Mammalian Target of Rapamycin (mTor) Mediates Tau Protein Dyshomeostasis: IMPLICATION FOR ALZHEIMER DISEASE*

Previous evidence from post-mortem Alzheimer disease (AD) brains and drug (especially rapamycin)-oriented in vitro and in vivo models implicated an aberrant accumulation of the mammalian target of rapamycin (mTor) in tangle-bearing neurons in AD brains and its role in the formation of abnormally hyperphosphorylated tau. Compelling evidence indicated that the sequential molecular events such as the synthesis and phosphorylation of tau can be regulated through p70 S6 kinase, the well characterized immediate downstream target of mTor. In the present study, we further identified that the active form of mTor per se accumulates in tangle-bearing neurons, particularly those at early stages in AD brains. By using mass spectrometry and Western blotting, we identified three phosphoepitopes of tau directly phosphorylated by mTor. We have developed a variety of stable cell lines with genetic modification of mTor activity using SH-SY5Y neuroblastoma cells as background. In these cellular systems, we not only confirmed the tau phosphorylation sites found in vitro but also found that mTor mediates the synthesis and aggregation of tau, resulting in compromised microtubule stability. Changes of mTor activity cause fluctuation of the level of a battery of tau kinases such as protein kinase A, v-Akt murine thymoma viral oncogene homolog-1, glycogen synthase kinase 3β, cyclin-dependent kinase 5, and tau protein phosphatase 2A. These results implicate mTor in promoting an imbalance of tau homeostasis, a condition required for neurons to maintain physiological function.     

Microbiological Identification and Analysis of Swine Lungs Collected from Carcasses in Swine Farms, China

The primary objective of this 3 years study was to determine the prevalence of porcine pathogens of the lungs of swine in swine farms in southern China. A total of 5,420 samples were collected from 200 swine farms. The bacterium that was most commonly isolated was Streptococcus suis, with 10.24 % of the samples being positive, 114 lungs (2.1 %) were positive for pseudorabies virus and 263 (4.85 %) were positive for classical swine fever virus; much lower than positive for PRRSV (15.1 %, p = 0.023) and PCV2 (13.8 %, p = 0.038). lungs that were positive for PRRSV and/or PCV-2 have significantly increased odds of being positive for any of the S. suis (9.79 vs. 0.44 %, p = 0.003).     

PEGylated human catalase elicits potent therapeutic effects on H1N1 influenza-induced pneumonia in mice

Therapeutic recombinant human catalase (rhCAT) can quench infection-induced reactive oxygen species (ROS), thereby alleviating the associated tissue damage. Although the intranasal route is efficient to deliver native rhCAT to the lung, the therapeutic effect is limited by rapid elimination from the blood. In this study, we modified rhCAT with the active polymer, polyethylene glycol monomethyl ether (PEG)-5000, and analyzed the pharmacokinetics of PEGylated rhCAT in mice. The high tetra-PEGylation ratio was about 60 %, and PEGylation prolonged the half-life of rhCAT in serum (75 vs. 13.5 min for native rhCAT). The protective effects of PEG-rhCAT were investigated in a mouse model of influenza virus A (H1N1)-associated pneumonia. PEG-rhCAT was more effectively delivered than native rhCAT and was associated with higher survival ratio, less extensive lung injuries, reduced ROS levels, and lower viral replication. Collectively, these findings indicate that PEGylation can enhance the therapeutic efficacy of native rhCAT and suggest that PEGylated rhCAT may represent a novel complement therapy for H1N1 influenza-induced pneumonia.     

Structure-based design and synthesis of potent benzothiazole inhibitors of interleukin-2 inducible T cell kinase (ITK)

Inhibition of the non-receptor tyrosine kinase ITK, a component of the T-cell receptor signalling cascade, may represent a novel treatment for allergic asthma. Here we report the structure-based optimization of a series of benzothiazole amides that demonstrate sub-nanomolar inhibitory potency against ITK with good cellular activity and kinase selectivity. We also elucidate the binding mode of these inhibitors by solving the X-ray crystal structures of several inhibitor-ITK complexes.     

Phylogeography of Sophora davidii (Leguminosae) across the ‘Tanaka‐Kaiyong Line’, an important phytogeographic boundary in Southwest China

The ‘Tanaka‐Kaiyong Line’ (TKL) is a major phytogeographic boundary in Southwest China, separating East Asia's Sino‐Himalayan and Sino‐Japanese Floras. However, little is known about the importance of this boundary in promoting intraspecific phylogeographic subdivision and divergence. Using chloroplast (cpDNA) and nuclear‐intron (nDNA) sequence data, we reconstructed the population history of Sophora davidii, a drought‐tolerant riparian shrub widely distributed on either side of the TKL. Specifically, we aimed at testing two long‐standing explanations for possible vicariant events across the TKL: (i) Late Pliocene (c. 3 Ma) geological uplift of the eastern Qinghai‐Tibetan Plateau (QTP) or (ii) a sharp environmental gradient associated with the establishment of different monsoon regimes on either side of the TKL during the (Late) Pleistocene. Our genealogical analyses detected a major west–east split in cpDNA, geographically largely consistent with the TKL, and dated to c. 1.28 Ma (95% HPD: 0.21–2.96 Ma), hence postdating the latest phase of eastern QTP uplift. Furthermore, integrating cpDNA phylogeographic patterns with mismatch analyses, we found multiple refugial isolation and long‐term demographic stability of populations in the west (Hengduan Mountain Range) compared with extensive range expansions in the east, possibly during the last glacial period(s) and followed by differentiation into regional sublineages (southeast: Yunnan‐Guizhou Plateau vs. northeast: Qinling Mts./Loess Plateau). Although nuclear differentiation was less marked, the geographical pattern of nDNA haplotypes provided some further indication of the species' eastward expansion, possibly from source populations located just east of the TKL (lower Jinshajiang region). Overall, the present data reject the geological (tectonic) explanation for the TKL and, instead, provide supportive evidence for its role as a climatically driven barrier to present‐day plant dispersal. In addition, our study highlights changing temperatures and vegetation types during the last glacial period(s), along with aspects of regional topography, to be important determinants of the glacial eastward expansion of S. davidii. In consequence, our study lends support to a ‘glacial out‐of‐Hengduan Mts’. hypothesis for the xerophytic‐riparian flora of Southwest China, which in turn is inconsistent with the traditional view of the TKL as a ‘classical’ vicariant‐biogeographic boundary.     

Open-Channel Structures of the Human Glycine Receptor α1 Full-Length Transmembrane Domain

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Hexokinase from grape berries: its prokaryotic expression, polyclonal antibody preparation and biochemical property analyses

Two full-length hexokinase (HXK, EC 2.7.1.1) cDNAs, VvHXK1 with 1,413 bp and VvHXK2 with 1,458 bp were cloned from grape berries (Vitis vinifera L. Cabernet Sauvignon). VvHXK1 and VvHXK2 genes sequence from grape berries were deposited in GenBank under the accession number JN118544 and JN118545, respectively. The homology of the amino acid of VvHXK1 or VvHXK2 was very similar to ‘Pinot Noir’ grape HXK sequence, their similarties were 99.36 % and 98.97 %, respectively. More intuitive phylogenetic tree showed that the homology of amino acid sequence VvHXK1 with melon CmHXK1 was 86 %, and VvHXK2 homology with rice OsHXK3 was 83 %. The HXK proteins were successfully expressed in plasmid pET-30a (+) vectors in Escherichia coli BL21 (DE3) pLysS. The expressed proteins were purified using Ni-NTA agarose column and used to produce HXK1 antibody and conducted HXK protein blotting analysis. The results,suggested that one polypeptide band of about 51 kDa HXK protein can be detected in grape berries, HXK protein level was the highest during early grape berry development, but the lowest from 50d to 60d during development. Biochemical analysis of two hexokinase isozymes indicated that glucose was the optimal substrate of HXK, The isoelectric points of the two isozymes were 5.8 and 5.6, respectively. And the optimum pH was about 8.0. These results provide a substantial basis for the further studies of functions of grape HXKs to manipulate sugar content of grape berries.     

傅家边丘陵山地滨梅根围AM真菌与土壤酶活性的关系

为揭示AM真菌对宿主滨梅（Prunus maritima）的作用特点及对根部土壤酶活性的影响,于2009年4月、7月和10月分别从江苏傅家边丘陵山地滨梅根围分0～10、10～20、20～30、30～40、40～50 cm 5个土层采集土壤样品,观察滨梅AM菌根结构,测定了AM真菌侵染率、孢子密度、土壤磷酸酶、脲酶活性及有效磷、碱解氮含量,着重分析了AM真菌与土壤酶活性之间的关系。结果表明,滨梅能与AM真菌形成良好的共生关系,共生体为泡囊-丛枝结构;AM真菌侵染率和孢子密度分别在7月份和10月份最高,均出现在0～20 cm土层,并随土层加深而下降;AM真菌侵染率与土壤酸性磷酸酶、中性磷酸酶、碱磷酸酶活性显著正相关,而与脲酶活性无相关性;AM真菌孢子密度与碱性磷酸酶、脲酶活性呈极显著正相关关系;孢子密度与土壤有效磷、土壤碱解氮含量显著正相关,但AM真菌侵染率仅与土壤有效磷含量显著正相关;孢子密度与菌根侵染率之间无相关性。可见,滨梅AM真菌侵染率与孢子密度有明显的时空分布并与土壤因子尤其是某些土壤酶活性密切相关,且AM菌根的形成是滨梅适应丘陵山地干旱贫瘠环境的有效对策之一。     

Construction of novel chimeric proteins through the truncation of SEC2 and Sak from Staphylococcus aureus

It is an usual clinical phenomenon that cancer patients are prone to thrombosis. Until now, there have been no efficient methods or appropriate drugs to prevent and cure tumor thrombus. Therefore, the construction of a bifunctional chimeric protein for the treatment of cancer, complicated with thrombosis, is of great significance. Utilizing the superantigenic activity of staphylococcal enterotoxin C2 (SEC2) and the thrombolytic activity of staphylokinase (Sak), Sak-linker-SEC2 and SEC2-linker-Sak were constructed which had good anti-tumor and thrombolytic activities at the same time. Due to the intrinsic emetic activity of SEC2 and high molecular weight (MW) of chimeric proteins (44?kDa), their clinical applications will be restricted. In this study, novel chimeric proteins including ΔSEC2–ΔSak and ΔSak–ΔSEC2 were constructed through the truncation of SEC2 and Sak without 9-Ala linker and His-tag. Compared with the former, both the truncated proteins preserved nearly the same anti-tumor and thrombolytic activities. In addition, their MWs were only 29?kDa and their immunoreactivities were slightly lower than that of Sak-linker-SEC2 and SEC2-linker-Sak, respectively. Therefore, the novel chimeric proteins possessed merits and characteristics, such as low MS, low immunogenicity, and difunctionality which the former had not. It will be of great interest if the above-mentioned proteins can be used to cure Trousseau syndrome in clinic.