壳聚糖涂膜对菜用大豆荚采后衰老和品质的影响

以"南农二号"菜用大豆(Glycine max)荚为试材,研究不同浓度的壳聚糖涂膜对菜用大豆荚采后衰老、品质和腐烂的影响.结果表明,1.0%和1.5%壳聚糖处理可显著降低豆荚的呼吸强度、蒸腾失水、多酚氧化酶(PPO)及过氧化物酶(POD)活性,保持较低的膜透性、丙二醛(MDA)含量和较高的叶绿素、Vc及还原糖含量,提高豆荚的苯丙氨酸解氨酶(PAL)活性和促进木质素合成,减少豆荚腐烂发生,从而延长贮藏期.     

胞外ATP对壳聚糖诱导的ROS和PAL活性变化的影响

以烟草BY-2悬浮细胞为材料,探讨了胞外ATP对壳聚糖引起的活性氧(reactive oxygen species,ROS)水平和苯丙氨酸解氨酶(phenylalanine ammonia-lyase,PAL)活性变化的影响。结果表明,5~20μg·mL^-1壳聚糖处理导致了烟草悬浮细胞细胞内ROS水平逐渐增加;壳聚糖也导致了PAL活性的增加,其活性在15μg·mL^-1壳聚糖处理下达到峰值,此后有所降低。10~40μmol·L^-1外源ATP处理未引起烟草悬浮细胞内ROS水平和PAL活性的显著变化。细胞外ATP水平则随壳聚糖浓度的增加而逐渐下降。本文进一步分析了细胞外ATP对壳聚糖引起的ROS水平和PAL活性变化的影响。结果显示,外源施加20μmol·L^-1ATP可以有效降低壳聚糖诱导的烟草悬浮细胞ROS水平上升,同时外源ATP也明显减缓了壳聚糖所诱导的PAL活性的上升。上述结果表明,细胞外ATP水平能够影响壳聚糖引起的ROS水平和PAL活性的变化。     

壳聚糖对红松幼苗多酚积累和抗氧化防御酶的诱导作用

为探究壳聚糖诱导促进红松(Pinus koraiensis)多酚合成的生理调控机制, 以红松幼苗为实验材料, 在DCR培养基中添加不同浓度的壳聚糖, 诱导8天后测定多酚和原花青素的含量, 筛选有利于多酚积累的最佳的壳聚糖浓度。随后测定最佳浓度壳聚糖诱导下红松幼苗中多酚物质积累量、防御酶活性和多酚合成途径关键酶活性随时间的变化。结果显示: 50-200 mg·L^-1壳聚糖可以有效地提高多酚和原花青素的积累量。壳聚糖浓度为100 mg·L^-1时诱导效果最佳, 多酚积累量可以达到(9.91 ± 0.68) mg·g ^-1鲜质量, 是对照组的1.64倍; 原花青素积累量可以达到(2.52 ± 0.11) mg·g ^-1鲜质量, 是对照组的1.53倍。100 mg·L^-1壳聚糖诱导下红松幼苗防御相关酶(超氧化物歧化酶、过氧化物酶、过氧化氢酶)和多酚合成关键酶(苯丙氨酸转氨酶、肉桂酸4-羟化酶)迅速做出响应, 活性显著高于对照组。壳聚糖能够显著地激活红松幼苗的防御反应和苯丙烷代谢途径, 从而促进抗氧化物质多酚的合成与积累, 有利于提高红松幼苗的抵抗力。     

由废弃淡水龙虾壳制备的羧甲基壳聚糖在草莓保鲜过程中的生理作用

利用废弃淡水龙虾壳制备得到的羧甲基壳聚糖应用到草莓的保鲜,为如何有效地解决废弃淡水龙虾壳提供了解决途径.实验结果显示不同浓度的羧甲基壳聚糖处理的草莓腐烂率下降,同时延缓了维生素C水平的下降趋势,其中0.2%浓度的羧甲基壳聚糖的作用效果最为明显.在保鲜过程中,测定草莓果肉中超氧化物歧化酶 (SOD)、过氧化物酶 (POD)、过氧化氢酶(CAT)的活性和脂质过氧化物丙二醛(MDA)的含量的变化,发现经过处理的草莓在保鲜过程中,其SOD,POD,CAT酶活性明显提高,MDA的水平同时显著下降,结果说明羧甲基壳聚糖的保鲜作用可能通过提高抗氧化水平来实现.     

柑橘精油及壳聚糖复合处理对橄榄果实采后生理和耐贮性的影响

为探究柑橘精油(0.25%、0.75%、1.5%)和壳聚糖(0.25%、0.75%、1.5%)复合处理对橄榄(Canarium album)果实采后生理和耐贮性的影响，以鲜食橄榄品种(系)‘檀香’、‘梅埔2号’为材料，对采后贮藏期间的腐烂率、褐变指数、相对电导率、呼吸强度、丙二醛含量、内源抗氧化物质谷胱甘肽含量、过氧化物酶(POD)、抗坏血酸过氧化物酶(APX)活性进行测定，筛选橄榄果实复合保鲜剂的最佳浓度组合。结果表明，与对照相比，所有柑橘精油和壳聚糖复合处理均可有效降低橄榄果实在贮藏期间腐烂率、褐变指数和丙二醛的积累，抑制呼吸作用，保持较高的内源抗氧化物质谷胱甘肽含量和POD、APX活性。因此柑橘精油及壳聚糖处理能够有效延长橄榄果实的保鲜时间，提高耐贮性，其中以1.5%柑橘精油和1.5%壳聚糖组合的保鲜效果最佳。     

桦褶孔菌漆酶固定化及其对染料的降解

采用壳聚糖交联法和海藻酸钠-壳聚糖包埋交联法固定化桦褶孔菌产生的漆酶,探讨最佳固定化条件,固定化漆酶的温度,pH稳定性及操作稳定性,并以两种固定化酶分别对4种染料进行了降解.结果表明:(1)壳聚糖交联法固定化漆酶的最佳条件为:壳聚糖2.5%,戊二醛7%,交联时间2h,固定化时间5h,给酶量1g壳聚糖小球:1mL酶液(1U/mL),固定化效率56%;(2)海藻酸钠-壳聚糖包埋交联法固定化漆酶的最佳条件为:海藻酸钠浓度4%,壳聚糖浓度0.7%,氯化钙浓度5%,戊二醛浓度0.6%,给酶量4mL 4%海藻酸钠:1mL酶液(1U/mL),固定化效率高达86%;(3)固定化的漆酶相比游离漆酶有更好的温度和pH稳定性;(4)比较两种固定化漆酶,海藻酸钠-壳聚糖包埋交联法固定化酶的温度及酸度稳定性要优于壳聚糖固定化酶,但可重复操作性要弱于后者,两者重复使用8次后的剩余酶活比率分别为71%及64%;(5)两种固定化酶对所选的4种不同结构的合成染料均有较好的降解效果,其中壳聚糖固定化酶对茜素红的降解效果及重复使用性极佳,重复降解40mg/L的茜素红10次,降解率仍保持在100%.     

产壳聚糖酶菌株的筛选、鉴定及酶学特性分析

【目的】利用筛选培养基,从福建沿海潮间带泥样中分离筛选产壳聚糖酶的菌株,并研究菌株的产酶特性。【方法】通过形态学观察,结合26S rDNA序列进行分类鉴定,采用DNS法测定酶活力。【结果】筛选得到产壳聚糖酶的菌株KQ-1002与草酸青霉(Penicillium oxalicum)的同源性为99%,并初步鉴定为青霉属的一种。发酵培养的最适温度为30°C,最适碳源为1.0%水溶性壳聚糖,最适氮源为1.87%(NH4)2SO4,最适pH为6.0。该菌株液体发酵培养72 h产壳聚糖酶活性最高,经优化后最高产酶量为18 U/mL。纯化后的壳聚糖酶经SDS-PAGE分析其分子量约40 kD。酶促反应最适pH为5.0,最适反应温度为55°C,Km值为1.293 g/L。在离子浓度为1.0×10 3mol/L时,金属离子Cu2+、Hg2+、Ag+对酶的活性均有强烈的抑制作用。壳聚糖酶对不同底物及脱乙酰度的壳聚糖具有不同的降解作用。【结论】筛选获得产壳聚糖酶的真菌菌株KQ-1002的壳聚糖酶活力经优化后提高了约7倍,是一株具有研究和应用潜力的产壳聚糖酶菌株。     

纤维素酶解制取高活性壳聚糖

研究了不同条件纤维素酶降解壳聚糖的特性 ,结果表明 5 0U/ g底物 纤维素酶 5 0℃酶解 8h的壳聚糖的抑菌活性最好。壳聚糖的平均分子量从 780ku降为 2 10ku。对酶解的壳聚糖用葡聚糖G 10 0凝胶分离 ,有效抑菌的分子片断其分子量为 130ku。     

壳聚糖在香蕉果实贮藏保鲜上的应用效果

以巴西香蕉果实为材料,研究了不同浓度（0、0．5％、1．0％、2．0％）的壳聚糖溶液对香蕉果实贮藏保鲜的影响。结果表明：壳聚糖处理可明显延缓香蕉果实的软化进程和病情指数的升高;同时维持了果实较低的细胞膜透性、多酚氧化酶（PPO）活性和较高的苯丙氨酸解氨酶（PAL）、β-1,3-葡聚糖酶（GUN）的活性。在四种处理中,以2％壳聚糖处理效果最好。表明壳聚糖对香蕉果实的贮藏保鲜效应可能与其调控病害相关酶的活性有关。     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

Effects of Cordyceps sinensis on testosterone production in normal mouse Leydig cells.

The stimulatory effect of Cordyceps sinensis (CS) on MA-10 mouse Leydig tumor cell steroidogenesis was previously demonstrated in our laboratory. In the present studies, we further determined the effect of CS on steroidogenesis in purified normal mouse Leydig cells. Different concentrations of CS (0.1-10 mg/ml) were added to Leydig cells without or with human chorionic gonadotropin (hCG) (50 ng/ml), and the steroid production was determined by radioimmunoassay (RIA). The results illustrated that CS stimulated normal mouse Leydig cell steroidogenesis in a dose-dependent relationship. CS at 3 mg/ml significantly stimulated testosterone production (p<0.05). Concerning the temporal relationship, CS at 3 mg/ml stimulated maximal testosterone production between 2 to 3 hr. Interestingly, hCG-stimulated testosterone productions were suppressed by CS in a dose-dependent relationship. CS also reduced dbcAMP-stimulated testosterone productions, which indicated that CS affected signal transduction pathway of steroidogenesis after the formation of cyclic AMP. Moreover, cycloheximide inhibited CS-treated mouse Leydig cell testosterone production, suggesting that new protein synthesis was required for CS-stimulated steroidogenesis.     

Effect of cigarette smoking on nitric oxide, structural, and mechanical properties of mouse arteries

Cigarette smoking (CS) is a major risk factor for vascular disease. The aim of this study was to quantitatively assess the influence of CS on mouse arteries. We studied the effect of short-term (6 wk) and long-term (16 wk) CS exposure on structural and mechanical properties of coronary arteries compared with that of control mice. We also examined the reversibility of the deleterious effects of CS on structural [e.g., wall thickness (WT)], mechanical (e.g., stiffness), and biochemical [e.g., nitric oxide (NO) by-products] properties with the cessation of CS. The left and right coronary arteries were cannulated in situ and mechanically distended. The stress, strain, elastic modulus, and WT of coronary arteries were determined. Western blot analysis was used to analyze endothelial NO synthase (eNOS) in the femoral and carotid arteries of the same mice, and NO by-products were determined by measuring the levels of nitrite. Our results show that the mean arterial pressure was increased by CS. Furthermore, CS significantly increased the elastic modulus, decreased stress and strain, and increased the WT and WT-to-radius ratio compared with those of control mice. The reduction of eNOS protein expression was found only after long-term CS exposure. Moreover, the NO metabolite was markedly decreased in CS mice after short- and long-term exposure of CS. These findings suggest that 16 wk of CS exposure can cause an irreversible deterioration of structural and elastic properties of mouse coronary arteries. The decrease in endothelium-derived NO in CS mice was seen to significantly correlate with the remodeling of arterial wall.     

Chitosan-Mediated siRNA Delivery <Emphasis Type="Italic">In Vitro</Emphasis>: Effect of Polymer Molecular Weight,Concentration and Salt Forms

The aim of this study was to investigate chitosan/siRNA complexes formulated with various chitosan salts (CS) including chitosan aspartate (CS-Asp), chitosan glutamate (CS-Glu), chitosan acetate (CS-Ac), and chitosan hydrochloride (CS-HCl) for in vitro siRNA delivery into stable and constitutive enhanced green fluorescent protein (EGFP)-expressing HeLa cells. The CS/siRNA complexes were characterized by 2% agarose gel electrophoresis and investigated for their transfection efficiency in stable and constitutive EGFP-expressing HeLa cells. The cytotoxicity of the complexes was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The formation of complexes CS/siRNA is mainly dependent on the weight ratio, whereas salt form and molecular weight has less effect. The particle sizes of the complete complexes were in the range of 270–373 nm except the complete complex of CS-Ac, with a slightly positive charge of less than 2 mV. The ability of CS to transfer functionally active siRNA into cell culture is mainly dependent on the weight ratio and molecular weight of CS whereas salt form of CS has less effect. The high gene-silencing efficiency was observed with low MW of CS (20 kDa) and high weight ratio of 32. Over 80% average cell viabilities were observed for CS/siRNA complexes in all weight ratios comparison to untreated cells. This study suggests CS salts have the potential to be used as safe siRNA delivery vectors.     

海马内NA能神经损毁对抗急性低氧诱发皮质酮分泌

本工作观察了６羟多巴胺（６ｈｙｄｒｏｘｙｄｏｐａｍｉｎｅ,６ＯＨＤＡ）损毁大鼠腹侧海马去甲肾上腺素能神经对急性低氧诱发皮质酮分泌的影响。结果显示,吸入１０４％Ｏ２３０ｍｉｎ后血浆皮质酮水平显著升高,６ＯＨＤＡ注入腹侧海马致使海马内去甲肾上腺素（ＮＡ）含量降低（－３８５％）;血浆皮质酮水平也较未损毁组为低（－３３２％）。吸入１０４％Ｏ２后,皮质酮对低氧刺激的反应性升高现象消失。结果提示：海马内ＮＡ可能参与急性低氧应激引发血浆皮质酮分泌的调节活动。     

小牛血清对鸡传染性法氏囊病病毒增殖的抑制机制

本试验研究了小牛血清(CS)对法氏囊炎病毒(IBDV)蚀斑形成的抑制机制。CS与鸡胚细胞(CEF)作用后,CS中的抑制因子能被细胞吸收。这说明血清中的抑制因子可附着在CEF上。细胞预先用CS处理,则吸附病毒的能力明显降低。还发现,若把CS加入琼脂培养液中,则能抑制IBDV的蚀斑形成。这说明CS能抑制病毒对其周围细胞的感染。CS对IBDV蚀斑形成的抑制机制,不是由于抑制因子直接中和了病毒,而是因为抑制因子附着在细胞表面,占据了细胞的病毒受体,从而阻止了病毒附着于细咆,以致抑制了病毒蚀斑的形成。     

Conditioned augmentation of natural killer cell activity. Independence from nociceptive effects and dependence on interferon-beta

Injection of mice with 20 micrograms polyinosinic: polycytidylic acid (Poly I:C) after exposure to camphor odor results in a conditioned augmentation of natural killer cell (NK) activity. In this study, we show that the conditioned response is not the result of nociceptive stimulation and that interferon-beta (IFN), but not IFN-alpha can replace Poly I:C as the unconditioned stimulus (US). Two conditioned stimuli (CS) were used with equivalent results. A combination CS consisting of a novel taste (saccharin) and a 125 mg/kg injection of LiCl that induces gastric upset was paired with a Poly I:C or IFN-beta (1 X 10(4) IU) injection. This resulted in an augmentation of NK activity when the conditioned animals were reexposed to the saccharin-LiCl CS. In addition, an identical conditioned response was elicited when a camphor odor CS was paired with either of these US. To test whether the conditioned response might be an artifact not detected by our controls, a mock conditioning experiment was performed, which assessed the differential effect of multiple exposures to the saccharin-LiCl CS without a CS/US pairing. The mock conditioned group was significantly suppressed relative to saline treated controls, whereas the mock nonconditioned group and the mock conditioned group that was not reexposed to the CS after conditioning did not show significant suppression. This indicates that the augmentation observed in the conditioned group after CS/US pairing was not the result of exposure to the CS itself. Small doses of Poly I:C (5 micrograms or 2.5 micrograms) given on days 3 and 5 (or on day 5 only) to boost NK activity had the effect of increasing the magnitude of the conditioned response measured on day 6. In addition, an identical conditioned response was observed when the interval between the CS/US pairing and the later CS exposures was changed, which places the test for the conditioned response either on day 6 (CS given on days 3 and 5) or day 10 (CS given on days 7 and 9). These results show that the observed conditioned enhancement of NK activity in conditioned animals is not caused by any nociceptive properties of the CS itself and is dependent on the IFN-beta produced after Poly I:C injection in the conditioned paradigm.     

Kinetics of cyclosporine A-induced inhibition of succinate-coenzyme Q dehydrogenase in rat renal cortical mitochondria

The kinetic mechanism of succinate-coenzyme Q dehydrogenase (Complex II) inhibition by cyclosporine A (CS) on rat renal cortical mitochondria was investigated. CS showed two modes of inhibition of Complex II of the mitochondrial electron transport system: (a) a mixed linear noncompetitive inhibition of resting succinate-limited and ADP-stimulated respirations suggesting that CS binds to Complex II at a different site than the substrate, affecting the dissociation constant for the enzyme-substrate complex and (b) a competitive inhibition of the DNP-stimulated electron transport system suggesting competition with the oxidized form of a component of Complex II. CS action to renal mitochondrial Complex II limits its function, an effect which may be related to CS nephrotoxicity.     

Effect of suramin on growth of androgen-responsive mouse tumor (Shionogi carcinoma 115) and its autonomous subline (Chiba subline 2)

Suramin has been shown to inhibit the binding of various growth factors to their receptors. Shionogi Carcinoma 115 cells (SC 115 cells) and Chiba Subline 2 cells (CS 2 cells) are clones of an androgen-responsive mouse tumor cell and its autonomous subline, respectively. Since the growth of SC 115 and CS 2 cells are assumed to be regulated by their own fibroblast growth factor (FGF)-like growth factors, the present study was undertaken to examine the effect of suramin on these cells. Suramin inhibited the growth of SC 115 and CS 2 cells in a dose dependent manner. The inhibition of suramin was reversible up to 50 micrograms/ml. Suramin reversibly changed the shape of these cells from fibroblast-like to polygonal and epithelial-like ones, and inhibited 3H-thymidine incorporation into these cells which was evoked by acidic and basic FGFs, and conditioned medium obtained from CS 2 cells. The binding of 125I-basic FGF to SC 115 and CS 2 cells was inhibited by suramin. However, suramin had no effect on growth factor production and the hst-1 gene expression on CS 2 cells. In conclusion, suramin inhibited the autocrine and paracrine growth of SC 115 and CS 2 cells by blocking the binding of autocrine growth factors to their receptors.     

Secondary dormancy in Avena fatua: Effect of temperature and after-ripening

To evaluate the effect of after-ripening on secondary dormancy induction in pure genetic lines of Avena fatua L., seed samples were periodically removed from standard conditions of storage and the caryopses then subjected to anoxia. Anoxia did not induce secondary dormancy in SH430, a line characterized by no primary dormancy at harvest maturity; secondary dormancy was induced in caryopses of other lines that had been after-ripened to over-come primary dormancy ranging in duration from a few days (CS40, CS166) to several months (AN51, AN127). Germination response to low GA₃ concentrations indicated that secondary dormancy in CS40 and CS166 was less intense than in AN51 and AN127. The longer the period of dry after-ripening prior to anoxia treatment, the lower the intensity of secondary dormancy induced. After a period of dry after-ripening, which was characteristic for each line, anoxia became an ineffective dormancy-inducing treatment. Caryopses selected for their response to dormancy induction by anoxia were subjected to temperatures from 5 to 35°C to investigate the effect of low (5 to 18°C) and high (20 to 35°C) temperatures on both thermo- and secondary dormancy induction. SH430 was not responsive to any treatment, while CS40, CS166 and AN51 were induced into a thermo-dormancy at temperatures above 20°C and CS166 and AN51 were induced into secondary dormancy by anoxia at temperatures from 5 to 35°C. The effect of anoxia on secondary dormancy induction in a range of pure genetic lines is discussed with reference to primary dormancy, after-ripening and temperature.