川牛膝多糖的免疫活性及其水解活性片段分离研究

川牛膝多糖（CP）是从传统中药川牛膝（Cyathula officinalis Kuan）中提取的一种活性多糖，现代药理研究表明川牛膝多糖是川牛膝许多生物活性的物质基础。本实验室前期进行了川牛膝多糖的提取、分离、结构鉴定及其部分活性研究，发现川牛膝中多糖含量非常高，在对川牛膝多糖活性的初步研究中也证实了其具有免疫调节作用。我们为了进一步了解其免疫调节活性，并为构效关系的研究奠定基础，对其进行了如下研究： 1． 通过体外毒性检测、淋巴细胞增殖实验、NK细胞杀伤活性和腹腔巨噬细胞吞噬中性红活性测定，发现川牛膝多糖在10～300μg/mL浓度范围内，对细胞无毒性作用；能够促进LPS诱导的B淋巴细胞增殖(P<0.01)、增强NK细胞杀伤活性(P<0.05)和PMΦ吞噬中性红活性(P<0.01)，且随多糖浓度增高而增强；但其对ConA诱导的T淋巴细胞的增殖无促进作用(P>0.05)。 2． 通过正常小鼠体内淋巴细胞转化实验、迟发型变态反应分析、抗体生成细胞检测、碳粒廓清检测、腹腔巨噬细胞吞噬鸡红细胞活性和NK细胞活性测定，发现川牛膝多糖在适应性免疫方面能够促进SRBC免疫小鼠体内的抗体生成细胞的生成（P<0.01）和增强DNFB诱导的DTH（P<0.05），但对ConA诱导的脾淋巴细胞增殖无促进作用（P>0.05）；在固有免疫方面能够提高小鼠碳粒廓清速率（P<0.05），PMΦ吞噬 CRBC 活性（P<0.01）和NK细胞杀伤活性（P<0.05）。同时还发现其对由环磷酰胺(Cy)引起的白细胞数下降具有很好的抑制作用（P<0.01）。 3． 为了获得结构明确、均一的保留活性的川牛膝多糖片段，为其作用机制、构效关系研究提供关键研究材料，我们开展了“保留免疫活性的最小片段”的分离制备的初步研究。建立并优化了川牛膝多糖的酸水解条件，发现在6%的样品浓度，0.025mol/L的硫酸浓度，65℃的水解温度，水解时间为8min的条件下可以得到一系列连续的多糖片段；采用Bio-Gel P2 分子筛柱层析分离得到5个级分，通过体外淋巴细胞增殖实验、NK细胞活性测定、腹腔巨噬细胞吞噬中性红实验发现其中的一个片段仍保留较强的免疫活性，并测得其分子量约为2057Da，为保留免疫活性的最小片段的进一步分离奠定了基础。 Cyathula officinalis Kuan is a commonly-used Traditional Chinese Medicine (TCM) with a wide range of pharmacological activities. Modern pharmacological researches showed the polysaccharide extracted from it (CP) is an important component for many bioactivities of this TCM. In the previous studies, we found CP showed significant immuno-regulative activities. In order to evaluate this activity systematically and lay foundations for revealling its immuno-regulative machanisms and the Structure -Function relationship, we carried out the following research works: 1． The in vitro immunoactivities of CP were evaluated by using normal mice immunocytes with respects to cytotoxicity, lymphocytes proliferation, NK activity and the ability of peritoneal macrophage phagocytizing neutral red. The polysaccharide showed no cytotoxicity below the concentration of 300 μg/mL, and could promote B lymphocytes proliferation (P<0.01), enhance NK activity (P<0.05) and the ability of peritoneal macrophage phagocytizing neutral red (P<0.01) at the concentration of 10-300 μg/mL. The above effects were positively correlated with the concentration of the polysaccharides. But it could not promote T lymphocytes proliferation (P>0.05). 2． The in vivo immunoactivities of CP were observed on normal mice through the following indices: splenic lymphocyte transformation efficiency, delayed-type allergy, antibody-forming cells activity (AFC), rate of carbon clearance, rate of peritoneal macrophage phagocytizing chicken red blood cell (CRBC) and NK activity, and its influence on the decline of the mouse leucocyte count induced by Cy. The polysaccharide at medium-dose enhanced delayed-type allergy （P<0.05）and NK activity（P<0.05） and increased the rate of carbon clearance（P<0.05）, AFC activity（P<0.01） and the rate of peritoneal macrophage phagocytizing CRBC（P<0.01）. The polysaccharides also effectively resisted the decline of the mouse leucocyte count induced by Cy（P<0.01）. However, it couldn’t increase the splenic lymphocyte transformation efficiency（P>0.05）. 3． Attempting to isolate and prepare the minimal fragments retaining activity with identical structure for further studying on immuno-regulative mechanism and Structure-Function relationship, we carried out the study on hydrolysis of CP, isolation of hydrolysed fragments, and the activity evaluation of the isolated fragments. CP with concentration of 6% was hydrolysed at 65℃ for 8 min with sulfuric acid of 0.025 mol/L，then the hydrolysate was separated using Bio-Gel P2 chromatography, 5 portions of fragments were obtained. The immunoactivities of these fragments were evaluated by using normal mice immunocytes with respect to lymphocytes proliferation, NK activity and ability of peritoneal macrophage phagocytizing neutral red. One fragment with relative molecular mass of 2057Da was found retaining immunoactivity.

Biphasic regulation of H2O2 on angiogenesis implicated NADPH oxidase

ROS (reactive oxygen species) take an important signalling role in angiogenesis. Although there are several ways to produce ROS in cells, multicomponent non-phagocytic NADPH oxidase is an important source of ROS that contribute to angiogenesis. In the present work, we examined the effects of H2O2 on angiogenesis including proliferation and migration in HUVECs (human umbilical vein endothelial cells), new vessel formation in chicken embryo CAM (chorioallantoic membrane) and endothelial cell apoptosis, which is closely related to anti-angiogenesis. Our results showed that H2O2 dose-dependently increased the generation of O-2(-) (superoxide anion) in HUVECs, which was suppressed by DPI (diphenylene iodonium) and APO (apocynin), two inhibitors of NADPH oxidase. H2O2 at low concentrations (10 mu M) stimulated cell proliferation and migration, but at higher concentrations, inhibited both. Similarly, H2O2 at 4 nmol/cm(2) strongly induced new vessel formation in CAM, while it suppressed at high concentrations (higher than 4 nmol/cm(2)). Also, H2O2 (200 similar to 500 mu M) could stimulate apoptosis in HUVECs. All the effects of H2O2 on angiogenesis could be suppressed by NADPH oxidase inhibitors, which suggests that NADPH oxidase acts downstream of H2O2 to produce O-2(-) and then to regulate angiogenesis. In summary, our results suggest that H2O2 as well as O-2(-) mediated by NADPH oxidase have biphasic effects on angiogenesis in vitro and in vivo.

Integrated lectin affinity microfluidic chip for glycoform separation

Lectin affinity chromatography was miniaturized into a microfluidic format, which results in improvement of performance, as compared to the conventional method. A lectin affinity monolith column was prepared in the microchannel of a microfluidic chip. The porous monolith was fabricated by UV-initiated polymerization of ethylene dimethacrylate (EDMA) and glycidyl methacrylate (GMA) in the presence of porogeneities, followed by immobilization of pisum sativum agglutinin (PSA) on the monolith matrix. Using electroosmosis as the driven force, lectin affinity chromatographies of three kinds of glycoprotein, turkey ovalbumin (TO), chicken ovalbumin (CO), and ovomucoid (OM), were carried out on the microfluidic system. All the glycoproteins were successfully separated into several fractions with different affinities toward the immobilized PSA. The integrated system reduces the time required for the lectin affinity chromatography reaction to similar to3%, thus, the overall analysis time from 4 h to 400 s. Only 300 pg of glycoprotein is required for the whole separation process. Moreover, troublesome operations for lectin affinity chromatography are simplified.

Coupling of seagrass (Cymodocea nodosa) patch dynamics to subaqueous dune migratio

The coupling between patch dynamics - described by the patch growth (horizontal and vertical), patch mortality, and life-history of Cymodocea nodosa (Ucria) Aschers., and the disturbance caused by the migration of subaqueous dunes over the plants was examined in a shallow NW Mediterranean bay (Alfacs Bay) where this species maintains a patchy cover. C. nodosa shoots survived substantial burial rates (up to 2.4 mm/day) by growing vertically at rates proportional to, albeit four-fold slower than, burial rates. Patch death was caused by erosion as large subaqueous dunes migrated pass the plant patch. Patch growth was fastest over the progressing slope of the dunes ( similar to 2.5 m year super(-1)) and flowering was also stimulated by sand accretion. The time interval between the passage of consecutive dunes, which sets the time window available for patch development, ranged between 2 and 6 years. This time interval allowed C. nodosa to recolonize bare substrata, with patch formation occurring about half a year after the disturbance, and also allowed established shoots to complete their life-cycle and produce seeds and thus enable subsequent recolonization. The time windows available for patch development also set an upper limit to patch size of about 26 m. Significant cross correlations between dune topography and patch dynamics and plant flowering frequency provide evidence that the spatial heterogeneity in the vegetation is closely associated with the disturbance imposed by the migration of sand dunes. The migration of subaqueous dunes maintains C. nodosa in a continuous state of colonization involving spatially asynchronous patch growth and subsequent mortality, which is ultimately responsible for the characteristic patchy landscape of this Bay. 

鸡粪中活性有机酸的分离与鉴定

本文通过生物追踪实验法 ,研究了鸡粪中有机酸的生物活性成分 ,并从中筛选出了一种强活性物质 .通过红外光谱和质谱及生源关系初步确定其分子式为C36H56O18Na,名称为 3-O -D -葡萄糖 -6,1 -0 -葡萄糖酸 2 β ,2 ,2 0 -二羟基蛋甾酸钠

Preparation and mass spectrometric study of egg yolk antibody (IgY) against rabies virus

Rabies virus was used as the antigen to immunize laying chickens. Anti-rabies virus immunoglobulin Y(IgY) was isolated from yolks of the eggs laid by these chickens using a two-step salt precipitation and one-step gel filtration protocol. The purified IgY was reduced with dithiothreitol, and heavy chains (HC) and light chains (LC) were obtained. In addition, the purified IgY was digested with pepsin and the fragment with specific antigen binding properties (Fab) was produced. Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOFMS), the average molecular weights of IgY, HC, LC, and Fab were determined as 167 250, 65 105, 18 660, and 45,359 Da, respectively. IgY has two structural differences compared with mammalian IgGs. First, the molecular weight of the heavy chain of IgY is larger than that of its mammalian counterpart, while the molecular weight of the light chain of IgY is smaller. Second, upon pepsin digestion, anti-rabies virus IgY is degraded into Feb, in contrast to mammalian IgG, which has been reported to be degraded into F(ab')(2) under the same conditions. Copyright (C) 2001 John Wiley & Sons, Ltd.

A fibrinogen-related protein from bay scallop Argopecten irradians involved in innate immunity as pattern recognition receptor

The family of fibrinogen-related proteins (FREPs) is a group of proteins with fibrinogen-like domains. Many members of this family play important roles as pattern recognition receptors in innate immune responses. The cDNA of bay scallop Argopecten irradians FREP (designated as AiFREP) was cloned by rapid amplification of cDNA ends (RACE) method based on the expressed sequence tag (EST). The full-length cDNA of AiFREP was of 990 bp. The open reading frame encoded a polypeptide of 251 amino acids, including a signal sequence and a 213 amino acids fibrinogen-like domain. The fibrinogen-like domain of AiFREP was highly similar to those of mammalian ficolins and other FREPs. The temporal expression of AiFREP mRNA in hemolymph was examined by fluorescent quantitative real-time PCR. The mRNA level of scallops challenged by Listonella anguillarum was significantly up-regulated, peaked to 9.39-fold at 9 h after stimulation, then dropped back to 4.37-fold at 12 h, while there was no significant change in the Micrococcus luteus challenged group in all periods of treatment. The function of AiFREP was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli Rosetta gami (DE3). The recombinant AiFREP (rAiFREP) agglutinated chicken erythrocytes and human A, B, O-type erythrocytes. The agglutinating activities were calcium-dependent and could be inhibited by acetyl group-containing carbohydrates. rAiFREP also agglutinated Gram-negative bacteria E. coli JM109, L anguillarum and Gram-positive bacteria M. luteus in the presence of calcium ions. These results collectively suggested that AiFREP functions as a pattern recognition receptor in the immune response of bay scallop and contributed to nonself recognition in invertebrates, which would also provide clues for elucidating the evolution of the lectin pathway of the complement system. (C) 2008 Elsevier Ltd. All rights reserved.

cDNA cloning and mRNA expression of the translationally controlled tumor protein (TCTP) gene from Japanese sea perch (Lateolabrax japonicus)

A homologue of the lower vertebrates translationally controlled tumor protein (TCTP) was cloned from the marine fish Japanese sea perch (Lateolabrax japonicus) by the technology of homology cloning. The full-length cDNA sequence of the sea perch TCTP gene contained a 5' untranslated region (UTR) of 47 bp, a 3' UTR of 433 bp, and a putative open reading frame (ORF) of 510 bp encoding a polypeptide of 170 amino acids. The deduced amino acid sequence of the sea perch TCTP gene showed a high similarity to that of zebrafish, rohu, rabbit, chicken and human. Sequence analysis revealed there were a signature sequence of TCTP family, an N-glycosylation site, and five Casein kinase phosphorylation sites in the sea perch TCTP. The temporal expression of TCTP genes in healthy and lipopolysaccharide (LPS) challenged fishes was measured by semi-quantitative reverse transcription-PCR (RT-PCR). The results indicated that LPS could up-regulate the expression of sea perch TCTP in the examined tissues, including head-kidney, spleen and liver.

Influence of albumen on aggregation and O-2 evolution of protoplasm from Bryopsis hypnoides Lamourou

The extruded protoplasm from the coenocytic green alga, Bryopsis hypnoides Lamouroux, was able to reform a cell wall and develop further into a mature alga in seawater. In this paper, the influence of albumen on the ability of aggregation and on the photosynthesis of protoplasm was examined. Results show that the protoplasm of B. hypnoides could aggregate in either albumen or chicken egg, which is similar to that in seawater. However unlike in seawater, the aggregation from B. hypnoides in albumen and chicken egg failed to develop into a mature individual. Interestingly, the protoplasm of B. hypnoides could maintain its photosynthetic O-2 evolution in albumen and chicken egg, while the time in chicken egg was longer than that in albumen.