人参叶水溶性多糖——杂多糖P_N的纯化与结构初步确定

 从人参叶中提取的水溶性多糖经分离纯化得杂多糖P_N。P_N的分子量约为190万,单糖组成为阿拉伯糖、鼠李糖、木糖、半乳糖醛酸、半乳糖、葡萄糖及少量未知糖,单糖的摩尔比依次为8.1:0.8:1.0:1.6:12.5:4.1(未知糖除外)。经超离心分析,琼脂糖4B柱分析,玻璃纤维纸电泳和醋酸薄膜电泳鉴定等证明P_N为均一组份。经果胶酶降解,部分酸水解,高碘酸盐氧化,Smith降解,甲基化及其产物气相色谱(GLC)、气相色谱-质谱联用(GLC-MS)等结构分析表明P_N为多分支结构,分子的主链主要是由β-(1→3)连接的半乳糖组成,并在4—0和6—0上带有分支,平均每三个半乳糖有二个分支。     

人重组卵泡刺激素和表皮生长因子对性未成熟小鼠卵母细胞和颗粒细胞复合体体外发育的作用

卵泡刺激素（FSH）对有腔卵泡和排卵前卵泡的促生命作用已被普遍接受,但关于其对腔前卵泡发育的作用报道结果不尽相同,关于表皮生长因子（EGF）对腔前卵泡的作用尚不确切,本研究目的在于探讨人重组卵泡刺激素（rechFSH)和EGF对早期卵泡发育的作用,利用胶原酶消化法从12日龄的小鼠卵巢中分离得到卵母细胞－颗粒细胞复合体（OGCs)(Fig.1)。体外每孔30－40个培养物并分别添加胎牛血清（FBS）,rechFSH和EGF。培养物每4天测量卵母细胞和OGCs直径,并每天照相,结果显示,rechFSH显著促进小鼠OGCs 及其卵母细胞的体外发育,这一作用可被EGF进一步增强（P＜0．05）（Fig.2),但到第八天培养结束时,培养后的OGCs卵母细胞要显著小于体内期生长对照组（P＜0．05）（Fig.3),说明FSH和EGF在卵泡早期发育中起重要作用。     

血小板活化因子对大鼠卵巢颗粒细胞垂体腺苷酸环化酶激活肽mRNA表达的影响及调节机制

目的：探讨血小板活化因子（PAF）对大鼠卵巢颗粒细胞垂体腺苷酸环化酶激活肽（PACAP）mRNA表达的影响及其可能调节机制,旨在寻找PAF在卵巢中的作用靶点。方法：原代培养卵巢颗粒细胞,用放射免疫分析（RIA）及逆转录．聚合酶链反应（RT-PCR）方法检测颗粒细胞雌二醇分泌情况及其PACAP mRNA表达变化．结果：PAF对PACAP mRNA表达无明显影响,但与hCG共同作用可促进PACAP mRNA的表达：Forkolin可使PACAP mRNA表达升高。结论：PAF可通过对hCG的允许作用间接促进大鼠卵巢颗粒细胞PACAP mRNA表达,hCG的促进作用可能是通过cAMP—PKA途径介导的。     

处理方式不同的颗粒细胞和卵泡液对牛卵母细胞体外受精后卵裂率和囊胚率的影响

将牛的卵母细胞置于添加有不同预处理颗粒细胞及含有卵泡液的培养液或不舍有卵泡液的培养液中进行体外成熟、受精及胚胎发育培养,研究了颗粒细胞、卵泡液及颗粒细胞与卵泡液交互作用对牛卵母细胞成熟、受精后卵裂率、囊胚率的影响。2178枚卵母细胞体外成熟受精后胚胎发育的对比观察结果表明：颗粒细胞、卵泡液及颗粒细胞与卵泡液交互作用对卵母细胞成熟受精后胚胎的卵裂具有显著影响（P〈0．05）;颗粒细胞对囊胚的发育有显著影响（P〈0．05）;卵泡液及颗粒细胞与卵泡液交互作用对囊胚的发育无显著影响（P〉0．05）。不同因素对卵裂率、囊胚率的影响表现为：颗粒细胞因素〉培养液因素〉培养液×颗粒细胞交互作用。结论：TCM199培养液中添加卵泡液和单层颗粒细胞组成的培养系统用于牛卵母细胞体外成熟及胚胎发育的效果较好。共培养体系中的单层颗粒细胞用经酶消化分散处理后在培养箱中孵育10min的颗粒细胞替代时,胚胎的发育效果并不受影响。     

人参多糖对环磷酰胺的增效减毒作用

目的探讨人参多糖（ginseng polysaccharide,GPS）对化疗药物环磷酰胺（cyclophosphamide,CTX）抗肿瘤的增效减毒作用。方法建立小鼠肝癌H22实体瘤模型,随机分组,设模型对照组（生理盐水）、环磷酰胺组（30 mg/kg）、人参多糖给药组（7.5、15、30 mg/kg剂量组）和联合给药组（GPS 7.5、15、30 mg/kg,CTX 30 mg/kg）;人参多糖尾静脉注射,CTX腹腔给药,连续给药10 d;测定抑瘤率、血液学指标、脾脏系数和胸腺系数,评价人参多糖对环磷酰胺的增效减毒作用。结果 GPS、GPS＋CTX各组肿瘤生长明显低于模型对照组（P〈0.01）,GPS中、高剂量＋CTX联合给药组肿瘤生长明显低于CTX组（P〈0.05,P〈0.01）。GPS高剂量＋CTX组与CTX组相比,体重、脾脏指数和胸腺指数明显升高（P〈0.05）。结论人参多糖对化疗药物环磷酰胺抗小鼠肝癌H22肿瘤具有增效减毒作用。     

人参果胶中蛋白质与多糖关系的研究

从人参（Panax ginseng)热水根取物中,分离出两个蛋白多糖级份（PA,PB）,琼脂糖4B凝胶柱层析结果表明,二者均为单一分布峰,通过氨基酸分析,气相色谱分析,β-消去反应,部分酸水解,果胶酶水解,聚丙烯酰胺凝胶圆盘电泳及等电聚焦电泳等生化分析,确定PA,PB均含有苏氨酸与多糖中糖残基以－O－糖甙键相连的共价结合蛋白质;另外PB中尚存在另一种形式蛋白质,其中精氨酸等碱性氨基酸丰富（占PB中蛋白质59．9％）,圆盘电泳结果表明：这部分蛋白质与多糖中的半乳糖醛酸残基靠静电力结合,等电点为9．1。     

人参果胶中蛋白质与多糖关系的研究

从人参（Panax ginseng)热水根取物中,分离出两个蛋白多糖级份（PA,PB）,琼脂糖4B凝胶柱层析结果表明,二者均为单一分布峰,通过氨基酸分析,气相色谱分析,β-消去反应,部分酸水解,果胶酶水解,聚丙烯酰胺凝胶圆盘电泳及等电聚焦电泳等生化分析,确定PA,PB均含有苏氨酸与多糖中糖残基以－O－糖甙键相连的共价结合蛋白质;另外PB中尚存在另一种形式蛋白质,其中精氨酸等碱性氨基酸丰富（占PB中蛋白质59．9％）,圆盘电泳结果表明：这部分蛋白质与多糖中的半乳糖醛酸残基靠静电力结合,等电点为9．1。     

玉屏风多糖合生元对热应激草鱼免疫性能的影响

为研究不同浓度的玉屏风多糖合生元对热应激草鱼免疫性能的影响, 选360尾健康草鱼Ctenopharyngodon idella (20±2) g, 随机分为4组。其中Ⅰ组为对照组, 试验组(Ⅱ、Ⅲ和Ⅳ组)在饲喂基础日粮的基础上分别添加0.08%、0.12%和0.20%玉屏风多糖, 并在饲喂前按复合益生菌体积(mL)/饲料重(g)为 1%的剂量添加复合益生菌拌料, 整个试验期为21d。在饲养试验结束后, 对草鱼进行热应激48h处理, 在0h、6h、24h和48h的4个时间点采集样品并测定血清、肝脏生化指标和肝脏相关基因表达量。结果表明: Ⅲ组草鱼血清中谷丙转氨酶(ALT)和谷草转氨酶(AST)在各时间点均显著低于对照组(P<0.05); 同时, 草鱼肝脏中超氧化物歧化酶(SOD)、总抗氧化能力(T-AOC)、谷胱甘肽过氧化物酶(GSH-Px)和谷胱甘肽还原酶(GR)在各时间点均不同程度高于对照组, 其中SOD在0h和24h(P<0.05), GR在0h和 48h均显著高于对照组(P<0.05), 而MDA均低于对照组, 其中在24h和48h显著低于对照组(P<0.05); 草鱼肝脏中Nrf2、Cu/Zn-SOD、GSH-Px、GR、HSP70和MHC Ⅱ相对表达量在各时间点均不同程度高于对照组, 其中GR和MHC II相对表达量在各时间点均显著高于对照组(P<0.05), 而Keap1和IL-8在各时间点均在不同程度低于对照组, 其中Keap1在各时间点均显著低于对照组(P<0.05), IL-8均差异不显著(P>0.05)。总之, 随着热应激时间的延长, 草鱼的血清和肝脏生化指标、肝脏相关基因表达量均产生了不同程度的变化, 说明热应激后草鱼产生了强烈的应激反应。在饲料中添加0.12%的玉屏风多糖合生元最有利于提高草鱼的抗氧化应激能力, 有效缓解肝脏氧化损伤, 具有较好的免疫调节作用。     

冷应激大鼠睾丸细胞AR和ABP的检测

目的为研究冷应激大鼠睾丸细胞雄激素受体和雄激素结合蛋白的变化,本实验检测了睾丸细胞雄激素受体和雄激素结合蛋白的含量。方法采用放射配体—3H-睾酮结合分析法。结果冷应激实验组与对照组比较,雄激素受体最大结合量(单位:fmol/mgDNA)为143.38±9.47和87.46±15.11;雄激素结合蛋白(单位:fmol/mgpro.)为34.98±8.16和12.94±3.63;差异有显著性,P<0.05。结论冷应激导致睾酮水平下降的同时,反馈地引起其受体浓度增加的变化是机体代偿适应性反应。     

Effects of cold stress on glutathione and related enzymes in rat erythrocytes

Effects of acute and chronic cold stress on glutathione and related enzymes in rat erythrocytes were investigated. Blood from both cold-acclimated (CA) and cold-adapted (CG) rats had significantly lower concentrations of glutathione than blood from control animals. Superoxide dismutase activity was increased significantly in CA rats and tended to rise in CG rats. Activity of glutathione peroxidase in erythrocytes was inconsistent in that it tended to increase in CA rats but decreased significantly in CG rats. The results may imply that CG rats suffered deleterious effects of hydrogen peroxide. On the other hand, there were marked decreases in glutathione peroxidase and glutathione reductase activities in acutely cold-exposed rats in conjunction with unchanged levels of glutathione. In all treatments the state of riboflavin metabolism was estimated to be adequate, since no increases were observed in the erythrocyte glutathione reductase activity coefficient.     

急性冷应激对牦牛乳腺上皮细胞 HSP70 mRNA 表达的影响

本文主要研究了急性冷应激对牦牛乳腺上皮细胞热休克蛋白７０ （Heat stress protein,ＨＳＰ７０） 表达量的影响。采用实时荧光定量ＰＣＲ 技术,以急性冷刺激１０℃ 为典型研究环境,分析了ＨＳＰ７０ ｍＲＮＡ 表达的变化规律。结果显示,乳腺上皮细胞在１０℃分别冷处理２ ｈ、４ ｈ、６ ｈ 和８ ｈ,其ＨＳＰ７０ ｍＲＮＡ 的表达量变化均不显著（Ｐ ＞０. ０５）;分别在１０℃冷处理２ ｈ、４ ｈ、６ ｈ 和８ ｈ,再复温培养４ ｈ,ＨＳＰ７０ ｍＲＮＡ 的表达量均极显著增加（Ｐ ＜０. ０１）,于６ ｈ 达到峰值;在１０℃先冷处理４ ｈ,然后分别复温２ ｈ、４ ｈ、６ ｈ 和８ ｈ,ＨＳＰ７０ ｍＲＮＡ 的表达量亦均显著增加（Ｐ ＜０. ０１）,并于４ ｈ 达到峰值。结论：急性冷应激诱导牦牛乳腺上皮细胞ＨＳＰ７０ 表达量的增加不是发生在冷处理过程中,而是发生在复温过程中,并且在一定范围内随冷处理时间的增长表达量增高。     

Beneficial developmental acclimation in reproductive performance under cold but not heat stress

Thermal plasticity can help organisms coping with climate change. In this study, we analyse how laboratory populations of the ectotherm species Drosophila subobscura, originally from two distinct latitudes and evolving for several generations in a stable thermal environment (18 °C), respond plastically to new thermal challenges. We measured adult performance (fecundity traits as a fitness proxy) of the experimental populations when exposed to five thermal regimes, three with the same temperature during development and adulthood (15-15 °C, 18-18 °C, 25-25 °C), and two where flies developed at 18 °C and were exposed, during adulthood, to either 15 °C or 25 °C. Here, we test whether (1) flies undergo stress at the two more extreme temperatures; (2) development at a given temperature enhances adult performance at such temperature (i.e. acclimation), and (3) populations with different biogeographical history show plasticity differences. Our findings show (1) an optimal performance at 18 °C only if flies were subjected to the same temperature as juveniles and adults; (2) the occurrence of developmental acclimation at lower temperatures; (3) detrimental effects of higher developmental temperature on adult performance; and (4) a minor impact of historical background on thermal response. Our study indicates that thermal plasticity during development may have a limited role in helping adults cope with warmer - though not colder - temperatures, with a potential negative impact on population persistence under climate change. It also emphasizes the importance of analysing the impact of temperature on all stages of the life cycle to better characterize the thermal limits.     

The effect of adaptation to cold and re-adaptation to room temperature on the level of glutathione in rat tissues

1. Glutathione (GSH) was assayed in plasma, liver and IBAT in control (22±1°C) and cold adapted rats (45 days in 5±1°C), and in rats cold adapted and brought back to room temperature after 1, 3, 7 and 15 days.

2. Adaptation to the cold led to reduced GSH in the liver and plasma while the level in intrascapular brown adipose tissue (IBAT) was increased in comparison to controls.

3. On the first day of re-adaptation, plasma GSH was similar to the control level, as were hepatic levels on the first and fifteenth day, but GSH in IBAT remained higher even after fifteen days.

Effect of oxygen supply on cell growth and saponin production in bioreactor cultures of Panax ginseng

The effects of oxygen supply within the range 20.8–50% (using pure oxygen and air), on cell cultures of Panax ginseng were investigated in a balloon-type bubble bioreactor (5 L capacity, containing 4 L Murashige and Skoog medium, supplemented with 7.0 mg L⁻¹ indolebutyric acid, 0.5 mg L⁻¹ kinetin and 30 g L⁻¹ sucrose). A 40% oxygen supply was found to be optimal for the production of both cell mass and saponin yielding values of 12.8 g (DW) L⁻¹, 4.5 mg (g DW)⁻¹ on day 25, respectively. Low (20.8%, 30%) and high (50%) oxygen concentration supplies were unfavorable to cell growth and saponin accumulation. The results indicate that oxygen supplementation to bioreactor-based ginseng cultures was beneficial for biomass accumulation and saponin production.     

The effect of osmotic stress on the cell volume, metaphase II spindle and developmental potential of in vitro matured porcine oocytes

Porcine animal models are used to advance our understanding of human physiology. Current research is also directed at methods to produce transgenic pigs. Cryobanking gametes and embryos can facilitate the preservation of valuable genotypes, yet cryopreserving oocytes from pigs has proven very challenging. The current study was designed to understand the effects of anisotonic solutions on in vitro matured porcine oocytes as a first step toward designing improved cryopreservation procedures. We hypothesized that the proportion of oocytes demonstrating a normal spindle apparatus and in vitro developmental potential would be proportional to the solution osmolality. Oocytes were incubated for 10 min at 38 degrees C in various hypo- or hypertonic solutions, and an isotonic control solution and then assessed for these two parameters. Our results support the hypothesis, with an increasing proportion of spindles showing a disrupted structure as the levels of anisotonic exposure diverge from isotonic. Only about half of the oocytes maintained developmental potential after exposure to anisotonic solutions compared to untreated controls. Oocyte volume displayed a linear response to anisotonic solutions as expected, with an estimated relative osmotically inactive cell volume of 0.178. The results from this study provide initial biophysical data to characterize porcine oocytes. The results from future experiments designed to determine the membrane permeability to various cryoprotectants will allow predictive modeling of optimal cryopreservation parameters and provide a basis for designing improved cryopreservation procedures.     

Formation of early-light-inducible-protein complexes and status of xanthophyll levels under high light and cold stress in barley (Hordeum vulgare L.)

In our previous work we found considerable accumulation of early light-inducible proteins (ELIPs) in barley during adaptation to combined high light and cold stress, an accumulation which occurred preferentially in the apical part of the leaves (M.-H. Montané et al., 1997, Planta 202: 293–302). Here we studied, under the same conditions, the effect of adaptation on the composition of thylakoid membrane proteins and pigments, particularly xanthophylls and chlorophyll, and their distribution within the barley leaf. It was observed that high light fluxes appeared to favour the trimerization of the light-harvesting complex of photosystem II (LHC II) whereas cold appeared to favour the monomers of LHC II. High light, cold or the combination of both factors had only a small effect on the protein composition of the thylakoid membranes except for the proteins of LHC II which were found to decrease under high light to a greater extent at 25 °C than at 5 °C. The total xanthophyll-cycle carotenoid content increased linearly with cellular development, the highest amount being observed in the apical part of the leaf. Cold and high light acted synergistically to induce less than a doubling in the amount of total xanthophylls, while chlorophylls a and b remained nearly constant. The fraction consisting of antheraxanthin plus zeaxanthin was up to 4- to 5-fold higher at 5 °C than at 25 °C. As determined previously (Montané et al. 1997), the same conditions caused a 15-fold increase in the accumulation of ELIPs. Consequently, neither the distribution of total xanthophylls nor that of antheraxanthin plus zeaxanthin along the leaf followed the same pattern as ELIP. Thus, the accumulation of xanthophylls cannot be stoichiometrically correlated with that of ELIPs. Using electrophoresis in the presence of decylmaltoside, we could demonstrate for the first time that ELIPs of 13.5 kDa are contained in high-molecular-mass complexes of >100 kDa, which are located in the unstacked stroma lamellar region of the thylakoid membranes. Received: 6 April 1998 / Accepted: 26 January 1999