HIV-1 TAT-mediated protein transduction and subcellular localization using novel expression vectors

Several novel prokaryotic and eukaryotic expression vectors were constructed for protein transduction and subcellular localization. These vectors employed an N-terminal stretch of 11 basic amino acid residues (47-57) from the human immunodeficiency virus type 1 (HIV-1) TAT protein transduction domain (PTD) for protein translocation and cellular localization. The vectors also contained a six-histidine (His(6)) tag at the N- or C-terminus for convenient purification and detection, and a multiple cloning site for easy insertion of foreign genes. Some heterologous genes including HSV-TK, Bcl-rambo, Smac/DIABLO and GFP were fused in-frame to TAT PTD and successfully overexpressed in Escherichia coli. The purified TAT-GFP fusion protein was able to transduce into the mammalian cells and was found to locate mainly in the cytosol when exogenously added to the cell culture medium. However, using a transfection system, mammalian-expressed TAT-GFP predominantly displayed a nuclear localization and nucleolar accumulation in mammalian cell lines. This discrepancy implies that the exact subcellular localization of transduced protein may depend on cell type, the nature of imported proteins and delivery approach. Taken together, our results demonstrate that a TAT PTD length of 11 amino acids was sufficient to confer protein internalization and its subsequent cellular localization. These novel properties allow these vectors to be useful for studying protein transduction and nuclear import.     

Carbon accumulation and distribution in Pinus massoniana and Cunninghamia lanceolata mixed forest ecosystem in Daqingshan, Guangxi, China

Carbon accumulation and distribution were studied at three sampling plots in a 13-year-old mixed planatation of Pinus massoniana and Cunninghamia lanceolata in Daqingshan, Guangxi, China. The results showed that carbon content varied with tissues and tree species, but the total carbon content of Pinus massoniana was higher than that of Cunninghamia lanceolata. The average tissue carbon contents of Pinus massoniana were: wood (58.6%) > root (56.3%) > branch (51.2%) > bark (49.8%) > leaf (46.8%), while those of Cunninghamia lanceolata were: bark (52.2%) > leaf (51.8%) > wood (50.2%) > root (47.5%) > branch (46.7%). The carbon contents of the soil (at a depth of 60cm) ranged from 1.45% to 1.84% with an average of 1.70%. Carbon contents were higher in the surface soil (0–20cm) than in the deep layer (below 20cm). The average carbon contents were the highest for trees (51.1%), followed by litter (48.3%), shrubs (44.1%), and herbs (33.0%). The biomass of the trees in the three plots ranged from 85.35 t hm^-2 to 101.35 t hm^-2 with an average of 93.83 t hm^-2, in which 75.7%–82.6% was Pinus massoniana. The biomass of the understory was 2.10–3.95 t hm^-2 with an average of 2.72 t hm^-2, while the standing stock of ground litter was 5.49–7.91 t hm^-2 with an average of 6.75 t hm^-2. The carbon storage in the mixed plantation reached the maximum in the soil layer (69.02%), followed by vegetation (29.03%), and standing litter (1.82%). The carbon storage in the tree layer occupied 23.90% of the total ecosystem and 97.7% of the vegetation layer. Pinus massoniana accounted for 65.39% of the total carbon storage in the tree layer. Tissue carbon storage was directly related to the corresponding amount of biomass. Trunks had the highest carbon storage, accounting for 53.23% of the trees in Pinus massoniana and 55.57% in Cunninghamia lanceolata, respectively. Roots accounted for about 19.22% of the total tree carbon. The annual net productivity of the mixed plantation was 11.46 t hm^-2a^-1, and that of sequestered carbon was 5.96 t hm^-2a^-1, which was equivalent to fixing CO₂ of 21.88 t hm^-2a^-1. The plantation was found to be an important sink of atmospheric CO₂.     

Pan‐Cancer analyses of Necroptosis‐Related genes as a potential target to predict immunotherapeutic outcome

Necroptosis is a unique programmed death mechanism of necrotic cells. However, its role and specific mechanism in cancer remain unclear, and a systematic pan‐cancer analysis of necroptosis is yet to be conducted. Thus, we performed a specific pan‐cancer analysis using The Cancer Genome Atlas and Genotype‐Tissue Expression databases to analyse necroptosis expression in terms of cancer prognosis, DNA methylation status, tumour mutative burden, microsatellite instability, immune cell infiltration in different types of cancer and molecular mechanisms. For the first time, we explored the correlation between necroptosis and immunotherapy prognosis. Thus, our study provides a relatively comprehensive understanding of the carcinogenicity of necroptosis in different types of cancer. It is suggested that necroptosis can be used to evaluate the sensitivity of different patients to immunotherapy and may become a potential target for tumour immunotherapy.     

Preparation and characterization of a soluble nitrate reductase from Azotobacter chroococcum

Summary The assimilatory nitrate reductase of the N₂-fixing bacterium Azotobacter chroococcum has been prepared in a soluble form from cells grown with nitrate as the nitrogen source, and some of its properties (electron donors and cofactors, K _mvalues for substrates, molecular weight, inhibitors, activators, etc.) have been studied. The enzyme is of an inducible nature and can exist in two interconvertible forms, either active or inactive.Tungstate very efficiently inhibits growth of the microorganism in media with nitrate. When either nitrite or ammonia are substituted for nitrate as the nitrogen source, growth is unaffected by tungstate concentrations which otherwise completely suppress growth on nitrate. Tungstate interferes by decreasing the cellular level of nitrate reductase activity, preventing, as a consequence, utilization of nitrate.     

Adenovirus structural protein IIIa is involved in the serotype specificity of viral DNA packaging

The packaging of the adenovirus (Ad) genome into a capsid displays serotype specificity. This specificity has been attributed to viral packaging proteins, the IVa2 protein and the L1-52/55K protein. We previously found that the Ad17 L1-52/55K protein was not able to complement the growth of an Ad5 L1-52/55K mutant virus, whereas two other Ad17 packaging proteins, IVa2 and L4-22K, could complement the growth of Ad5 viruses with mutations in the respective genes. In this report, we investigated why the Ad17 L1-52/55K protein was not able to complement the Ad5 L1-52/55K mutant virus. We demonstrate that the Ad17 L1-52/55K protein binds to the Ad5 IVa2 protein in vitro and the Ad5 packaging domain in vivo, activities previously associated with packaging function. The Ad17 L1-52/55K protein also associates with empty Ad5 capsids. Interestingly, we find that the Ad17 L1-52/55K protein is able to complement the growth of an Ad5 L1-52/55K mutant virus in conjunction with the Ad17 structural protein IIIa. The same result was found with the L1-52/55K and IIIa proteins of several other Ad serotypes, including Ad3 and Ad4. The Ad17 IIIa protein associates with empty Ad5 capsids. Consistent with the complementation results, we find that the IIIa protein interacts with the L1-52/55K protein in vitro and associates with the viral packaging domain in vivo. These results underscore the complex nature of virus assembly and genome encapsidation and provide a new model for how the viral genome may tether to the empty capsid during the encapsidation process.     

Comparative genomic analysis of the pPT23A plasmid family of Pseudomonas syringae

Members of the pPT23A plasmid family of Pseudomonas syringae play an important role in the interaction of this bacterial pathogen with host plants. Complete sequence analysis of several pPT23A family plasmids (PFPs) has provided a glimpse of the gene content and virulence function of these plasmids. We constructed a macroarray containing 161 genes to estimate and compare the gene contents of 23 newly analyzed and eight known PFPs from 12 pathovars of P. syringae, which belong to four genomospecies. Hybridization results revealed that PFPs could be distinguished by the type IV secretion system (T4SS) encoded and separated into four groups. Twelve PFPs along with pPSR1 from P. syringae pv. syringae, pPh1448B from P. syringae pv. phaseolicola, and pPMA4326A from P. syringae pv. maculicola encoded a type IVA T4SS (VirB-VirD4 conjugative system), whereas 10 PFPs along with pDC3000A and pDC3000B from P. syringae pv. tomato encoded a type IVB T4SS (tra system). Two plasmids encoded both T4SSs, whereas six other plasmids carried none or only a few genes of either the type IVA or type IVB secretion system. Most PFPs hybridized to more than one putative type III secretion system effector gene and to a variety of additional genes encoding known P. syringae virulence factors. The overall gene contents of individual PFPs were more similar among plasmids within each of the four groups based on T4SS genes; however, a number of genes, encoding plasmid-specific functions or hypothetical proteins, were shared among plasmids from different T4SS groups. The only gene shared by all PFPs in this study was the repA gene, which encoded sequences with 87 to 99% amino acid identityamong 25 sequences examined. We proposed a model to illustrate the evolution and gene acquisition of the pPT23A plasmid family. To our knowledge, this is the first such attempt to conduct a global genetic analysis of this important plasmid family.     

Volatile organic compound fate in phytoremediation applications: natural and engineered systems

Unique sampling techniques have generated a new understanding regarding the fate of volatile organic compounds (VOCs) in phytoremediation systems. Tissue sampling and diffusion traps were used to determine how VOCs are transported in and diffuse from vegetation, particularly woody species. These techniques were then utilized to observe how plants interact with different contaminated media, showing transport of contaminants occurs from the vadose zone (vapor phase) as well as the saturated zone (aqueous phase). Data was gathered in laboratory studies, in native vegetation, and in engineered phytoremediation systems. The findings reveal that diffusion from the xylem tissues to the atmosphere is a major fate for VOCs in phytoremediation applications. Linking VOCs' fate with groundwater hydraulics, mass removal rates from contaminant plumes can be estimated. These techniques were also utilized to observe the impact of engineered plant/microbe systems, which utilize recombinant, root-colonizing organisms to selectively degrade compounds and subsequently alter the fate of VOCs and other organic compounds. The genetically enhanced rhizoremediation methods pose a novel approach that may allow for biodegradation of compounds that formerly were considered recalcitrant.     

时间序列修订对森林二氧化碳通量的影响

对长白山阔叶红松林2003年生长季的涡动相关实测时间序列进行了去倾修订与超声风速仪倾斜修订,并分析了不同修订方法对森林CO2通量计算值的影响．结果表明,基于未修订时间序列计算得到的森林CO2通量(Fcraw)被高估．线性与非线性去倾对Fcraw的修订量分别为1．6％、1．8％,两者差异很小．平面拟合坐标变换与流线坐标变换对Fcraw的修订量分别为3．7％、4．7％,两者差异较大．对线性去倾后的时间序列分别进行流线坐标变换与平面拟合坐标变换,二者对Fcraw的修订量分别为5．5％与4．6％．建议对时间序列进行线性去倾与平面拟合坐标变换综合修订．     

城市生态用地分类及其规划的一般原则

城市生态用地的规划布局是城市规划的难点之一,也是急待解决的问题.本文根据城市生态系统的特点,给出了城市生态用地定义,指出城市生态用地同时具有自然属性和社会属性,依据这个特点,将城市生态用地划分为服务型生态用地和功能型生态用地两大类型,并结合具体的城市生态规划对各种类型的生态用地规划进行了定性分析.