玉米AGPase基因启动子的克隆及鉴定

以玉米基因组DNA为模板,通过PCR技术扩增得到了腺苷二磷酸葡萄糖焦磷酸化酶AGPase基因编码区上游1912 bp的启动子(AGPasep)序列.该序列包含TATA-box,CAAT-box等一些高等植物特有的启动子基本核心序列,推断其为一种新的启动子.为了验证该序列是否具有启动子功能,构建了含有此序列的植物表达载体pCAM-AGPasep,通过农杆菌介导法转化玉米愈伤组织进行瞬时表达.GUS染色结果表明,该序列可以驱动GUS基因的表达,具有启动子功能.     

Tbx18 regulates development of the epicardium and coronary vessels

The epicardium and coronary vessels originate from progenitor cells in the proepicardium. Here we show that Tbx18, a T-box family member highly expressed in the proepicardium, controls critical early steps in coronary development. In Tbx18^−/− mouse embryos, both the epicardium and coronary vessels exhibit structural and functional defects. At E12.5, the Tbx18-deficient epicardium contains protrusions and cyst-like structures overlying a disorganized coronary vascular plexus that contains ectopic structures resembling blood islands. At E13.5, the left and right coronary stems form correctly in mutant hearts. However, analysis of PECAM-1 whole mount immunostaining, distribution of SM22α^lacZ/+ activity, and analysis of coronary vascular casts suggest that defective vascular plexus remodeling produces a compromised arterial network at birth consisting of fewer distributing conduit arteries with smaller lumens and a reduced capacity to conduct blood flow. Gene expression profiles of Tbx18^−/− hearts at E12.5 reveal altered expression of 79 genes that are associated with development of the vascular system including sonic hedgehog signaling components patched and smoothened, VEGF-A, angiopoietin-1, endoglin, and Wnt factors compared to wild type hearts. Thus, formation of coronary vasculature is responsive to Tbx18-dependent gene targets in the epicardium, and a poorly structured network of coronary conduit vessels is formed in Tbx18 null hearts due to defects in epicardial cell signaling and fate during heart development. Lastly, we demonstrate that Tbx18 possesses a SRF/CArG box dependent repressor activity capable of inhibiting progenitor cell differentiation into smooth muscle cells, suggesting a potential function of Tbx18 in maintaining the progenitor status of epicardial-derived cells.     

Entropy-driven mechanism of an E3 ligase

Ubiquitin-like modifications are macromolecular chemistry for which our understanding of the enzymatic mechanisms is lacking. Most E3 ligases in ubiquitin-like modifications do not directly participate in chemistry but are thought to confer allosteric effects; however, the nature of the allosteric effects has been elusive. Recent molecular dynamics simulations suggested that an E3 binding enhances the population of the conformational states of the E2·SUMO thioester that favor reactions. In this study, we conducted the first temperature-dependent enzyme kinetic analysis to investigate the role of an E3 on activation entropy and enthalpy. The small ubiquitin-like modifier (SUMO) E3, RanBP2, confers unusually large, favorable activation entropy to lower the activation energy of the reaction. Mutants of RanBP2, designed to alter the flexibilities of the E2·SUMO thioester, showed a direct correlation of their favorable entropic effects with their ability to restrict the conformational flexibility of the E2·SUMO thioester. While the more favorable activation entropy is consistent with the previously suggested role of E3 in conformational selection, the large positive entropy suggests a significant role of solvent in catalysis. Indeed, molecular dynamics simulations in explicit water revealed that the more stable E2·SUMO thioester upon E3 binding results in stabilization of a large number of bound water molecules. Liberating such structured water at the transition state can result in large favorable activation entropy but unfavorable activation enthalpy. The entropy-driven mechanism of the E3 is consistent with the lack of structural conservation among E3s despite their similar functions. This study also illustrates how proteins that bind both SUMO and E2 can function as E3s and how intrinsically unstructured proteins can enhance macromolecular chemistry in addition to their known advantages in protein--protein interactions.     

Effects of culture conditions on osteogenic differentiation in human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hBMMSCs) must differentiate into osteogenic cells to allow for successful bone regeneration. In this study, we investigated the effects of different combinations of three soluble osteogenic differentiation-inducing factors [L-ascorbic acid (AC), beta-glycerophosphate (betaG), and bone morphogenic protein-2 (BMP-2)] and the presence of a hydroxyapatite (HA) substrate on hBMMSC osteogenic differentiation in vitro. hBMMSCs were cultured in medium containing various combinations of the soluble factors on culture plates with or without HA coating. After 7 days of culture, alkaline phosphatase (ALP) activity, calcium deposition, and osteoprotegerin (OPG) and osteopontin (OPN) expression were measured. The effects of individual and combined factors were evaluated using a factorial analysis method. BMP-2 predominantly affected expression of early markers of osteogenic differentiation (ALP and OPG). HA had the highest positive effect on OPN expression and calcium deposition. The interaction between AC, betaG, and HA had the second highest positive effect on ALP activity.     

Association of mitochondrial haplogroup D and risk of esophageal cancer in Taihang Mountain and Chaoshan areas in China

Both the Taihang Mountain area in north-central China and Chaoshan area in the southeastern littoral of China are areas with high risk of esophageal cancer (EC). Our previous study confirmed that populations from the two areas might share similar matrilineal backgrounds and found that mitochondrial DNA (mtDNA) haplogroup D, especially subhaplogroups D4a and D5a, might be genetic background markers of EC in Chaoshan area. Here, to further determine whether D4a, D5a, and D might be susceptibility markers for EC in the two high-risk areas, we performed a case–control study with larger samples and analyzed the distributions of these three haplogroups in subjects (controls [n = 898] and patients [n = 768]) from the two areas. D4a haplogroup was significantly associated with increased risk of EC in Taihang Mountain subjects, especially women. D5 haplogroup was associated with EC at the general population level in the Taihang Mountain area and in subjects ≤ 60 years, especially women ≤ 60 years, in the Chaoshan area. D haplogroup was associated with EC only in subjects ≤ 60 years, especially men ≤ 60 years, in the Chaoshan area. D4a and D5 showing positive association with EC in the Taihang Mountain area became the predominant subhaplogroups of D in Chaoshan controls. In conclusion, D, D4a, and D5 haplogroups might be susceptibility markers for EC in the two high-risk areas in China, particularly D4a and D5 for the Taihang Mountain area and D and D5 for the Chaoshan area.     

家蚕多聚泛素基因的电子克隆、序列分析及表达谱预测

具选择性蛋白质降解功能的泛素在昆虫生长发育过程中起着重要的调控作用。本研究采用电子克隆的方法钓取家蚕基因组中多聚泛素基因序列,命名为Bm-polyUB(GenBank登录号:AADK01019318),并进行序列分析和电子表达谱预测。序列分析表明,该编码区长3426bp,编码1141个氨基酸残基的15聚体,预测分子量和等电点分别为127.97kD和7.33,二级结构中α-螺旋、延伸带、β-转角和无规则卷曲各占17.09%、32.78%、14.37%和35.76%,亚细胞定位于细胞质、细胞核和线粒体各占43.5%、34.8%和21.7%,无前导肽、信号肽和跨膜区;多重序列比对显示,15个泛素单体基因序列间同源性和遗传距离分别介于89.0%～100.0%和0.000～0.120,其中Bm-UB3与Bm-UB10、Bm-UB5与Bm-UB7及Bm-UB10与Bm-UB14的序列相同;遗传多样性分析可见,共检出33个多态性位点,共生成12个单倍型,单倍型多样性(Hd=0.962)、平均核苷酸差异数(K=7.495)、核苷酸多样性(Pi=0.03287)、密码子有效值(ENC=41.62361)、偏爱指标(CBI=0.4960)和χ2检验计算(χ2=0.713)显示泛素单体间遗传多样性较丰富且呈现较强的密码子偏爱性;电子表达谱预测结果表明Bm-polyUB基因在家蚕中高表达的组织和发育阶段所占比例显著高于低表达。本文的研究结果可为进一步开展该基因进化机制、表达特性和生理功能等方面的实验研究提供基础资料。     

Circular RNA circDVL1 inhibits clear cell renal cell carcinoma progression through the miR-412-3p/PCDH7 axis

Clear cell renal cell carcinoma (ccRCC) is a primary kidney cancer with high aggressive phenotype and extremely poor prognosis. Accumulating evidence suggests that circular RNAs (circRNAs) play pivotal roles in the occurrence and development of various human cancers. However, the expression, clinical significance and regulatory role of circRNAs in ccRCC remain largely unclear. Here we report that circDVL1 to be reduced in the serums and tissues from ccRCC patients, and to negatively correlate with ccRCC malignant features. Overexpression of circDVL1 inhibits proliferation, induces G1/S arrest, triggers apoptosis, and reduces migration and invasion in different ccRCC cells in vitro. Correspondingly, circDVL1 overexpression suppresses ccRCC tumorigenicity in a mouse xenograft model. Mechanistically, circDVL1 serves as a sponge for oncogenic miR-412-3p, thereby preventing miR-412-3p-mediated repression of its target protocadherin 7 (PCDH7) in ccRCC cells. Collectively, our results demonstrate that circDVL1 exerts tumor-suppressive function during ccRCC progression through circDVL1/miR-412-3p/PCDH7 axis, and suggest that circDVL1 could be a novel diagnostic and prognositc marker and therapeutic target for ccRCC.     

A role for Rho kinase in vascular contraction evoked by sodium fluoride

Agonist and depolarization-induced vascular smooth muscle contractions involve the activation of Rho-kinase pathway. However, there are no reports addressing the question whether this pathway is involved in NaF-induced vascular contractions. We hypothesized that Rho-kinase plays a role in vascular contraction evoked by sodium fluoride in rat aortae. In both physiological salt solution and calcium-free solution with 2 mM EGTA, cumulative addition of NaF increased vascular tension in concentration-dependent manners. Effects of Rho-kinase inhibitor (Y27632) on phosphorylation of myosin light chain (MLC20) and myosin targeting subunit (MYPT1(Thr696)) of myosin light chain phosphatase as well as NaF-induced contractions were determined using isolated tissue and the Western blot experiments. Y27632 inhibited NaF-induced contractions in a concentration-dependent manner. NaF increased phosphorylation of MLC20 and MYPT1(Thr696), which were also inhibited by Y27632. However, MLCK inhibitor (ML-7) or PKC inhibitor (Ro31-8220) did not inhibit the NaF-induced contraction. These results indicate that activation of Rho-kinase and the subsequent phosphorylation of MYPT1(Thr696) play important roles in NaF-induced contraction of rat aortae.