Disruption of the FEN-1/PCNA interaction results in DNA replication defects, pulmonary hypoplasia, pancytopenia, and newborn lethality in mice

The interaction between flap endonuclease 1 (FEN-1) and proliferation cell nuclear antigen (PCNA) is critical for faithful and efficient Okazaki fragment maturation. In a living cell, this interaction is probably important for PCNA to load FEN-1 to the replication fork, to coordinate the sequential functions of FEN-1 and other enzymes, and to stimulate its enzyme activity. The FEN-1/PCNA interaction is mediated by the motif (337)QGRLDDFFK(345) of FEN-1, such that an F343AF344A (FFAA) mutant cannot bind to PCNA but retains its nuclease activities. To determine the physiological roles of the FEN-1/PCNA interaction in a mammalian system, we knocked the FFAA Fen1 mutation into the Fen1 gene locus of mice. FFAA/FFAA mouse embryo fibroblasts underwent DNA replication and division at a slower pace, and FFAA/FFAA mutant embryos displayed significant defects in growth and development, particularly in the lung and blood systems. All newborn FFAA mutant pups died at birth, likely due to pulmonary hypoplasia and pancytopenia. Collectively, our data demonstrate the importance of the FEN-1/PCNA complex in DNA replication and in the embryonic development of mice.     

一株纤维化纤维微细菌的生物学特性及其对几种苯环类化合物的利用研究

本文针对一株纤维化纤维微细菌Cellulosimicrobium cellulans Ha8菌株开展了生物学特性和苯环类物质代谢能力研究.该菌株革兰氏阳性,长杆状,培养后期逐渐变为短杆状;能固氮,水解蛋白质,液化明胶,利用淀粉、纤维素和果胶,分解几丁质;在pH 6.0～9.0和20℃～40℃范围内生长较好;能利用苯甲酸、苯酚、二甲苯、苯丙烯酸和二苯胺为唯一碳源进行生长,对这几种苯环类物质浓度的耐受范围分别为0 mmol/L～30 mmol/L、0 mmol/L～8 mmol/L、0 mmol/L～30 mmol/L、0 mmol/L～15 mmol/L和0 mmol/L～40 mmol/L,但不能利用2,4-二硝基苯酚、邻硝基酚、邻甲氧基酚、氨基苯磺酸、邻苯二酚和邻菲罗啉为唯一碳源生长.     

单级自养脱氮系统亚硝化菌株的分离、鉴定及定性

[目的]研究单级自养脱氮系统中亚硝化菌株的培养及代谢特征,为系统运行操控提出理论指导.[方法]从单级白养脱氮系统活性污泥中采集微生物样品,经过4次富集和分离过程,最终获得一株亚硝化能力较强的菌株Nl,通过显微镜观察及16S rDNA序列分析鉴定该菌株,研究摇瓶装量、pH、温度及底物浓度对其代谢过程的影响.[结果]该菌株与Nitrosomonas sp.NL7(AY958677)、Nitrosomonas AS1(EF016119)、Nitrosomonas sp.Is32(AJ621027)相似性分别为97%、96%和96%,对氧气需求量存在较严格要求,pH及温度对其氨氧化活性具有明显的影响,最适条件分别为pH8.0和30℃,在氨氮浓度80-800 mg/L较宽的底物浓度范围内具有活性,当氨氮浓度高达800 mg/L时,其氨氧化活性没有受到明显的抑制.[结论]该N1菌株为亚硝化单胞菌(Nitrosomonas sp.),与已有报道的其它亚硝化菌相比,N1对氨氮有较强降解能力及较广的浓度适应范围.     

Growing season ecosystem respirations and associated component fluxes in two alpine meadows on the Tibetan Plateau

From 30 June to 24 September in 2003 ecosystem respiration （Re） in two alpine meadows on the Tibetan Plateau were measured using static chamber- and gas chromatography- （GC） based techniques. Simultaneously, plant removal treatments were set to partition Re into plant autotrophic respiration （Ra） and microbial heterotrophic respiration （Rh）. Results indicated that Re had clear diurnal and seasonal variation patterns in both of the meadows. The seasonal variability of Re at both meadow sites was caused mainly by changes in Ra, rather than Rh. Moreover, atthe Kobresia humilis meadow site （K_site）, Ra and Rh accounted for 54% and 46% of Re, respectively. While at the Potentilla fruticosa scrub meadow （P_site）, the counterparts accounted for 61% and 39%, respectively. T test showed that there was significant difference in Re rates between the two meadows （t = 2.387, P = 0.022）. However, no significant difference was found in Rh rates, whereas a significant difference was observed in Ra rates between the two meadows. Thus, the difference in Re rate between the two meadows was mainly attributed to plant autotrophic respirations. During the growing season, the two meadows showed relatively low Q10 values, suggesting that Re, especially Rh was not sensitive to temperature variation in the growing season. Additionally, Re and Rh at the K_site, as well as Rh at the Psite was negatively correlated with soil moisture, indicating that soil moisture would also play an important role in respirations.     

Dynamic modeling for shear stress induced ATP release from vascular endothelial cells

A dynamic model is proposed for shear stress induced adenosine triphosphate (ATP) release from endothelial cells (ECs). The dynamic behavior of the ATP/ADP concentration at the endothelial surface by viscous shear flow is investigated through simulation studies based on the dynamic ATP release model. The numerical results demonstrate that the ATP/ADP concentration against time at endothelium-fluid interface predicted by the dynamic ATP release model is more consistent with the experimental observations than that predicted by previous static ATP release model.     

Treatment of brewery wastewater using anaerobic sequencing batch reactor (ASBR)

Brewery wastewater was treated in a pilot-scale anaerobic sequencing batch reactor (ASBR) in which a floating cover(@) was employed. Long time experiments showed that the reactor worked stably and effectively for COD removal and gas production. When the organic loading rate was controlled between 1.5 kg COD/m3 d and 5.0 kg COD/m(3)d, and hydraulic retention time one day, COD removal efficiency could reach more than 90%. Sludge granulation was achieved in the reactor in approximately 60 days, which is much less than the granulation time ever reported. In addition, high specific methanogenic activity (SMA) for formate was observed. The study suggests that the ASBR technology is a potential alternative for brewery wastewater treatment.     

Induction of oocyte-like cells from mouse embryonic stem cells by co-culture with ovarian granulosa cells

Abstract In vitro derivation of oocytes from embryonic stem (ES) cells has the potential to be an important tool for studying oogenesis as well as advancing the field of therapeutic cloning by providing an alternative source of oocytes. Here, we demonstrate a novel, two-step method for inducing mouse ES cells to differentiate into oocyte-like cells using mouse ovarian granulosa cells. First, primordial germ cells (PGCs) were differentiated within the embryonic body (EB) cells around day 4 as defined by the expression of PGC-specific markers and were distinguished from undifferentiated ES cells. Second, day 4 EB cells were co-cultured with ovarian granulosa cells. After 10 days, these cells formed germ cell colonies as indicated by the expression of the two germ cell markers Mvh and SCP3. These cells also expressed the oocyte-specific genes Fig α, GDF-9 , and ZP1-3 but not any testis-specific genes by RT-PCR analysis. EB cultured alone or cultured in granulosa cell-conditioned medium did not express any of these oocyte-specific markers. In addition, EB co-cultured with Chinese hamster ovary (CHO) cells or cultured in CHO cell-conditioned medium did not express all of these oocyte-specific markers. Immunocytochemistry analysis using Mvh and GDF-9 antibodies confirmed that some Mvh and GDF-9 double-positive oocyte-like cells were generated within the germ cell colonies. Our results demonstrate that granulosa cells were effective in inducing the differentiation of ES cell-derived PGCs into oocyte-like cells through direct cell-to-cell contacts. Our method offers a novel in vitro system for studying oogenesis; in particular, for studying the interactions between PGCs and granulosa cells.     

Effects of type I interferons on the adjuvant properties of plasmid granulocyte-macrophage colony-stimulating factor in vivo

While administration of granulocyte-macrophage colony-stimulating factor (GM-CSF) can induce the local recruitment of activated antigen-presenting cells at the site of vaccine inoculation, this cellular recruitment is associated with a paradoxical decrease in local vaccine antigen expression and vaccine-elicited CD8+ T-cell responses. To clarify why this cytokine administration does not potentiate immunization, we examined the recruited cells and expressed inflammatory mediators in muscles following intramuscular administration of plasmid GM-CSF in mice. While large numbers of dendritic cells and macrophages were attracted to the site of plasmid GM-CSF inoculation, high concentrations of type I interferons were also detected in the muscles. As type I interferons have been reported to damp foreign gene expression in vivo, we examined the possibility that these local innate mediators might decrease plasmid DNA expression and therefore the immunogenicity of plasmid DNA vaccines. In fact, we found that coadministration of an anti-beta interferon monoclonal antibody with the plasmid DNA immunogen and plasmid GM-CSF restored both the local antigen expression and the CD8+ T-cell immunogenicity of the vaccine. These data demonstrate that local innate immune responses can change the ability of vaccines to generate robust adaptive immunity.     

Efficient gene delivery into mammalian cells mediated by a recombinant baculovirus containing a whispovirus ie1 promoter, a novel shuttle promoter between insect cells and mammalian cells

Recombinant baculoviruses have emerged as a new gene delivery vehicle for mammalian cells. Thus, a shuttle promoter that mediates gene expression in both insect and mammalian cells will facilitate the development of a baculovirus vector-based mammalian cell gene delivery vehicle. This study described the generation of three recombinant baculoviruses with an EGFP reporter gene under the control of the white spot syndrome virus (WSSV) ie1 promoter, or either of two control promoters, the baculovirus early-to-late (ETL) promoter and polyhedrin promoter. The resulting recombinant baculoviruses were used to infect insect Sf9 cells and transduce several mammalian cell lines to test the expression of EGFP. We found that the WSSV ie1 promoter displayed a strong promoter activity in both insect and mammalian cells, and showed a stronger promoter activity than the ETL promoter in some mammalian cell lines. The activity of the WSSV ie1 promoter, but not the ETL promoter, can be enhanced by sodium butyrate, a histone deacetylase inhibitor. A transient plasmid transfection assay indicated that the WSSV ie1 promoter activity in mammalian cells is independent of baculovirus gene expression, differing from the ETL promoter, which was shown to be baculovirus-dependent. This study demonstrates, for the first time, that the WSSV ie1 promoter can function as a baculovirus-independent shuttle promoter between insect cells and mammalian cells. This novel shuttle promoter will facilitate the application of baculovirus-based vectors in gene expression, gene therapy, and non-replicative vector vaccines.