Genetic heterogeneity and ploidy level analysis among different gynogenetic clones of the polyploid gibel carp

Background: Some triploid and tetraploid clones have been identified in the gynogenetic gibel carp, Carassius auratus gibelio Bloch, by karyotypic and cytologic analyses over many years. Further, 5-20% males and karyotypic diversity have been found among their natural and artificial populations. However, the DNA contents and the relation to their ploidy level and chromosome numbers have not been ascertained, and whether normal meiosis occurs in spermatogenesis needs to be determined in the different clones. Methods: The sampled blood cells or sperms were mixed with blood cells from chicken or individual gibel carp and fixed in 70% pre-cooled ethanol overnight at 4degreesC. The mixed cell pellets were washed 2-3 times in 1x phosphate buffered saline and then resuspended in the solution containing 0.5% pepsin and 0.1 M HCl. DNA was stained with propidium iodide solution (40 mug/mL) containing 4 kU/ml RNase. The measurements of DNA contents were performed with Phoenix Flow Systems. Results: Triploid clones A, E, F, and P had almost equal DNA content, but triploid clone D had greater DNA content than did the other four triploid clones. DNA content of clone M (7.01 +/- 0.15 pg/nucleus) was almost equal to the DNA content of clone D (5-38 +/- 0.06 pg/nucleus) plus the DNA content of common carp sperm (1.64 +/- 0.02 pg/nucleus). The DNA contents of sperms from clones A, P, and D were half of their blood cells, suggesting that normal meiosis occurs in spermatogenesis. Conclusions: Flow cytometry is a powerful method to analyze genetic heterogeneity and ploidy level among different gynogenetic clones of polyploid gibel carp. Through this study, four questions have been answered. (a) The DNA content correlation among the five triploid clones and one multiple tetraploid clone was revealed in the gibel carp, and the contents increased with not only the ploidy level but also the chromosome number. (b) Mean DNA content was 0.052 pg in six extra chromosomes of clone D, which was higher than that of each chromosome in clones A, E, F, and P (about 0.032 pg/ chromosome). This means that the six extra chromosomes are larger chromosomes. (c) Normal meiosis occurred during spermatogenesis of the gibel carp, because DNA contents of the sperms from clones A, P, and D were almost half of that in their blood cells. (d) Multiple tetraploid clone M (7.01 +/- 0.15 pg/nucleus) contained the complete genome of clone D (5.38 +/- 0.06 pg/nucleus) and the genome of common carp sperm (1.64 +/- 0.02 pg/nucleus). Cytometry Part A 56A:46-52, 2003. (C) 2003 Wiley-Liss, Inc.

人载脂蛋白APOA5的克隆表达纯化及相互作用蛋白研究

人类的载脂蛋白A5（apolipoprotein A5，APOA5）是一个新近发现的载脂蛋白家族成员。它在血浆中的含量比其他载脂蛋白低1-2个数量级，但能显著影响血浆三酰甘油水平，对血脂代谢具有重要意义，可以作为降血脂药物治疗中一个强有力的潜在靶标。 由于APOA5在血浆中含量低，直接从血浆中分离纯化很困难，国内一直没有报道简易可靠的纯化方法。为进一步研究APOA5的生物学特性，探讨其与TG代谢中的其它关键成分之间的相互关系，揭示其在脂类代谢相关疾病中的重要地位，必须有大量的蛋白和抗体用于基础研究。因此本研究首先利用基因工程技术，诱导表达纯化APOA5蛋白，免疫动物制备多克隆抗体，为进一步研究人肝脏细胞中APOA5的相互作用蛋白，研究APOA5蛋白在肝脏细胞中的功能奠定基础。 为了深入研究APOA5在肝脏中如何行使功能，我们采用细菌双杂交技术寻找与APOA5相互作用的蛋白因子。并采用Pull-down技术，免疫荧光及免疫共沉淀技术进一步确证其在体外和体内的相互作用关系，为进一步阐明APOA5在体内的生理功能提供了新的线索。 第一部分 APOA5基因的克隆、原核表达、纯化及其多克隆抗体的制备 本研究首先应用基因克隆技术，从人肝癌细胞系SMMC-7721的cDNA中扩增出1.1 kb的ApoA5基因全长序列。然后将其克隆至表达载体pThioHisD，构建原核表达载体pTH-APOA5。该重组质粒转化至大肠杆菌 BL21（DE3），成功实现人APOA5融合蛋白在大肠杆菌中的表达。经发酵得到高效表达的融合蛋白。 融合蛋白在 IPGT 诱导下以包涵体的形式大量表达。利用融合蛋白上的一段组氨酸序列，用镍离子亲和柱进行纯化和复性后，获得较高纯度的人APOA5融合蛋白。利用该融合蛋白免疫新西兰大耳白兔，获得了高效价的兔抗人APOA5多克隆抗体，Western Blot结果显示此多克隆抗体与APOA5特异性结合。 第二部分 细菌双杂交筛选与APOA5相互作用的蛋白 本实验首先构建了pBT-APOA5重组质粒，经双酶切、PCR和测序鉴定证明重组诱饵质粒构建成功，并进行了表达、自激活鉴定。Western Blot鉴定证实报告菌株中表达了分子量为 68 kD左右的重组融合蛋白，与预测的分子量APOA5（41 kD）/lamda cI （27 kD）一致。自激活实验证明诱饵蛋白不能单独激活报告基因，可用于筛选人肝脏cDNA文库。经过双重抗性筛选和回复筛选，分离出10个阳性克隆。对结果进行生物信息学分析，得到7个与APOA5相互作用的蛋白，其中BI1为细胞凋亡调节因子；ATP6、CYTB、ND2、COX-1为线粒体表达蛋白； ALB、TTR为血清蛋白。 第三部分 APOA5与BI1相互作用的确证 首先构建了BI1的原核表达载体pGEX-5X-3-BI1，利用Pull-down实验检测了APOA5与BI1在体外具有相互作用。然后构建了BI1的真核表达载体pCDNA3.1-HA-BI1和APOA5的真核表达载体pCDNA3.1-APOA5，并验证其表达。通过免疫荧光细胞内共定位研究发现，靶蛋白APOA5主要分布于胞浆，与BI1在HEK293细胞有共定位，即APOA5与BI1存在相互作用的可能。最后利用免疫共沉淀手段，在HEK293细胞中确证了靶蛋白APOA5与BI1在体内的相互作用。 上述研究结果，为深入研究APOA5在体内的生物学功能提供了新的思路。 Apolipoprotein A5 (APOA5) is a newly discovered protein belongs to apolipoprotein family. APOA5’s concentration is 1-2 orders of magnitude lower than other apolipoproteins in the circulation. APOA5 significantly affected plasma triglyceride levels, which is important on lipid metabolism. APOA5 has strong potential to be used as a hypolipidemic drug target. Large amount of APOA5 protein and antibodies are needed in basic research, such as biological characteristics study of the APOA5, its relationship with other key components in TG metabolism, its role played in Lipid metabolism-related diseases. Due to its low concentration in plasma, separation and purification of APOA5 from the plasma is very difficult. Until now no report on simple and reliable method for purification has been published in China. In this study, we firstly got APOA5 recombinant protein using genetic engineering technology. The purified recombinant protein was used to immunize rabbits to get antiserum. It is important for further study of the APOA5 protein-interacting protein. And it lays the foundation for studing APOA5 function in liver. In order to study APOA5 function in liver, we used bacterial two-hybrid technology to find the APOA5 protein interactor. Pull-down, immunofluorescence and immunoprecipitation techniques were used to further confirm the interaction between APOA5 with its interactor in vitro and in vivo. All of these stdudies provided new clues on its physiological functions in vivo. Part I: Cloning, prokaryotic expression, purification and polyclonal antibody preparation of APOA5 First of all, we amplified APOA5 CDS sequence from the human hepatoma cell line SMMC-7721, and subcloned into Expression vector pThioHisD, and got the recombinants named pTH-APOA5. The plasmid was transformed to BL21 (DE3). E. coli BL21(DE3) cells bearing the pTH-APOA5 plasmid were cultured and APOA5 protein synthesis was induced by the addition of IPTG. Recombinant protein was expression in the form of inclusion. Inclusion bodies were dissolved in phosphate-buffered saline containing 8 M urea and 40 mM imidazole, then applied to a Ni2+ affinity column, and were eluted in a buffer containing 4 M urea and 200 mM imidazole. Fractions containing the APOA5 protein were pooled and dialyzed against buffer containing phosphate-buffered saline. Antiserum to recombinant human APOA5 was generated by immuning rabbit. Western Blot showed that this antiserum specific binding with APOA5. Part II Two-hybrid system screening protein interactions with the APOA5 The coding sequence of human APOA5 was amplified using synthetic oligonucleotide primers from pTH-APOA5 vector and was subcloned into the pBT plasmidc to yield pBT-APOA5 vector. DNA sequencing was performed to verify that no unwanted mutations occurred during the process of plasmid vector construction. We verified recombinant protein expression and tested self-activation by pBT-APOA5 prior to screening. Western Blot verified inducing a 68 kD band, consistent with the predicted molecular weight (APOA5 41 kD, lamda cI 27 kD). pBT-APOA5 can be used for screening human liver cDNA library because it can not self-activation. Totally 10 positive clones were isolated. The nucleotide sequence of the positive clones were determined and compared to NCBI nucleotide sequence databases. We got 7 protein which interact with APOA5, included BI1(Apoptosis regulator); ATP6, CYTB, ND2, COX-1(Mitochondrial protein) and ALB, TTR(Serum protein). Part III Confirming of interaction between APOA5 with BI1 pGEX-5X-3-BI1 vector was subcloned at first. Pull-down experiments were used to detect the interaction between APOA5 with BI1 in vitro. Later, pCDNA3.1-HA-BI1 and pCDNA3.1-APOA5 were subcloned. Through immunofluorescence co-localization study, we found APOA5 mainly distributed in the cytoplasm. APOA5 is co-localization with BI1 in HEK293 cells. Finally, we verified interaction between APOA5 with BI1 in vivo through immunoprecipitation.

用PBS作为悬浮介质代替PVP用于红细胞变形性测量的可能性评估

<正> 用激光衍射仪测量RBC的变形性一般要求所用缓冲液的粘度高于18CP,以其获得稳定的,具有可比性的数据,常用的是不同浓度的右旋糖酐溶液。这在实用中带来两个问题;(1)粘度太高,故实验时红细胞所受剪切应力远高于生理水平;(2)价格昂贵,不利于临床推广。因此,我们试图寻求一种可以代替右旋糖酐溶液的新的缓冲液。本文中用了两种缓冲液,即PBS和15％PVP和四种不同的红细胞组成的悬浮液,用

Subsurface damage of Nd-doped phosphate glasses in optical fabrication

We present a destructive method for detecting and measuring subsurface damage of Nd-doped phosphate glasses. An instrument based on the dimple method - a destructive method - was developed. Subsurface damage depth produced in each fabrication procedure was obtained. We extend the surface roughness-subsurface damage relation to Nd-doped phosphate glasses. The constant ratio of subsurface damage and surface roughness was obtained as well. We also analyse the relation of abrasive size and subsurface damage experimentally. From a measurement of the surface roughness or abrasive size, one can obtain an accurate estimate of the damage layer thickness that must be eliminated by polishing or subsequent grinding operations. (C) 2007 Elsevier GmbH. All rights reserved.

Effect of Fe impurity on the optical loss of Nd-doped phosphate laser glass

The optical loss coefficient at 1053-nm wavelength, influenced by Fe ions in N31-type Nd-doped phosphate laser glass, was determined precisely and analyzed in detail. It is found that the optical loss coefficient per unit of Fe concentration (cm^(-1)/ppmw) increases with Fe concentration in the range of 0---300 ppmw, but it approaches a constant as the Fe concentration is larger than 300 ppmw. Such a concentration effect is due to a shift in the redox equilibrium between Fe3+ and Fe2+ ions in the glass. The effect of oxygen pressure, temperature, and variable valence states of other metal ions in glass samples on the optical loss is also discussed.

Effect of network modifiers on spectroscopic properties of erbium-doped phosphate glasses

The integrated absorption cross section Sigma(abs), I peak emission cross section sigma(cmi), Judd-Ofeld intensity parameters Omega(iota) ( t = 2,4,6), and spontaneous emission probability A(R) of Er3+ ions were determined for Erbium doped alkali and alkaline earth phosphate glasses. It is found the compositional dependence of sigma(emi) 5 almost similar to that of Sigma(abs), which is determined by the sum, of Omega(1) (3 Omega(2) + 10 Omega(4) + 21 Omega(6)). In addition, the compositional dependence of Omega(1) was studied in these glass systems. As a result, compared with. Omega(4) and Omega(6) the Omega(2) has a stronger compositional dependence on the ionic radius and content of modifers. The covalency of Er-O bonds in phosphate glass is weaker than that in silicate glass, germanate glass, aluminate glass, and tellurate glass, since Omega(6) of phosphate glass is relatively large. A(R) is affected by the covalency of the Er3+ ion sites and corresponds to the Omega(6) value.

Er3+ and Yb3+ codoped phosphate laser glass for high power flashlamp pumping

A novel Vb(3+)-Er-(3+) codoped phosphate glass for high power flashlamp pumping and high repetition rate laser at 1.54 mu m, designated EAT5-2, is developed. The weight-loss rate of is 1.3 x 10(-5) gcm(-2) h(-1) in boiling water, which is comparable to Kigre's QX-Er glass. Some spectroscopic parameters are analysed by Judd-Ofelt theory and McCumber theory The emission cross section is calculated to be 0.73 x 10(-20) cm(2). The thermo-mechanical properties of EAT5-2 are modified after an ion-exchange chemical strengthening process in a KNO3/NaNO3 molten salt bath. The thresholds for optical damage from the flashlamp pumping are tested on glass rods. A repetition rate of 15 Hz is achieved for chemically strengthened glass. The laser experimental results at. 1.54 mu m from flashlamp pumping are also reported.

Ridge optical waveguide in an Er3+/Yb3+ co-doped phosphate glass produced by He+ ion implantation combined with Ar+ ion beam etching

This paper reports on the fabrication and characterization of a ridge optical waveguide in an Er3+/Yb3+ co-doped phosphate glass. The He+ ion implantation (at energy of 2.8 MeV) is first applied onto the sample to produce a planar waveguide substrate, and then Ar+ ion beam etching (at energy of 500 eV) is carried out to construct rib stripes on the sample surface that has been deposited by a specially designed photoresist mask. According to a reconstructed refractive index profile of the waveguide cross section, the modal distribution of the waveguide is simulated by applying a computer code based on the beam propagation method, which shows reasonable agreement with the experimentally observed waveguide mode by using the end-face coupling method. Simulation of the incident He ions at 2.8 MeV penetrating into the Er3+/Yb3+ co-doped phosphate glass substrate is also performed to provide helpful information on waveguide formation.

Gain characteristics of Er3+-doped phosphate glass fibres

An erbium-doped phosphate glass fibre has been drawn by the rod-in-tube technique in our laboratory. The gain for the Er3+-doped phosphate glass fibre with different pump powers and with different input signal wavelengths is investigated. The 2.2-cm-long fibre, pumped by a single-mode 980-nm fibre-pigtailed laser diode, can provide a net gain per unit length greater than 1.8dB/cm. The pump threshold is about 50 mW at the wavelength of 1534 nm, and below 70 mW at 1550 nm. The gain linewidth of the Er3+-doped phosphate glass fibre is greater than 34 nm and can cover the C band in optical communication networks.