Transgenic mice for cre-inducible overexpression of the Cul4A gene

The Cullin 4A(Cul4A) gene is important in cell survival, development, growth, and cell cycle control and is amplified in breast and hepatocellular cancers. Recently, we reported that Cul4A plays an oncogenic role in the pathogenesis of mesothelioma. An important strategy for studying Cul4A in different tissues is targeted overexpression of this gene in vivo. Studies of Cul4A in mice have been restricted to the loss-of-function studies using Cul4A knockout mice; gain-of-function studies of Cul4A using transgenic mice have not been reported. We, therefore, generated a gain-of-function transgenic mouse model that overexpresses Cul4A in a Cre-dependent manner. Before Cre recombination, these mice express LacZ during development in most adult tissues. After Cre-mediated excision of the LacZ reporter, the transfected Cul4A gene is expressed along with a C-terminal Myc-tag in different tissues. In this study, Cre-excision was induced in mouse lungs by inhalation of an adenovirus vector encoding Cre recombinase. This mouse model provides a valuable resource for investigating the significance of Cul4A overexpression in various tissues.     

Phenology shifts at start vs. end of growing season in temperate vegetation over the Northern Hemisphere for the period 1982–2008

Changes in vegetative growing seasons are dominant indicators of the dynamic response of ecosystems to climate change. Therefore, knowledge of growing seasons over the past decades is essential to predict ecosystem changes. In this study, the long‐term changes in the growing seasons of temperate vegetation over the Northern Hemisphere were examined by analyzing satellite‐measured normalized difference vegetation index and reanalysis temperature during 1982–2008. Results showed that the length of the growing season (LOS) increased over the analysis period; however, the role of changes at the start of the growing season (SOS) and at the end of the growing season (EOS) differed depending on the time period. On a hemispheric scale, SOS advanced by 5.2 days in the early period (1982–1999) but advanced by only 0.2 days in the later period (2000–2008). EOS was delayed by 4.3 days in the early period, and it was further delayed by another 2.3 days in the later period. The difference between SOS and EOS in the later period was due to less warming during the preseason (January–April) before SOS compared with the magnitude of warming in the preseason (June–September) before EOS. At a regional scale, delayed EOS in later periods was shown. In North America, EOS was delayed by 8.1 days in the early period and delayed by another 1.3 days in the later period. In Europe, the delayed EOS by 8.2 days was more significant than the advanced SOS by 3.2 days in the later period. However, in East Asia, the overall increase in LOS during the early period was weakened in the later period. Admitting regional heterogeneity, changes in hemispheric features suggest that the longer‐lasting vegetation growth in recent decades can be attributed to extended leaf senescence in autumn rather than earlier spring leaf‐out.     

The EGFP/hERG fusion protein alter the electrophysiological properties of hERG channels in HEK293 cells

EGFP (enhanced green fluorescent protein) tagged to either the N (amino)-terminus [EGFP/hERG (human ether-a-go-go-related gene)] or C (carboxyl)-terminus (hERG/EGFP) of hERG channel is used to study mutant channel protein trafficking for several years. However, it has been reported that the process can alter hERG channel properties. The aim of the study was to determine whether EGFP tagged to N-terminus of hERG channels would alter the cellular localizations and the electrophysiological properties of hERG channels compared with untagged hERG channels. The hERG channels tagged with or without EGFP were transiently expressed in HEK (human embryonic kidney) 293 cells using a lipofectamine method. HEK 293 cells expressing pCDNA3-hERG or pEGFP-hERG were double immunolabelled with anti-hERG and anti-calnexin (an ER marker protein) followed with FITC- and TRITC (tetramethylrhodamine β-isothiocyanate)-labelled secondary antibodies, respectively. Confocal laser scanning microscope was used to observe the cellular localization of EGFP-tagged hERG channels and untagged hERG channels. Patch-clamp technique was used to record whole cell currents. We found that the EGFP/hERG fusion protein and untagged hERG channels were both expressed not only on the cell surface membrane but also in the cytoplasm of HEK293 cells. The EGFP/hERG appeared to influence the hERG channel gating properties, including reduction of the peak tail current density, more rapid inactivation process, faster recovery from inactivation and faster deactivation kinetics compared with untagged hERG channels. Our results suggest that the EGFP/hERG channel alter the electrophysiological properties of hERG channel, but it does not seem to alter the cellular location of hERG channels. Thus, EGFP tagging to N-terminus might be used for research of subcellular location of hERG channels but not for the channel electrophysiological properties.     

Maternal inheritance of plastids and mitochondria in Cycas L. (Cycadaceae)

Cycas is often considered a living fossil, thereby providing a unique model for revealing the evolution of spermatophytes. To date, the genetic inheritance of these archaic plants is not fully understood. The present study seeks to document the process of organelle inheritance in an interspecific cross of Cycas species. Extranuclear organelle DNA from chloroplasts and mitochondria was analyzed using both polymerase chain reaction-restriction fragment length polymorphism analysis and microscopy. Here, we show that the chloroplasts and mitochondria in the progeny of interspecific crosses between Cycas taitungensis and Cycas ferruginea were exclusively inherited from the female parent. Epifluorescence microscopic analyses of the pollen cells from Cycas elongata indicated that there was a significant degradation of organelle DNA in male reproductive cells following maturation; the DNA fluorescent signals were only seen after pollen mitosis two, but not detectable at mature stage. Lack of organelle DNA fluorescent signal in prothallial cells was confirmed by the absence of plastids and mitochondria in electronic microscopic images. In conclusion, these data suggest that the maternal plastid and mitochondrial inheritance in Cycas, native to the old world, are the same as seen in seed plants.     

Production of ��-poly-l-lysine using a novel two-stage pH control strategy by Streptomyces sp. M-Z18 from glycerol

The production of ε-poly-l-lysine (ε-PL) by Streptomyces sp. M-Z18 from glycerol was investigated in a 5-L jar-fermenter. Batch fermentations by Streptomyces sp. M-Z18 at various pH values ranging from 3.5 to 4.5 were studied. Based on the analysis of the time course of specific cell growth rate and specific ε-PL formation rate, a novel two-stage pH control strategy was developed to improve ε-PL production by shifting the culture pH from 3.5 to 3.8 after 36 h of cultivation. By applying the strategy, the maximal ε-PL concentration and productivity had a significant improvement and reached 9.13 g L⁻¹ and 4.76 g L⁻¹ day⁻¹, respectively, compared with those in one-stage pH control process where the pH value is controlled at 3.5 (7.83 g L⁻¹ and 3.13 g L⁻¹ day⁻¹). Fed-batch fermentation with two-stage pH control strategy was also applied to produce ε-PL; final ε-PL concentration of 30.11 g L⁻¹ was obtained, being 3.3-fold greater than that of batch fermentation. To our knowledge, it is the first report on production of ε-PL from glycerol in fermenter scale and achievement of high ε-PL production with two-stage pH control strategy.     

改良CCI大鼠痛行为学检测与损伤区自发放电活动再观察

目的：应用改良CCI模型研究外周神经损伤后痛觉过敏和自发放电各自特征及相互关系。方法：雄性SD大鼠,随机分为CCI组和Sham组,分别于术前1天和术后1、4、7、9、11、14天测定机械刺激缩足反射阈值和热缩腿反射潜伏期,同时选取术侧机械刺激缩腿反射阈值低于4g或者术侧和健侧热缩腿反射潜伏期差异大于2s的CCI模型大鼠观察术后4-14天损伤区自发放电活动。结果：神经纤维损伤后,机械痛敏和热痛敏随时间表现为逐渐增强,同时在损伤区观察到三类放电模式：整数倍放电﹑阵发放电和周期放电。结论：术后大鼠机械痛阈和热痛阈逐渐降低,机械痛敏的产生和损伤区自发放电活动关系密切,不同的放电模式可能代表不同的传入信息。     

中国特有植物血水草RAPD反应体系的优化

为建立血水草的RAPD-PCR最佳反应体系,对影响血水草RAPD反应的Mg²⁺、模板DNA、dNTPs、引物浓度和Taq聚合酶用量等因素进行优化。结果表明：25 μL反应体系中含10×Buffer 2.5 μL,1.8 mmol·L^-1 Mg²⁺,2U Taq DNA聚合酶,50 ng模板,0.2 mmol·L^-1 dNTPs,1.6 μmol·L^-1引物。扩增程序为：94℃预变性2 min;预扩增：94℃变性20 s,36℃退火30 s,72℃延伸75 s,5个循环;扩增：94℃变性20 s,40℃退火30 s,72℃延伸60 s,40个循环;72℃保温20 min,4℃保存。所建立的血水草RAPD-PCR反应体系具有标记位点清晰、反应系统稳定、检测多态性能力强、重复性好等特点,可以较好地应用于血水草的遗传多样性分析研究。     

Structure analysis of a new psychrophilic marine protease

A new psychrophilic marine protease was found from a marine bacterium Flavobacterium YS-80 in the Chinese Yellow Sea. The protease is about 49 kD with an isoelectric point about 4.5. It consists of 480 amino acids and is homologous to a psychrophilic alkaline protease (PAP) from an Antarctic Pseudomonas species. The protein was purified from the natural bacterium fermented and crystallized. Its crystal structure (PDB ID 3U1R) was solved at 2.0 Å by Molecular Replacement using a model based on PAP, and was refined to a crystallographic R_work of 0.16 and an R_free of 0.21. The marine protease consists of a two domain structure with an N-terminal domain including residues 37–264 and a C-terminal domain including residues 265–480. Similar to PAP, the N-terminal domain is responsible for proteolysis and the C-terminal is for stability. His186, His190, His196 and Tyr226 are ligands for the Zn²⁺ ion in the catalytic center. The enzyme''s Tyr226 is closer to the Zn²⁺ ion than in PAP and it shows a stronger Zn²⁺―Tyr-OH bond. There are eight calcium ions in the marine protease molecule and they have significantly shorter bond distances to their ligands compared to their counterparts in all three crystal forms of PAP. On the other hand, the loops in the marine protease are more compact than in PAP. This makes the total structure stable and less flexible, resulting in higher thermo stability. These properties are consistent with the respective environments of the proteases. The structural analysis of this new marine protease provides new information for the study of psychrophilic proteases and is helpful for elucidating the structure-environment adaptation of these enzymes.     

Characterization of family IV UDG from Aeropyrum pernix and its application in hot-start PCR by family B DNA polymerase

Recombinant uracil-DNA glycosylase (UDG) from Aeropyrum pernix (A. pernix) was expressed in E. coli. The biochemical characteristics of A. pernix UDG (ApeUDG) were studied using oligonucleotides carrying a deoxyuracil (dU) base. The optimal temperature range and pH value for dU removal by ApeUDG were 55-65°C and pH 9.0, respectively. The removal of dU was inhibited by the divalent ions of Zn, Cu, Co, Ni, and Mn, as well as a high concentration of NaCl. The opposite base in the complementary strand affected the dU removal by ApeUDG as follows: U/C≈U/G>U/T≈U/AP≈U/->U/U≈U/I>U/A. The phosphorothioate around dU strongly inhibited dU removal by ApeUDG. Based on the above biochemical characteristics and the conservation of amino acid residues, ApeUDG was determined to belong to the IV UDG family. ApeUDG increased the yield of PCR by Pfu DNA polymerase via the removal of dU in amplified DNA. Using the dU-carrying oligonucleotide as an inhibitor and ApeUDG as an activator of Pfu DNA polymerase, the yield of undesired DNA fragments, such as primer-dimer, was significantly decreased, and the yield of the PCR target fragment was increased. This strategy, which aims to amplify the target gene with high specificity and yield, can be applied to all family B DNA polymerases.