hPeriod1_(PAS)结构域相互作用蛋白的筛选和研究

目的寻找与近日节律系统核心基因Period1(Per1)相互作用的新蛋白,揭示近日节律相关的可能信号通路。方法采用酵母双杂交方法,以人Per1的PAS结构域为诱饵,扫描人下丘脑cDNA文库,并通过体外转录、免疫共沉淀验证蛋白间相互作用;通过RT-PCR检测RACK1(receptors for activated C-k inase)表达的组织特异性及节律性;运用RNA i技术初步研究RACK1与Per1的信号联系。结果人下丘脑区域RACK1蛋白与Per1存在相互作用;RACK1在多器官组织保守表达,但其表达未呈现明显节律性;上调及下调Per1表达,RACK1的RNA水平无显著变化。结论细胞内接头分子RACK1是一种新的Per1作用蛋白,可能介导、调控Per1的功能。

层粘连蛋白受体Ⅰ与hPeriod1相互作用的研究

目的寻找与节律蛋白hPeriod1(hPer1)相互作用的蛋白,并对筛选出的层粘连蛋白受体Ⅰ(Lamr1)在节律系统中的功能作初步研究。方法分别构建pGAD rec脑/cDNA文库和pGBKT7/hPer1bH LH-PAS重组诱饵蛋白质粒,酵母双杂交筛选下丘脑里与hPer1相互作用的蛋白。利用RT-PCR技术检测新蛋白在脑、肺、心、肝、肾组织中的表达及在SH-SY5Y细胞中的节律性质。通过RNA干扰技术研究新蛋白与hPer1的关系。结果通过酵母双杂交筛选,证明Lamr1能与hPer1蛋白发生相互作用。该蛋白在脑、肺、心、肝、肾组织广泛表达,灰度比较显示经马血清诱导后的SH-SY5Y细胞的hPer1表达呈现近日节律,而Lamr1表达无节律变化。RNA干扰显示hPer1表达下调不影响Lamr1的表达。结论Lamr1能与hPer1蛋白发生相互作用,但其表达在RNA水平无节律变化。Lamr1参与细胞的黏附、转移和侵袭。其前体蛋白作为核蛋白参与细胞蛋白的合成。Lamr1可能参与hPer1相关的生物节律系统的信号传出。

生物钟Period1基因rs2585405、rs2253820和rs2735611位点多态性与原发性失眠的相关研究

探讨中国人群生物钟Period1(Per1)基因rs2585405、rs2253820和rs2735611位点单核苷酸多态性(single-nucleotide?polymorphisms,SNP)与原发性失眠(primary insomnia,PI)的相关性。方法:采用病例对照研究,选取157例PI者为实验组,并匹配133名健康者为对照组。采用MassARRAY SNP 基因分型技术检测Per1基因rs2585405、rs2253820和rs2735611位点多态性。结果:①Per1基因rs2585405、rs2253820和rs2735611位点多态性在PI和对照组的基因型分布均符合Hardy-Weinberg平衡(P>0.05)。②PI组和对照组Per1基因3个SNP位点的基因型和等位基因频率比较,均没有显著性差异(P>0.05)。③PI组和对照组Per1基因3个SNP位点进行单倍型分析,显示构建的3种单倍型之间均没有显著差异(p>0.05)。结论:我们选择标记的Per1基因3个SNP位点可能不是中国人群PI的易感位点。

胃癌组织中节律基因Period1和Period2的表达变化及意义

目的观察节律基因Period1和Period2在胃癌组织中的表达变化,并探讨其临床意义。方法选择138例胃癌患者,术中留取胃癌及癌旁组织,采用免疫组化法检测胃癌及癌旁组织中的Period1和Period2,实时荧光定量PCR法检测胃癌及癌旁组织中的Period1和Period2 mRNA。结果胃癌组织中Period1和Period2阳性表达率分别为23.2%、31.2%,均低于癌旁组织中的44.9%、64.5%(P均<0.01);胃癌组织中Period1 mRNA和Period2 mRNA相对表达量分别为1.05±0.10、1.09±0.12,均低于癌旁组织中的1.31±0.13、1.47±0.16(P均<0.01);Period1 mRNA表达与胃癌组织分化程度、浸润深度、淋巴结转移和Lauren分型有关(P均<0.05),Period2 mRNA表达与胃癌组织分化程度、浸润深度、TNM分期、淋巴结转移和Lauren分型均有关(P均<0.05);相关性分析显示,胃癌组织中Period1与Period2表达呈正相关(r=0.285,P<0.05),Period1 mRNA与Period2 mRNA表达呈正相关(r=0.379,P<0.05)。结论胃癌组织中节律基因Period1和Period2呈低表达,可能在胃癌的发生、发展中起重要作用。

肝细胞癌Period1表达与预后的关系

目的:探讨Period1在肝细胞癌(HCC)组织中的表达及意义。方法:用RT-PCR与Western blot法检测30例HCC组织与其癌旁肝组织及5例正常肝组织中Period1 mRNA与蛋白表达;甲基化特异性PCR检测HCC组织与癌旁肝组织中Period1启动子甲基化水平;免疫组化检测67例HCC组织及其癌旁肝组织石蜡切片中Period1蛋白的表达;分析Period1的表达与HCC患者临床病理特征及预后的关系。结果:HCC组织Period1 mRNA和蛋白的表达水平均较癌旁肝组织与正常肝组织明显降低(均P<0.05),而两者在癌旁肝组织与正常肝组织间差异无统计学意义(均P>0.05);HCC组织Period1启动子甲基化水平较其癌旁肝组织明显增高(均P<0.05)。HCC组织石蜡切片中Period1阳性表达率为47.8%,而癌旁肝组织阳性表达率为83.6%,差异有统计学意义(P<0.05);Period1表达与HCC分化程度、结节数目、有无静脉浸润有关(均P<0.05);Period1阳性表达HCC患者中位复时间与中位生存时间均明显短于阳性者(均P<0.05)。结论:Period1在肝癌中表达下调可能与HCC的发生发展和侵袭转移密切相关;启动子甲基化可能是Period1表达下调的重要机制。

Period1、Period2对胶质瘤细胞的生物节律性调控作用

目的研究大鼠胶质瘤细胞内生物钟基因Period1(Per1)、Per2mRNA及蛋白的周期表达规律。方法将体外培养的大鼠胶质瘤C6细胞接种于SD大鼠右侧尾状核区,在12h光照和12h黑暗的标准昼夜节律环境下,采用RT-PCR和免疫组织化学方法分别检测成瘤后4、8、12、16、20、24h肿瘤组织中Per1、Per2 mRNA和蛋白的表达。结果在大鼠胶质瘤组织中,Per1、Per2mRNA及蛋白产物呈节律性表达,其表达周期为12h的超日节律。Per1mRNA和蛋白的高表达时间点均位于4、16h,低表达时间点位于12、24h;Per2mRNA和蛋白的高表达时间点位于12、24h,低表达时间点位于8、20h。结论在SD大鼠体内,胶质瘤细胞的Per1、Per2基因呈12h的超日节律表达,提示生物钟基因的表达异常与胶质瘤的发生发展具有相关性。

PER1与RACK1蛋白作用位点分析

目的筛选人下丘脑视交叉上核(SCN)区域内与PERIOD1(PER1)相互作用的新蛋白,研究RACK1与PER1的作用特点,明确其结合的关键结构域。方法采用酵母双杂交方法,筛选得到人SCN区域内与PER1-PAS结构域相互作用的新蛋白,并构建5种表达不同长度RACK1片段的酵母文库质粒与PER1诱饵质粒共转染酵母AH109进行杂交,通过营养缺陷筛选、报告基因检测获得阳性克隆,并采用体外转录、免疫共沉淀实验证实阳性克隆蛋白间的相互作用。结果酵母双杂交筛选得到人SCN区域内表达RACK1蛋白的克隆,重组RACK1表达质粒与PER-PAS诱饵质粒进行酵母双杂交筛选后得到三个阳性克隆:RACK1(WD1-7)、RACK1(WD4-7)和RACK1(WD5-7),β-半乳糖苷酶测试阳性证实报告基因表达,免疫共沉淀结果显示阳性克隆与PER1蛋白间存在相互作用。结论RACK1与PER1存在直接相互作用,RACK1含有7个WD40结构域,本研究发现与PER1结合的最小区域位于、、三个WD40结构域,提示其碳端序列为其结合的关键部位。

PMA诱导C6细胞rPer1,rDbp基因的近日节律表达

目的寻找一种新的体外节律诱导剂以研究C6神经胶质瘤细胞近日节律基因的表达情况。方法采用PMA(phorbol12-myristate13-acetate),100nmol/L)及50%的马血清刺激体外培养的小鼠NIH-3T3细胞,RT-PCR检测不同时间点mPer1mRNA的表达水平,比较两者的诱导效率;PMA刺激体外培养的大鼠C6神经胶质瘤细胞,RT-PCR检测不同时间点rPer1,rDbpmRNA的表达水平。结果PMA及50%的马血清均能诱导NIH-3T3细胞mPer1的近日节律表达且诱导效率近似;并首次发现C6细胞的rPer1,rDbp基因存在着近日节律振荡。结论PMA是一种有效的体外节律诱导剂;经PMA诱导后,C6细胞的节律基因存在近日振荡,为体外研究授时因子对近日节律的导引机制提供依据。

光线诱导下大鼠体内Gnb2l1基因及其蛋白节律性表达的研究

目的:观察Gnb2l1基因及其蛋白在大鼠体内是否存在节律性表达。方法:12D/12L光暗循环下,采用半定量RT-PCR的方法观察大鼠脑组织及肝脏组织的Gnb2l1,Per1 mRNA的变化规律;采用Western Blot方法检测大鼠脑组织及肝脏组织中Gnb2l1编码蛋白、PER1蛋白在不同时间点的变化。结果:12D/12L光暗循环下,大鼠脑组织及肝脏组织中Gnb2l1 mRNA和节律基因Per1mRNA及其蛋白产物均呈近日节律。结论:在大鼠体内,Gnb2l1基因及其编码的蛋白存在着近日振荡,其相位在节律基因Per1之前。由于Gnb2l1基因编码的蛋白RACK1是一种重要的接头分子,参与了PKC信号通路,而Per1基因的启动子又可以被PKC通路激活,加之前期试验证实RACK1能与PER1蛋白相互作用,因此RACK1蛋白很可能在有Per1参与的节律基因分子振荡环路的反馈机制中起到了重要的作用。

Diurnal expression of circadian clock genes period 1 and period 3 in Pelteobagrus vachellii

Circadian clock genes are crucial for generating and sustaining most rhythmic daily functions in the animal kingdom,which entrain the rhythms of biochemical,physiological,and behavioural processes.To better understand the molecular oscillations of the circadian rhythms in darkbarbel catfish(Pelteobagrus vachellii),we isolated and characterized two circadian clock genes in P.vachellii,period 1(per1),and period 3(per3).The circadian clock gene per1 was found to encode a 1428-amino acid polypeptide,including PER-ARNT-SIM(PAS)dimerisation domains,a PAS-associated C-terminal motif(PAC),a short mutable domain(S/M),and a nuclear export signal(NES).The 4902-bp per3 cDNA includes an open reading frame encoding a 1292-amino acid residue polypeptide with a PER-ARNT-SIM(PAS)domain,cytoplasmic localisation domain(CLD),interaction site(TIS),and a nuclear localisation signal(NLS).The per1 and per3 gene was constitutively expressed in all examined tissues.Moreover,per1 expression within a light/dark cycles showed rhythmic expression in the diencephalon,brain,liver and intestine,with the acrophase at 15:15,12:52,7:51 and 12:55,respectively.Daily expression of per3 was rhythmic in the diencephalonbrain,liver and intestine,with the acrophase at 8:15,9:54,10:39,and 10:25 h,respectively.These findings expand our understanding of circadian mechanism at the molecular level in this species.

Development and validation of an ocean wave retrieval algorithm for VV-polarization Sentinel-1 SAR data

The purpose is to study the accuracy of ocean wave parameters retrieved from C-band VV-polarization Sentinel-1Synthetic Aperture Radar（SAR） images, including both significant wave height（SWH） and mean wave period（MWP）, which are both calculated from a SAR-derived wave spectrum. The wind direction from in situ buoys is used and then the wind speed is retrieved by using a new C-band geophysical model function（GMF） model,denoted as C-SARMOD. Continuously, an algorithm parameterized first-guess spectra method（PFSM） is employed to retrieve the SWH and the MWP by using the SAR-derived wind speed. Forty-five VV-polarization Sentinel-1 SAR images are collected, which cover the in situ buoys around US coastal waters. A total of 52 subscenes are selected from those images. The retrieval results are compared with the measurements from in situ buoys. The comparison performs good for a wind retrieval, showing a 1.6 m/s standard deviation（STD） of the wind speed, while a 0.54 m STD of the SWH and a 2.14 s STD of the MWP are exhibited with an acceptable error.Additional 50 images taken in China＇s seas were also implemented by using the algorithm PFSM, showing a 0.67 m STD of the SWH and a 2.21 s STD of the MWP compared with European Centre for Medium-range Weather Forecasts（ECMWF） reanalysis grids wave data. The results indicate that the algorithm PFSM works for the wave retrieval from VV-polarization Sentinel-1 SAR image through SAR-derived wind speed by using the new GMF C-SARMOD.

Role of the ACTH/MC2R System in the Hair Cycle in Mice

Adrenocorticotropin hormone (ACTH), which is secreted in response to psychological stress, plays an important role in the hair cycle. This study examined the mechanism by which ACTH affects the hair cycle using mice deficient in melanocortin receptor-2(MC2R-/-), which is a main receptor for ACTH. We observed the hair cycle using female MC2R-/- mice at 15 weeks old and five days old to determine whether there were any age-dependent differences. The 15-week-old MC2R-/- mice showed the anagen phase for all mice. On the other hand, all of the MC2R+/+ mice showed the telogen phase at the same age. Moreover, in the five-day-old mice, the hair growth of the MC2R-/- mice occurred earlier than in the MC2R+/+ mice. Both the 15-week-old and five-day-old MC2R-/- mice had higher levels of ACTH and alpha-melanocyte stimulating hormone in the blood than did the MC2R+/+ mice. In addition, in the 15-week-old MC2R-/- mice, the hair cycle shifted to the telogen phase following the administration of a cyclic guanosine monophosphate (cGMP) inhibitor and MC1R/MC5R inhibitor. In the five-day-old MC2R-/- mice, the hair growth was slowed by the administration of corticosterone. These results suggest that the ACTH/MC2R system has an important role in the hair cycle.

mRNA of Expression of Per 1 in Mice Bone Marrow Mesenchymal Stem Cells Irradiated by Red Laser

A core group of circadian genes regulate the circadian rhythms in mammalian cells. However, the mammalian cellular circadian rhythm in photobiomodulation remains unknown. A lot of evidence has shown that >20% of expressed mRNAs in bone stissues oscillate in a circadian manner. The aim of this paper is to investigate the mRNA expression of period 1 (per 1) in murine bone marrow-derived mesenchymal stem cells (BMSCs) which were irradiated by 635 nm red laser light. The cells were seeded in 35mm tissue-culture dishes at a density of 8 × 104 cells/dish and cultured in Dulbecco’s modi?ed Eagle’s medium (DMEM). BMSCs were irradiated once by 635 nm red light with radiation energies 0 J/cm2, 1 J/cm2, 4 J/cm2. mRNA expression of per 1 via Semi-Quantitative Real-time RT-PCR at 0h, 6 h, 12 h, 18 h, 24 h. The genes displayed a oscillatory period of nearly 24 hours. And 635 m laser light changed the mRNA expression of per 1. We conclude that red light irradiation can affect the circadian rhythm of BMSCs.

Multi-symplectic method for the generalized(2+1)-dimensionalKdV-mKdV equation

In the present paper, a general solution involv- ing three arbitrary functions for the generalized （2＋1）- dimensional KdV-mKdV equation, which is derived from the generalized （1＋1）-dimensional KdV-mKdV equa- tion, is first introduced by means of the Wiess, Tabor, Carnevale （WTC） truncation method. And then multi- symplectic formulations with several conservation laws taken into account are presented for the generalized （2＋1）- dimensional KdV-mKdV equation based on the multi- symplectic theory of Bridges. Subsequently, in order to simulate the periodic wave solutions in terms of rational functions of the Jacobi elliptic functions derived from thegeneral solution, a semi-implicit multi-symplectic scheme is constructed that is equivalent 1：o the Preissmann scheme. From the results of the numerical experiments, we can con- clude that the multi-symplectic schemes can accurately sim- ulate the periodic wave solutions of the generalized （2＋1）- dimensional KdV-mKdV equation while preserve approxi- mately the conservation laws.

Deoxyribozymes inhibit the expression of periodl gene in vitro

To investigate the effect of two deoxyribozymes targeting period1 (per1) mRNA in vitro for exploring a novel gene therapy approach about circadian rhythm diseases, the specific deoxyribozymes targeting per1 were designed and synthesized chemically following MFold analysis according to its mRNA secondary structure. per1 RNA fragments were prepared by in vitro transcription of pcDNA3.1(+)-per1164:256. The cleavage reactions containing deoxyribozymes and per1 RNA fragments were performed under certain conditions. With the transfection tech- nique mediated by LipofectAMINETM, pcDNA3-per1 and DRz164 or DRz256 were introduced into NIH3T3 cells. The effects of deoxyribozymes on per1 were studied by reverse tran- script-polymerase chain reaction (RT-PCR) and flow cytometry (FCM). When deoxyribozymes and RNA transcripts were incubated under the adopted conditions at 37℃ for 2 h, about 63% of per1164:256 RNA transcripts were cleaved by DRz164 and about 50.5% by DRz256. After co- transfecting pcDNA3-per1 with DRz164 or DRz256, the expression of per1 mRNA was de- creased, as indicated by RT-PCR semi-quantity analysis. FCM analysis showed that Per1 protein was inhibited. Both DRz164 and DRz256 targeting per1 have the specific cleavage activity to- ward per1 mRNA in vitro and can highly block the expression of per1 gene in cellular milieu.

Bifurcations of Fine 3-point-loop in Higher Dimensional Space

By using the linear independent fundamental solutions of the linearvariational equation along the heteroclinic loop to establish a suitable local coordinate system insome small tubular neighborhood of the heteroclinic loop, the Poincaré map is constructed to studythe bifurcation problems of a fine 3–point loop in higher dimensional space. Under some transversalconditions and the non–twisted condition, the existence, coexistence and incoexistence of2–point–loop, 1–homoclinic orbit, simple 1–periodic orbit and 2–fold 1–periodic orbit, and thenumber of 1–periodic orbits are studied. Moreover, the bifurcation surfaces and existence regionsare given. Lastly, the above bifurcation results are applied to a planar system and an insidestability criterion is obtained.

Characterization of microstructure in high strength Mg_(96)Y_3Zn_1 alloy processed by extrusion and equal channel angular pressing

The Mg96Y3Zn1 alloy processed by extrusion and equal channel angular pressing （ECAP） was investigated. It was found that the Mg96Y3Zn1 alloy processed by extrusion and ECAP obtained ultrafine grains and exhibited excellent mechanical properties. After ECAP, the average grain size of Mg96Y3Zn1 alloy was refined to about 400 nm. The highest strengths with yield strength of 381.45 MPa and ultimate tensile strength of 438.33 MPa were obtained after 2 passes at 623 K. The high strength of Mg96Y3Zn1 alloy was due to the strengthening by the grain refinement, the long period stacking （LPS） structure, solid solution, fine Mg24Y5 particles, and nano-scale precipitates. It was found that the elongation was decreased with pass number increasing. It was because that the cracks were preferentially initiated and propagated in the interior of X-phase during the tensile test.