moreover, wogonin-induced Nrf2 activation could suppress NF-κB-regulated up-regulation of pro-inflammatory cytokines. Ultimately, we provided in vivo evidence that wogonin activated Nrf2 signalling, potentiated anti-oxidative enzymes and inhibited NF-κB-regulated pro-inflammatory signalling. Taken together, this study demonstrates that wogonin can be the potential therapeutic agent for alleviating liver injury in sepsis by simultaneously ameliorating oxidative stress and inflammatory response through the activation of Nrf2.