CpG island hypermethylation of multiple tumor suppressor genes associated with loss of their protein expression during rat lung carcinogenesis induced by 3-methylcholanthrene and diethylnitrosamine

The epigenetic mechanisms underlying the tumorigenesis caused by polycyclic aromatic hydrocarbons and nitrosamine compounds such as 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN) are currently unknown. We reported previously that dynamic changes in DNA methylation occurred during MCA/DEN-induced rat lung carcinogenesis. Here, we used the same animal model to further study the evolution of methylation alterations in tumor suppressor genes (TSGs) DAPK1, FHIT, RASSF1A, and SOCS-3. We found that none of these genes were methylated in either normal or hyperplasia tissue. However, as the severity of the cancer progressed through squamous metaplasia and dysplasia to carcinoma in situ (CIS) and infiltrating carcinoma, so methylation became more prevalent. Particularly dramatic increases in the level of methylation, the average number of methylated genes, and the incidence of concurrent methylation in three genes were observed in CIS and infiltrating carcinoma. Similar but less profound changes were seen in squamous metaplasia and dysplasia. Furthermore, methylation status was closely correlated to loss of protein expression for these genes, with protein levels markedly declining along the continuum of carcinogenesis. These results suggest that progressive CpG island hypermethylation leading to inactivation of TSGs might be a vital molecular mechanism in the pathogenesis of MCA/DEN-induced multistep rat lung carcinogenesis.     

Two novel bocaparvovirus species identified in wild Himalayan marmots

Bocaparvovirus (BOV) is a genetically diverse group of DNA viruses and a possible cause of respiratory, enteric, and neurological diseases in humans and animals. Here, two highly divergent BOVs (tentatively named as Himalayan marmot BOV, HMBOV1 and HMBOV2) were identified in the livers and feces of wild Himalayan marmots in China, by viral metagenomic analysis. Five of 300 liver samples from Himalayan marmots were positive for HMBOV1 and five of 99 fecal samples from these animals for HMBOV2. Their nearly complete genome sequences are 4,672 and 4,887 nucleotides long, respectively, with a standard genomic organization and containing protein-coding motifs typical for BOVs. Based on their NS1, NP1, and VP1, HMBOV1 and HMBOV2 are most closely related to porcine BOV SX/1-2 (approximately 77.0%/50.0%, 50.0%/53.0%, and 79.0%/54.0% amino acid identity, respectively). Phylogenetic analysis of these three proteins showed that HMBOV1 and HMBOV2 formed two distinctly independent branches in BOVs. According to these results, HMBOV1 and HMBOV2 are two different novel species in the Bocaparvovirus genus. Their identification expands our knowledge of the genetic diversity and evolution of BOVs. Further studies are needed to investigate their potential pathogenicity and their impact on Himalayan marmots and humans.     

新疆蛾类区系初步分析

对新疆蛾类标本鉴定和资料整理,共得到蛾类12科245属549种。其中种类最多的是夜蛾科Noctuidae136属364种,物种数量超过20种的分别有卷蛾科Tortricidae 24属36种,天蛾科Sphingidae 13属30种,灯蛾科Arctiidae 19属25种和木蠹蛾科Cossidae 8属25种;物种数超过10种的有尺蛾科Geometridae 13属18种,螟蛾科Pyralidae 14属17种和舟蛾科Notodontidae 8属17种;其余几个科种类均少于10种。在世界动物地理中,新疆蛾类以古北界为主,种数占到了434种(79.05%),其他动物地理区划组成类型仅115种(20.95%)。属级区系类型与种级基本一致,古北界162种,占总数的66.12%;古北界+东洋界共有58属,占23.67%,两种分布型占总数的89.79%。在中国动物地理中,新疆蛾类与蒙新区关系最为密切,共计245种(44.63%),其次是蒙新与华北共有56种(10.20%),蒙新与青藏共有52种(9.47%),其他动物区系组成较少。属级的区系类型以蒙新区为主,与同处古北界的东北区、华北区和青藏区关系最为密切。     

牛生长激素基因在马铃薯中的表达

将牛生长激素基因ｃＤＮＡ 与Ｐａｔａｔｉｎ ＣｌａｓｓＩ启动子及ＮＯＳ３终止子连接,构建了表达载体ｐＰＢＧＴ． 用直接法将表达载体转入农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍ ｅｆａｃｉｅｎｓ） ＬＢＡ４４０４（ｐＲＡＬ４４０４）菌株, 用此菌株转化马铃薯（Ｓｏｌａｎｕｍ ｔｕｂｅｒｏｓｕｍ ）得到再生植株． 经ＮＰＴⅡ活性检测,总ＤＮＡ ＰＣＲ和Ｓｏｕｔｈｅｒｎ ｂｌｏｔ证明目的基因已整合到马铃薯基因组中．ＲＮＡ 点杂交和Ｗｅｓｔｅｒｎ ｂｌｏｔ表明牛生长激素基因已在转基因马铃薯块茎中转录和表达     

Knockdown of OsHox33, a member of the class III homeodomain-leucine zipper gene family, accelerates leaf senescence in rice

The class III homeodomain-leucine zipper (HD-Zip III) gene family plays important roles in plant growth and development, including regulation of apical embryo patterning, embryonic shoot meristem formation, leaf polarity, vascular development, and meristem function, with a particularly crucial function in leaf development. Although HD-Zip III members are highly conserved in land plants, previous studies, such as genetic analyses based on multiple mutants in Arabidopsis and other plants, suggest that various HD-Zip III family genes have evolved with distinct functions and pleiotropic effects on plant growth and development. In this study, we analyzed a HD-Zip III member, OsHox33, and demonstrated that it plays an important role in age-dependent leaf senescence in rice. We constructed two specific RNAi vectors derived from the 5′-end region and 3′-UTR of OsHox33 to knockdown its expression. Transgenic plants harboring either RNAi construct displayed similar phenotypes of precocious leaf senescence symptoms, suggesting that knockdown of OsHox33 accelerates leaf senescence in rice. pOsHox33::GUS fusion expression and RT-PCR revealed that OsHox33 is highly expressed in young organs, especially in young meristems such as shoot apical meristems, intercalary meristems, and young callus. In addition, real-time PCR indicated that OsHox33 was more highly expressed in young leaves than in old leaves. To further investigate OsHox33 function, we analyzed chloroplast ultrastructure in different-aged leaves of RNAi plants, and found that OsHox33 knockdown accelerated chloroplast degradation, which is consistent with RNAi phenotypes. Finally, real-time PCR studies showed that OsHox33 can regulate the expression of GS1 and GS2, two senescence-associated genes. Taken together, the work presented here provides new insights into the function of HD-Zip III members in plants.     

Modified Mouse Embryonic Stem Cell based Assay for Quantifying Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/β-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/β-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.     

大肠杆菌苹果酸合酶A的酶学和生理功能研究

苹果酸合酶是乙醛酸循环的关键酶之一。E.coli中苹果酸合酶A(malate synthase A,MSA)由aceB基因编码。根据E.coli基因组序列设计引物,利用PCR技术扩增aceB基因,并将其克隆入pET-29b(+),构建了重组表达质粒pET-MSA。经IPTG诱导,MSA在E.coliRosetta(DE3)中获得高效表达。纯化的MSA蛋白的分子量大小约为60 kDa,最适反应pH值和最适温度分别是pH值8.0、30℃。纯化的蛋白质在Mg2+存在时才能发挥最大的活性,其对乙酰辅酶A的Km和Vmax分别是8.07μM和3.6μM/min。此外构建了MSA和苹果酸合酶G(MSG)基因敲除菌株MG::ΔaceB和MG::ΔaceBΔglcB。研究发现缺少MSA的E.coli突变菌株在乙酸中的生长速率要比野生型菌株慢很多,表明MSA对大肠杆菌在乙酸中的生长起着重要作用。MSG虽然能部分补偿MSA的作用,但是包含MSA的乙醛酸旁路是更有效的乙醛酸代谢途径。     

A novel basic fibroblast growth factor delivery system fabricated with heparin-incorporated fibrin–fibronectin matrices for repairing rat sciatic nerve disruptions

Autologous nerve grafts are widely used in bridging critical gaps of peripheral nerves, but they remain associated with high morbidity of the donor site and lack of full recovery. As an alternative, we have focused on chitosan nerve conduits filled with a heparin-incorporated fibrin–fibronectin matrix serving as delivery systems for basic fibroblast growth factor (bFGF). The artificial nerve conduits were used for repairing sciatic nerve defects of 10 mm in adult rats. Three months post-operation, the conduction velocity recovery index (CVRI) and the muscle restoration rate (MRR) in animals of the experimental group were 32 ± 4.1 and 77.4 ± 7.9%, respectively, which were significantly higher than those of the PBS control group (17.8 ± 1.9 and 66.7 ± 6.5%), and similar to those of the autograft group (38.4 ± 3.9 and 81.3 ± 7.8%). These results were also consistent with the densities of regenerated axons in the three groups, which were demonstrated by histomorphological analysis.