Visualization of molecule interaction between antigen and antibody - One of ellipsometric imaging applications

It is to investigate molecule interactions between antigen and antibody with ellipsometric imaging technique and demonstrate some features and possibilities offered by applications of the technique. Molecule interaction is an important interest for molecule biologist and immunologist. They have used some established methods such as immufluorcence, radioimmunoassay and surface plasma resonance, etc, to study the molecule interaction. At the same time, experimentalists hope to use some updated technique with more direct visual results. Ellipsometric imaging is non-destructive and exhibits a high sensitivity to phase transitions with thin layers. It is capable of imaging local variations in the optical properties such as thickness due to the presence of different surface concentration of molecule or different deposited molecules. If a molecular mono-layer (such as antigen) with bio-activity were deposited on a surface to form a sensing surface and then incubated in a solution with other molecules (such as antibody), a variation of the layer thickness when the molecules on the sensing surface reacted with the others in the solution could be observed with ellipsometric imaging. Every point on the surface was measured at the same time with a high sensitivity to distinguish the variation between mono-layer and molecular complexes. Ellipsometric imaging is based on conventional ellipsometry with charge coupled device (CCD) as detector and images are caught with computer with image processing technique. It has advantages of high sensitivity to thickness variation (resolution in the order of angstrom), big field of view (in square centimeter), high sampling speed (a picture taken within one second), and high lateral resolution (in the order of micrometer). Here it has just shown one application in study of antigen-antibody interaction, and it is possible to observe molecule interaction process with an in-situ technique.

TNFa在鱼腥藻7120中的高效表达和对宿主光合作用的影响

蓝藻分子生物学的飞速发展已使其成为生物学的前沿。近几年来，以蓝藻为宿主的基因工程发展迅速，使转基因蓝藻已有希望制备药物或处理环境问题。但迄今为止，国内外用蓝藻表达外源基因的表达效率都不高。为了使转基因蓝藻在应用上产生较好的社会效益和经济效益，必须进一步提高外源基因在蓝藻中的表速效率，以及提高光合效率、加速生长。 本研究用人肿瘤坏死因子a（Human Tumor Necrosis Factora简称hTNFa）作为外源目的基因。它是由巨噬细胞和单核细胞受到刺激后产生的一种多功能蛋白质细胞因子。hTNFcc多种生物学效应并作为信号传导体，其中最引人注目的是它对肿瘤组织和肿瘤细胞直接地、特异性和广谱性地杀伤作用，极有希望制成抗癌剩的天然因子之一。但是用大肠杆菌得到的重组产物需要严格纯化，通常用于静脉注射，但由于毒副作用大，十几年来国内外一直停留在临床实验阶段，我们研究组建议用蓝藻为宿主表达hTNFa制备口服剂，来减缓毒副作用，已经得到了转基因鱼腥藻，并测得产物具有抑瘤的生物学活性。但是表达效率一直不高，并且它的表达对蓝藻生长有些抑制。 由于蓝藻是原核生物，基因的表达调控主要是在转录水平和翻译水平。因此，寻找在蓝藻中高效的启动子，改变SD序列的结构是提高外源基因在蓝藻中表达效率的有效手段。本研究将连有不同SD序列的TNFa cDNA克隆到穿梭表达载体pRL-489的启动子（PpsbA）下游，构建2个鱼腥藻7120的穿梭表达载体（pMD-489-TNF1，2），通过三亲接合转移法分别导八鱼腥藻7120细胞。用放射免疫法定量分析TNFa在转基因蓝藻中的表达效率。结果表明，有效地提高了TNFa在鱼腥藻7120中的表达。TNFa的表达量占总可溶性蛋白的2.1 - 2.9%和0.15%，表达效率分别提高到原来的21 - 29倍和1.5倍。 在培养转基因鱼腥藻中，观测到它们在形态和生理上都发生了变化，这反应了TNFcc基因的转入和表达对宿主光合作用的影响。 光学显微镜和扫描电子显微镜的观察发现：转基因鱼腥藻比野生型异形胞数目减少约30%。转入空质粒的营养细胞比野生型略大，转TNFa基因的鱼腥藻异形胞体积明显增加，而营养细胞比正对照和野生型小。到了生长后期，转TNFa因的鱼腥藻营养细胞体积明里增大，多与异形胞相当，有的甚至比异形胞大。转pMD-489-TNFI的鱼腥藻细胞内出现明显的空腔。通过透射电子显微镜的观察发现：转基因藻中的类囊体膜片屡结构更加明显。转基因藻和野生藻的生长曲线的比较表明，转入空质粒pRL-489对宿主的生长几乎没有影响，甚至还略快于野生型;TNFa的表达对细胞的生长有一定副作用，胞内TNFa的含量高时，细胞数增长缓慢，并且平台期时细胞数有一定下降。 从光合作用光强曲线的分析可见，转TNFa因的鱼腥藻有较低的光饱和点，暗示了TNFa的表达可以增强宿主对光的敏感性;同时，TNFa的转入使宿主的呼吸作用加强，几乎比野生型和转空质粒的正对照高一倍，显示了TNFa基因的转入和表达可能给宿主带来更大的代谢负荷;在光饱和点以上，几种藻的真实光合放氧能力大致相同，表明TNFa的表达没有破坏宿主的光合反应中心。 从室温吸收光谱分析可见，转基因蓝藻有相对较高的类胡萝卜素和叶绿素a蓝峰，转TNF谌因的鱼腥藻显示了藻蓝蛋白含量有所降低。因为蓝藻的主要天线色素为藻胆蛋白，藻蓝蛋白相对含量的下降可能与宿主对光更敏感有关。 从低温荧光发射光谱分析可见，转TNFa基因的鱼腥藻7120光系统II能量分配较高。可能是TNFa基因的转入提高了藻胆蛋白的吸收和传递光能的效率。 从叶绿素荧光动力学分析可见，鱼腥藻7120在生长的过程中PSII的活性存在一个变化的过程。TNFa的转入和表达在对数后期提高了宿主的光系统II原初光能转化效率。 从转基因藻光系统I和光系统II光合放氧活性分析与TNFa表达随培养时间变化曲线表明，转TNFcc基因鱼腥藻的光合放氧活性比野生型和正对照高，尤其是显著地提高了宿主的Psn活性。 用自然界中原来不存在的转基因鱼腥藻作上述研究表明：原来只存在于高等、异养的人类和哺乳动物中的TNFa基因，一旦转入最古老的放氧光合生物后，其表达可被调控;同时TNFa的表达又能影响宿主的光合作用。它提高了宿主对光的敏感性、光系统II的活性和对光能的利用率。这似乎都表明TNFa在蓝藻细胞中起信号传导体的作用。而且，这些数据的积累，还有助于我们优化培养条件，提高TNFa的表达效率，为产业化做好准备。

TMVA, a novel GPIb-binding protein, significantly prevents platelet microthrombi formation and prolongs discordant cardiac xenograft survival

In xenotransplantation, donor endothelium is the first target of immunological attack. Activation of the endothelial cell by preformed natural antibodies leads to platelet binding via the interaction of the glycoprotein (GP) Ib and von Willebrand factor (vWF). TMVA is a novel GPIb-binding protein purified from the venom of Trimeresurus mucrosquamatus. In this study, the inhibitory effect of TMVA on platelet aggregation in rats and the effect on discordant guinea pig-to-rat cardiac xenograft survival were investigated. Three doses (8, 20 or 40 mug/kg) of TMVA were infused intravenously to 30 rats respectively. Platelet aggregation rate was assayed 0.5, 12, and 24 h after TMVA administration. Wister rats underwent guinea pig cardiac cervical heterotopic transplantation using single dosing of TMVA (20 mug/kg, i.v., 0.5 h before reperfusion). Additionally, levels of TXB2 and 6-keto-PGF(1alpha) within rejected graft tissues were determined by radioimmunoassay. Treatment with TMVA at a dose of 20 or 40 mug/kg resulted in complete inhibition of platelet aggregation 0.5 h after TMVA administration. Rats receiving guinea pig cardiac xenografts after TMVA therapy had significantly prolonged xenograft survival. Histologic and immunopathologic analysis of cardiac xenografts in TMVA treatment group showed no intragraft platelet microthrombi formation and fibrin deposition. Additionally, the ratio of 6-keto-PGF(1alpha) to TXB2 in TMVA treatment group was significantly higher than those in control group. We conclude that the use of this novel GPIb-binding protein was very effective in preventing platelet microthrombi formation and fibrin deposition in a guinea pig-to-rat model and resulted in prolongation of xenograft survival. The increased ratio of PGI(2)/TXA(2) in TMVA treatment group may protect xenografts from the endothelial cell activation and contribute to the prolongation of xenograft survival.

Induction of hepatic cytochrome P4501A1/2B activity and disruption of thyroglobulin synthesis/secretion by mono-ortho polychlorinated biphenyl and its hydroxylated metabolites in rat cell lines

As the active metabolites of polychlorinated biphenyl (PCBs), hydroxylated polychlorinated biphenyls (OH-PCBs) are found in wildlife and human tissues. They have been proposed as main contributors for endocrine disruption of PCBs in living organisms. In this study, mono-ortho PCB 156 and its hydroxylated metabolites 4'-OH-PCB 159, 4'-OH-PCB 121, and 4'-OH-PCB 72 were selected to investigate the toxic effects on rat hepatoma H4IIE cell line and rat thyroid follicle FRTL-5 cell line at concentrations of 1, 10(2), 10(4) nM. 7-Ethoxyresorufin-O-deethylase (EROD) and 7-pentoxyresorufin-O-dealkylase (PROD) activities were determined with micro-EROD/PROD to indicate cytochrome P4501 A1 (CYP1A1) and cytochrome P4502B (CYP2B) induction in the H4IIE cell after exposure for 72 h. To assess thyroid disruption of these compounds, thyroglobulin concentrations also were detected inside FRTL-5 cell with immunocellularchemistry and in its medium with radioimmunoassay after exposure for 24 It. Significant inductions of EROD activity by PCB 156 at 102 and 104 nM (p < 0.05) were observed, but no effects by the three OH-PCBs in H4IIE cell line. 7-Pentoxyresorufin-O-dealkylase activities were induced only by 10(4) nM of PCB156 and the three OH-PCBs (p < 0.05). Meanwhile, significant increases of thyroglobulin concentrations were observed in the medium of FRTL-5 cell exposed to 4'-OH-PCB 121 and 4'-OH-PCB 72 at all of the test concentrations (p < 0.05), but not to the other compounds. The results demonstrated that mono-ortho PCBs mainly could be metabolized to hydroxylated metabolites through CYP1A1 instead of CYP2B. Moreover, after being metabolized, OH-PCBs still sustained the ability to induce PROD activity and did exhibit the disruption on thyroglobulin synthesis/excretion in rat cells.

Serum concentrations of gonadotropins and steroid hormones of Neophocaena phocaenoides asiaeorientalis in middle and lower regions of the Yangtze River

As a relatively isolated and unique freshwater population, the Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis) is the most endangered subpopulation of this species. The objective of this study was to improve our understanding of their reproductive endocrinology by measuring (radioimmunoassay) serum concentrations of FSH, LH, estradiol (E-2), progesterone (P-4) and testosterone (T-2) in free-ranging animals. Blood samples were collected from 66 Yangtze finless porpoises (41 males and 25 females) captured in the middle and lower reaches of the Yangtze River. Based on significant variation of serum T-2 concentrations in males with body length (BL) > 138 cm, we inferred that they were mature; in these animals, there were significant seasonal variations in serum T-2 concentrations, with the highest concentrations in March and April (502.0 +/- 319.8 ng/dL, mean +/- S.D.) and the lowest in December (79.4 +/- 83.2 ng/dL). Serum T-2 concentrations positively correlated with serum concentrations of LH and weakly correlated with serum concentrations of E-2, whereas there was a significant negative correlation between serum LH and FSH concentrations in males > 138 cm. The smallest apparently pregnant female porpoise had a BL of 130 cm. Serum P-4 concentrations ranged from 13.2 to 112.4 ng/mL (43.9 +/- 28.3 ng/mL) in pregnant females, and fluctuated under 1.0 ng/mL in non-pregnant females with BL > 130 cm. Serum LH concentrations were significantly higher in non-pregnant females > 130 cm versus those females < 130 cm. To our knowledge, this is the first study of endocrine-related maturity and seasonal breeding characteristics of the Yangtze finless porpoise. (c) 2006 Elsevier Inc. All rights reserved.

Reproductive seasonality and maturity of male Neophocaena phocaenoides asiaeorientalis in captivity: A case study based on the hormone evidence

To provide hormone evidence on reproductive seasonality and maturity of the Yangtze finless porpoise, the authors monitored the monthly variations of serum reproductive hormones of a male Yangtze finless porpoise in captivity via radioimmunoassay from 1997 to 2003. It was demonstrated that the immature animal had a serum T level of 28-101ngdL -1 (6552ngdL -1 ), reached puberty with the serum T concentration ranging between 250 and 590ngdL -1 (390130ngdL -1 ), and attained reproductive maturity when the serum T level went beyond 1120180ngdL -1 in the breeding season. The body length growth of the animal showed a pattern similar to the serum T variations during its adolescent period from the age of 4 to 6 years. Moreover, the serum T concentration of the male porpoise exhibited significant seasonal variations, indicating that its breeding season may start as early as March and end as late as September.

Estradiol stimulates growth hormone production in female goldfish

The effects of estradiol (E(2)) on growth hormone (GH) production was investigated in gonad-intact female goldfish. It was first necessary to generate a specific antibody for use in immunocytochemistry, Western, and dot-blot analyses of GH production. To accomplish this, grass carp GH (gcGH) cDNA was cloned by the reverse transcription polymerase chain reaction (RT-PCR) and expressed in Echerichia coli and a specific polyclonal antibody to recombinant gcGH was generated in the rabbit. In Western blot, the anti-gcGH antibody specifically immunoreacted with recombinant gcGH, purified natural common carp GH, and with a single 21.5-kDa GH form from pituitary extracts of grass carp, common carp, goldfish, and zebrafish but not salmon, trout, or tilapia. Intraperitoneal injection of the recombinant gcGH enhanced the growth rates of juvenile common carp demonstrating biological activity of this GH preparation. Electron microscopic studies showed that the anti-gcGH-I antibody specifically reacted with GH localized in the secretory granules of the goldfish somatotroph. Using anti-gcGH-I in a dot-blot assay, it was found that in vivo implantation of solid silastic pellets containing E(2) (100 mu g/g body weight for 5 days) increased pituitary GH content by 150% in female goldfish. In a second, independent study employing a previously characterized anticommon carp GH antibody for radioimmunoassay, it was found that E(2) increased pituitary GH content by 170% and serum GH levels by approximately 350%. The E(2)-induced hypersecretion of GH and increase in pituitary GH levels was not associated with changes in steady-state pituitary GH mRNA levels, suggesting that this sex steroid may enhance GH synthesis at the posttranscriptional or translational level. Previous observations indicate that GH can stimulate ovarian E(2) production. The present results show that E(2) can in turn stimulate GH production, indicating the existence of a novel pituitary GH-ovarian feedback system in goldfish. (C) 1997 Academic Press.

Inhibitory effects and mechanisms of high molecular-weight phlorotannins from Sargassum thunbergii on ADP-induced platelet aggregation

We evaluated the effects of high molecular-weight phlorotannins from Sargassum thunbergii (STP) on ADP-induced platelet aggregation and arachidonic acid (AA) metabolism in New Zealand white rabbits and Wistar rats. The inhibition of STP on platelet aggregation was investigated using a turbidimetric method, and the levels of the terminal products of AA metabolism were measured using the corresponding kits for maleic dialdehyde (MDA), thromboxane B-2 (TXB2) and 6-keto-prostaglandin F-1 alpha (6-keto-PGF(1 alpha)) by colorimetry and radioimmunoassay, as appropriate. We found that STP could inhibit ADP-induced platelet aggregation, and the inhibitory ratio was 91.50% at the STP concentration of 4.0 mg/mL. Furthermore, STP markedly affected AA metabolism by decreasing the synthesis of MDA (P < 0.01) and increasing the synthesis of 6-keto-PGF(1 alpha), thus changing the plasma TXB2/6-keto-PGF(1 alpha) balance when the platelets were activated (P < 0.01). Therefore, STP altered AA metabolism and these findings

Expression of human epidermal growth factor gene in cyanobacteria

The human epidermal growth factor (hEGF) is a small single-chain polypeptide of 53 amino acid residues. It can stimulate the proliferation of many cell types, mainly those of epidermal and epithelial tissues both in vivo and in vitro. A vector pRL-hEGF was constructed using plasmids pRL-489 and pUC-hEGF. The synthetic hEGF gene was recombined into the downstream of strong promoter psbA in plasmids pRL-489. Then, the vector was introduced into Synechococcus sp. PCC 7002 and Anabaena sp. PCC 7120 by triparental conjugative transfer. The transformation was confirmed by PCR amplification. The pRL-hEGF is thought to be retained as a plasmid form in the transgenic Anabaena sp. PCC 7120, since it can be recovered. However, it has been integrated into the chromosome of Synechococcus sp. PCC 7002 as there is no duplication origin in the pRL-hEGF in this cyanobacterium. and plasmid cannot be isolated from the Synechococcus sp. PCC 7002 either. The radioimmunoassay (RIA) proved that the hEGF gene has been expressed as the protein existed in these two strains of transgenic cyanobacteria, and the hEGF protein in Anabaena sp. PCC 7002 could be secreted into the medium.

中老年男性性激素水平与认知功能的关系

Abstract Objective: Study the relationship between sex hormones and cognitive function in aged male. Methods: The serum sex hormones including Free Testosterone(FT), Total Testosterone(TT), Estradiol(E2), Prolactin(PRL) and Luteotropic Hormone(LH) of 74 elderly men(47-75 years old, mean 58.73) were measured by radioimmunoassay. They were all applied the cognitive tests of episodic memory and verbal fluency. For the episodic memory, four experiments examined picture, digit and words memory separately. Four verbal fluency tests were performed in the middle of each episodic memory. The 74 men were divided into two groups according to the levels of their sex hormones respectively, with each group had 37 subjects. Data was analyzed by the software of SPSS 11.0. Results: Mean age and the mean years of formal education had no significant difference between the groups; Men in the group with higher levels of FT had significantly higher scores in the tests of picture memory and words memory；Men in the group with higher levels of TT had significantly higher scores in the tests of verbal fluency4, picture memory and words memory；Men in the group with higher levels of E2 had significantly higher scores in the tests of picture memory and verbal fluency3,4；There were no correlations between PRL or LH and the cognitive tests. Conclusions: 1 Sex hormones (especially FT, TT, E2) did have effect on the cognitive function in aged male, higher levels of hormones related with higher scores of some cognitive tests. 2 Among the cognitive functions, picture memory and words memory performance seemed being effected by the sex hormones most.