Characterization of humoral responses in mice immunized with plasmid DNAs encoding SARS-CoV spike gene fragments

The immunological characteristics of SARS-CoV spike protein were investigated by administering mice with plasmids encoding various S gene fragments. We showed that the secreting forms of S1, S2 subunits and the N-terminus of S1 subunit (residues 18-495) were capable of eliciting SARS-CoV specific antibodies and the region immediate to N-terminus of matured S1 protein contained an important immunogenic determinant for elicitation of SARS-CoV specific antibodies. In addition, mice immunized with plasmids encoding S1 fragment developed a Th1-mediated antibody isotype switching. Another interesting finding was that mouse antibodies elicited separately by plasmids encoding S1 and S2 subunits cooperatively neutralized SARS-CoV but neither the S1 nor S2 specific antibodies did, suggesting the possible role of both S1 and S2 subunits in host cell docking and entry. These results provide insights into understanding the immunological characteristics of spike protein and the development of subunit vaccines against SARS-CoV.     

鸡减蛋综合征病毒（EDSV—76）末端前体蛋白的基因结构分析

从中国发病鸡群中分离的鸡减蛋综合征病毒弱毒株ＡＡ－２,经常规方法提取其病毒核酸后,组建了完整的限制性内切酶ＰｓｔＩ及ＨｉｎｇⅢ水解片段的基因文库,并对其中ＨｉｎｄⅢ,－ＳａｃⅠ进行了序列测定。同源比较分析证明：其Ｌ链含编码病毒末端前体蛋白,容量为５８０个氨基酸残基的开放读码框架。     

Efficient mucosal vaccination mediated by the neonatal Fc receptor

Almost all infectious diseases are initiated at mucosal surfaces, yet intramuscular or subcutaneous vaccination usually provides only minimal protection at sites of infection owing to suboptimal activation of the mucosal immune system. The neonatal Fc receptor (FcRn) mediates the transport of IgG across polarized epithelial cells lining mucosal surfaces. We mimicked this process by fusing a model antigen, herpes simplex virus type-2 (HSV-2) glycoprotein gD, to an IgG Fc fragment. Intranasal immunization, together with the adjuvant CpG, completely protected wild-type, but not FcRn knockout, mice after intravaginal challenge with virulent HSV-2 186. This immunization strategy induced efficient mucosal and systemic antibody, B- and T-cell immune responses, with stable protection for at least 6 months after vaccination in most of the immunized animals. The FcRn-IgG transcellular transport pathway may provide a general delivery route for subunit vaccines against many mucosal pathogens.     

Design and comparison of gene-pyramiding schemes in animals

Marker-assisted gene pyramiding provides a promising way to develop new animal breeds or lines, in which genes responsible for certain favorable characters identified in different breeds or lines are incorporated. In consideration of features of animal populations, we proposed five schemes for pyramiding three genes, denoted Scheme A-E, and five schemes for pyramiding four genes, denoted Scheme F-J. These schemes are representative of the possible alternatives. We also provided an algorithm to compute the population sizes needed in each generation. We compared these schemes with respect to the total population size and the number of generations required under different situations. The results show that there is no scheme that is optimal in all cases. Among the schemes for pyramiding three genes from three lines (L1, L2 and L3), Scheme D (a three-way cross between the three lines are first performed, followed by a backcross to L1 and a subsequent intercross to obtain the desired genotype) has a significant advantage over the other schemes when the recombination rate between adjacent genes ranges from 0.1 to 0.4, while Scheme A (a two-way cross between L1 and L2 and a subsequent intercross are performed, followed by a cross with L3 and a subsequent intercross to obtain the desired genotype) is optimal when recombination rate is 0.5. Among schemes for pyramiding four genes from four lines (L1, L2, L3 and L4), Scheme I (seperately, a two-way cross between L1 and L2 (L3 and L4) followed by a backcross to L1 (L3) and a subsequent intercross are performed, then the offspring from the two sides are crossed and followed by a backcross to L1 and a subsequent intercross to obtain the desired genotype) is optimal when the recombination rate ranges from 0.1 to 0.4, while Scheme F (cross and subsequent intercross between the four lines are performed successively) is the optimal when the recombination rate is 0.5. We also disscuss how the animals' reproductive capacity, the probabilities of obtaining the desired genotypes and genetic distance between adjacent genes would affect the design of an optimal scheme.     

The tkNeo gene, but not the pgkPuro gene, can influence the ability of the beta-globin LCR to enhance and confer position-independent expression onto the beta-globin gene

Whether drug-selectable genes can influence expression of the beta-globin gene linked to its LCR was assessed here. With the tkNeo gene placed in cis and used to select transfected cells, the beta-globin gene was expressed fourfold lower when it was positioned upstream of the LCR rather than downstream. This difference did not occur when the pgkPuro gene replaced tkNeo. Moreover, the beta-globin gene situated upstream of the LCR was transcribed without position effects when it was cotransfected with a pgkPuro-containing plasmid, whereas cotransfection with a tkNeo plasmid gave measurable position effects. Previous results from transfected cells selected via a linked tkNeo gene suggested that the 3' end of the beta-globin gene has no impact on LCR-enhanced expression. Here, removal of the 3' end of the beta-globin gene resulted in lower and much more variable expression in both transgenic mice and cells cotransfected with pgkPuro. Together, the results suggest that tkNeo, but not pgkPuro, can strongly influence expression of the beta-globin gene linked to its LCR. The findings could partly explain why data on beta-globin gene regulation obtained from transfected cells have often not agreed with those obtained using transgenic mice. Hence, one must be careful in choosing a drug-selectable gene for cell transfection studies.     

Apolipoprotein A-II/A-I ratio is a key determinant in vivo of HDL concentration and formation of pre-beta HDL containing apolipoprotein A-II

Overexpression of human apolipoprotein A-II (apo A-II) in mice induced postprandial hypertriglyceridemia and marked reduction in plasma HDL concentration and particle size [Boisfer et al. (1999) J. Biol. Chem. 274, 11564-11572]. We presently compared lipoprotein metabolism in three transgenic lines displaying plasma concentrations of human apo A-II ranging from normal to 4 times higher, under ad libitum feeding and after an overnight fast. Fasting dramatically decreased VLDL and lowered circulating human apo A-II in transgenic mice; conversely, plasma HDL levels increased in all genotypes. The apo A-I content of HDL was inversely related to the expression of human apo A-II, probably reflecting displacement of apo A-I by an excess of apo A-II. Thus, the molar ratios of apo A-II/A-I in HDL were significantly higher in fed as compared with fasted animals of the same transgenic line, while endogenous LCAT activity concomitantly decreased. The number and size of HDL particles decreased in direct proportion to the level of human apo A-II expression. Apo A-II was abundantly present in all HDL particles, in contrast to apo A-I mainly present in large ones. Two novel findings were the presence of pre-beta migrating HDL transporting only human apo A-II in the higher-expressing mice and the increase of plasma HDL concentrations by fasting in control and transgenic mice. These findings highlight the reciprocal modifications of VLDL and HDL induced by the feeding-fasting transition and the key role of the molar ratio of apo A-II/A-I as a determinant of HDL particle metabolism and pre-beta HDL formation.     

Stress-induced RNASET2 overexpression mediates melanocyte apoptosis via the TRAF2 pathway in vitro

The recent genome-wide association study identified a link between vitiligo and genetic variants in the ribonuclease T2 (RNASET2) gene; however, the functional roles of RNASET2 in vitiligo pathogenesis or in melanocyte apoptosis have yet to be determined. The current study was designed to investigate the vitiligo-related expression pattern of RNASET2 and its molecular function involving apoptosis-related signaling proteins and pathways. The results showed overexpression of RNASET2 in epidermis specimens from 40 vitiligo patients compared with that from matched healthy controls. In addition, in vitro analyses indicated that overexpression of RNASET2 was inducible in cultured primary human melanocytes and keratinocytes by stress conditions, that is, exposure to UV irradiation, hydrogen peroxide, and inflammatory factors, respectively, and led to increased cell apoptosis via the tumor necrosis factor receptor-associated factor 2 (TRAF2)–caspases pathway through the physical interaction of RNASET2 with TRAF2. Thus, RNASET2 may contribute to vitiligo pathogenesis by inhibiting TRAF2 expression and, as such, RNASET2 may represent a potential therapeutic target of vitiligo.     

Soil respiration in Tibetan alpine grasslands: belowground biomass and soil moisture, but not soil temperature, best explain the large-scale patterns

The Tibetan Plateau is an essential area to study the potential feedback effects of soils to climate change due to the rapid rise in its air temperature in the past several decades and the large amounts of soil organic carbon (SOC) stocks, particularly in the permafrost. Yet it is one of the most under-investigated regions in soil respiration (Rs) studies. Here, Rs rates were measured at 42 sites in alpine grasslands (including alpine steppes and meadows) along a transect across the Tibetan Plateau during the peak growing season of 2006 and 2007 in order to test whether: (1) belowground biomass (BGB) is most closely related to spatial variation in Rs due to high root biomass density, and (2) soil temperature significantly influences spatial pattern of Rs owing to metabolic limitation from the low temperature in cold, high-altitude ecosystems. The average daily mean Rs of the alpine grasslands at peak growing season was 3.92 μmol CO(2) m(-2) s(-1), ranging from 0.39 to 12.88 μmol CO(2) m(-2) s(-1), with average daily mean Rs of 2.01 and 5.49 μmol CO(2) m(-2) s(-1) for steppes and meadows, respectively. By regression tree analysis, BGB, aboveground biomass (AGB), SOC, soil moisture (SM), and vegetation type were selected out of 15 variables examined, as the factors influencing large-scale variation in Rs. With a structural equation modelling approach, we found only BGB and SM had direct effects on Rs, while other factors indirectly affecting Rs through BGB or SM. Most (80%) of the variation in Rs could be attributed to the difference in BGB among sites. BGB and SM together accounted for the majority (82%) of spatial patterns of Rs. Our results only support the first hypothesis, suggesting that models incorporating BGB and SM can improve Rs estimation at regional scale.