均匀设计法优化发菜细胞悬浮培养条件

通过摇瓶发酵实验研究了培养温度、光照强度等培养条件对发菜细胞悬浮培养生物量和代谢产物发菜多糖累积的影响,通过均匀设计试验对培养条件进行了优化。结果表明:在培养温度24℃、培养基初始pH8.0、光照强度60μmol/(m2.s)、转速150r/min的条件下培养20d,发菜细胞生物量(细胞质量浓度)达到1.34g/L,胞外多糖产量达到208.32mg/L;与优化前相比,发菜细胞生物量和胞外多糖产量分别提高27.3%、111.17%。     

过氧化物酶作植物细胞培养物生长指示剂

爱达荷大学的K.Shetly及同事发现,在黄花蒿(Artemesia annua)、锦紫苏(Coleus blumei)、豌豆(Pisum sativum)和撒尔维亚(Salvia oflicinalis)悬浮细胞中,在培养的指数生长期与细胞干重成比例地产生胞外过氧化物酶.通过改变初始蔗糖浓度变更生长动力学时,过氧化物酶积累与干重增加间的相关性不改变.Shetly等推测:“胞外过氧化物酶     

不同化学物质对银耳孢子细胞膜通透性影响

为了提高外源基因表达胞外分泌水平,本研究通过测定不同化学物质对银耳孢子转化子细胞生物量、培养液电导率及胞外漆酶酶活的影响,探索不同化学物质对银耳孢子细胞膜通透性的影响。结果表明,浓度分别为1%、0.3%、0.2%和0.1%的二甲基亚砜、triton X‐100、氯化钾、两性霉素B均可显著提高电导率;同时银耳孢子胞外漆酶酶活分别提高至7.00U/L、6.67U/L、5.00U/L和5.00U/L,与对照达到显著差异。而表面活性剂(SDS)及有机溶剂(甲苯、戊二醛)则不利于银耳孢子的生长及漆酶的分泌。该研究结果显示,添加一定的化学物质对促进目标产物的外泌有较为明显的作用,这将为银耳孢子作为生物反应器高效表达外源基因提供科学依据。     

影响深红酵母和白地霉产生胞外蛋白和多肽的因素

采用白地霉2.498-47在以蛋白胨为氮源发酵时,只在开始pH3.2左右产生胞外蛋白,NH_4~+则无此限制,还有助于胞外多肽的产生。深红酵母2.280胞外蛋白的最大产率约在pH5.8;尿素作唯一氮源效果最好,与蛋白胨合并使用,可产胞外蛋白约800μg/ml。酵母膏对生长和产胞外蛋白很必要,不能用玉米浆和麦芽汁代替。用0.05N NaOH浸洗不同pH和氮源所培养的细胞都能浸出胞内蛋白,接近400μg/ml。发酵产生胞外蛋白自一日后开始,至二日接近最大量,以后逐渐减少。多肽产生起自第二日,五日未达到最大量。两株酵母菌都不表现蛋白酶活性,因此这个现象还不能解释。     

云南高原湖泊抚仙湖和星云湖的酵母菌胞外酶活性

【背景】高原湖泊因其海拔高、气压低、辐射强、氧气含量低,是一类特殊环境,而其中的微生物是高原湖泊生态系统物质循环与能量流动的重要参与者,其胞外酶活性的表现决定其适应这一特殊环境的方式与能力。【目的】对分离自云南高原湖泊抚仙湖和星云湖湖水的酵母菌进行产胞外酶活性的筛选,以期获得具有潜在应用价值的活性菌株。【方法】在5°C和25°C培养温度下,采用平板筛选法对两个湖泊酵母菌进行产胞外蛋白酶、纤维素酶、淀粉酶、脂肪酶、几丁质酶、木聚糖酶、植酸酶、菊粉酶、漆酶、锰依赖过氧化物酶和木质素过氧化物酶活性的筛选。【结果】抚仙湖和星云湖的所有测试酵母菌菌株至少都能产1种胞外酶,且主要产植酸酶、菊粉酶和淀粉酶;其次为脂肪酶、纤维素酶、木聚糖酶、锰依赖过氧化物酶和木质素过氧化物酶;产几丁质酶、蛋白酶和漆酶的酵母菌很少,星云湖酵母菌都不产漆酶。培养温度为5°C时,抚仙湖和星云湖的酵母菌产5种及5种以上胞外酶的活性菌株数均多于25°C。【结论】抚仙湖和星云湖的酵母菌产胞外酶菌株多样性丰富,胞外酶种类多样,产酶酵母菌可能参与高原湖泊生态系统的物质循环;筛选得到的产胞外酶菌株为开发与利用高原湖泊酶资源提供了良好的种质资源,具有进一步研究的价值。     

中性粒细胞胞外诱捕网(NETs)在肝细胞癌转移中的作用与机制研究

肝细胞癌(HCC)是一种炎症相关癌症,肿瘤免疫微环境在HCC的发生和发展中起关键作用。该文旨在研究中性粒细胞胞外诱捕网(NETs)在HCC转移中的作用及相关机制。ELISA和免疫组化方法检测HCC患者血清和肿瘤组织中的NETs水平以检测NETs与肝癌转移的相关性。在体外实验中,建立NETs与肝癌细胞系Hep3B和CSQT-2体外共培养模型,通过划痕实验和Transwell等实验,研究NETs对肝癌细胞迁移的影响。在体内实验中,建立尾静脉注射转移瘤模型并使用脂多糖诱导小鼠体内NETs形成,通过检测肝脏病理变化和肝脏Ki67蛋白水平等指标,研究NETs对肿瘤转移的作用。最后,为了探讨NETs影响HCC转移的机制,通过质谱的方法检测了NETs对细胞外基质的修饰,并检测了修饰的细胞外基质蛋白对整合素/FAK信号通路的影响。结果发现:高转移HCC患者肿瘤组织中髓过氧化物酶蛋白水平较高,且与早期HCC患者相比,晚期HCC患者血清中的MPO和中性粒细胞弹性蛋白酶水平升高。体外实验中, NETs与Hep3B和CSQT-2细胞共培养,可以促进Hep3B和CSQT-2细胞的迁移能力。体内实验中, NETs...     

胞外酸性与肿瘤的浸润转移

组织缺氧是实体瘤的一个主要特征,它引起肿瘤细胞胞外酸性环境的形成.肿瘤细胞通过质子感知的G蛋白偶联受体（G protein-coupled receptors,GPCRs）或质子感知的离子通道感知其胞外的酸性环境,并激活多条细胞内信号通路,影响细胞功能. 肿瘤最致命的方面在于其转移能力,肿瘤转移程度与肿瘤细胞迁移能力呈正相关. 因此,对胞外酸性与肿瘤细胞迁移扩散之间关系的深入研究将有助于发现更多新的抗肿瘤转移药物.本文就肿瘤酸性微环境的形成、肿瘤细胞的质子感知制、胞外酸性环境对肿瘤浸润转移的影响及如何将肿瘤pH调节应用于癌症治疗等方面的内容予以综述.     

一株假单胞菌(Pseudomonas sp．)产生的胞外脂酶

假单胞菌(Psendomonas sp．)生长在一定的培养条件中能产生胞外脂酶。 最适碳源为1．0％淀粉,氮源为1．0％蛋白胨。一些植物油,如橄榄油、糠油、菜油等能诱导脂酶的大量产生,诱导脂酶产生的橄榄油最适浓度为0．5％。无机离子在菌培养过程中对脂酶产率影响很大,K+、Na+、Mg2+、Ca2+等对脂酶产生有促进作用,而Mn2+、Ba2+、Zn2+、Fe3+、Co2+、Cu2+件等则抑制脂酶产生。非离子表面活性剂(tween、span及糖脂)能刺激胞外脂酶的产生。     

胞外体--免疫治疗中的"特洛伊木马"

胞外体是源于多种真核细胞的多泡体,通过后者与质膜融合释放到细胞外的一种膜性小囊泡,在多种生理过程中发挥作用。近年研究发现,由抗原提呈细胞分泌的胞外体富集MHCI/II类分子、协同刺激分子、热休克蛋白70和热休克蛋白90等多种生物活性分子于一身,像“特洛伊木马”一样,在体内外免疫调节中起关键作用。本文就胞外体的基本特征、生产纯化方法及其作为一种新型的亚细胞疫苗在抗肿瘤和抗病毒免疫中的应用前景予以综述。     

蛹虫草原生质体紫外诱变选育胞外多糖高产菌株

蛹虫草是重要的药食兼用两用真菌,具有较高的医用及经济价值。本文通过单因素和正交试验的方法研究了不同酶系统、酶解温度、酶解时间、渗透压稳定剂、菌龄对蛹虫草原生质体形成的影响,并对蛹虫草原生质体进行紫外诱变,以生物量和胞外多糖产量为指标选育胞外多糖高产菌株。结果表明：在30℃、1％溶壁酶＋0．5％蜗牛酶＋0．5％纤维素酶条件下,以甘露醇为渗透压稳定剂对4日龄蛹虫草菌丝酶解2h,原生质体产量可达到9．2×10^6个/mL。从150株诱变株中筛选出1株最佳正诱变株,编号为44#,经深层培养其生物量比出发菌株提高10％,胞外多糖产量提高84．3％,继代培养10代后,遗传稳定性良好。     

Sequential release of both basic and acidic isoperoxidases to the media of suspension cultured cells of Capsicum annuum

Summary The establishment of suspension cell cultures from trimmed cotyledons of pepper (Capsicum annuum L.) provides a new experimental system for studying the relationship between release of peroxidase (EC 1.11.1.7) into the free intercellular spaces and plant cell growth. In contrast with several other species, the total peroxidase activity in the medium increased continuously during the post-exponential growth phase of the pepper cell culture, and this was correlated with the growth inhibition of pepper cells cultivated in suspension. The increase in the peroxidase activity in the culture medium was the consequence of a differential release of isoperoxidases, prominently marked by a primary release of basic isoperoxidases, followed by a strong increase in the level of acidic isoperoxidases. Thus, pepper cells cultures constitute a new experimental system for studying the regulation of the sequential release of basic and acidic isoperoxidases, which occurs during the growth cessation of plant cells.     

Physiological Aspects of Biosynthesis of Lignin Peroxidases by Phanerochaete chrysosporium

Methods based on UV-visible diffuse reflectance spectroscopy were used to study the physiological aspects of lignin-peroxidase biosynthesis by Phanerochaete chrysosporium. Here we introduce the use of cytochrome aa₃ as an indicator of active fungal biomass and of its redox state to calculate the oxygen mass transport coefficient between the growth medium and the fungal cell interior. When lignin peroxidase biosynthesis was enhanced by the addition of Tween 80 or Tween 20 to the growth medium, a higher proportion of reduced cytochrome aa₃ and a higher oxygen diffusion barrier were observed compared with control cultures. In cultures supplemented with Tween 80 or Tween 20, a higher oxygen mass transport coefficient between the growth medium and the interior of the fungal cell was also found. The beginning of the lignin peroxidase activity in these cultures was found to coincide with a temporary cessation in the dry biomass increase and a reduction in the relative active-biomass concentration. During the lignin peroxidase activity, a decrease in the intracellular pH and an increase in the growth medium pH were determined in cultures supplemented with Tween 80.     

Kinetics of xanthan production when NH3-N limits biomass synthesis and glucose limits polysaccharide synthesis

The bacterium Xanthomonas campestris, which synthesizes the commercially important polysaccharide xanthan, was grown aseptically in 1.2 L fermenters using semicontinuous cell culture technique (d' = 0.0035 h-1). The effects of carbon-substrate concentration on xanthan production were investigated at three initial glucose concentrations (Go = 15, 20, 25 g/L). Cell biomass synthesis was nitrogen-limited by use of a chemically defined medium that contained NH3-N as the sole nitrogen source at a concentration where it was exhausted before glucose. A linear relationship between biomass synthesis and NH3-N depletion was observed. This relationship remained valid only until NH3-N exhaustion, after which biomass concentration slowly rose another 20 percent before declining. Another linear relationship was found between xanthan synthesis and glucose uptake. This relationship was unaffected by the disappearance of NH3-N and held through glucose exhaustion. The quasi-stoichiometric yield coefficients obtained for each linear relationship were not affected by G0-. Biomass synthesis kinetics showed no variation with G0 before NH3-N exhaustion; afterwards, cell biomass decline was delayed by increasing G0. Xanthan synthesis kinetics displayed no detectable response to depletion of NH3-N and plateauing of biomass concentration; however, there was a marked slow down in the net rate of xanthan synthesis and a drop in xanthan yield after cell biomass decline became noticeable.     

Production of biomass and ligninolytic enzymes by Pleurotus ostreatus in submerged culture.

The production of biomass and ligninolytic enzymes by Pleurotus ostreatus was analysed in synthetic medium with yeast extract and different glucose concentrations (0.5 - 20 g/l), at different pH (3.5-6.5) and incubation temperatures (23-32 degrees C). The best culture condition were: initial glucose concentration of 5 g/l, initial pH between 5.5-6.5 and incubation temperature between 26-29 degrees C. The saturation constant for glucose (Ks) was 1.75 g/l. The biomass concentration reached 8.6 g/l with a glucose addition of 20.0 g/l to the culture medium. The control of pH allowed an increment of 0.5 g/l of biomass concentration. The birreactor produced pellets with a homogeneous distribution of diameter size of 3.4 -/+ 0.2 mm. Approximately, 307 U/l of laccase and 0.41 U/l of manganese peroxidase were obtained in extracellular liquid medium and 0.015 U/g of laccase and 0.809 U/g of manganese peroxidase were obtained in solid substrate. Lignin peroxidase activity was not detected at any condition.     

Peroxidase as a developmental marker in plant tissue culture.

Peroxidase was studied as a developmental marker in pumpkin (Cucurbita pepo L.) callus lines and horse-radish (Armoracia lapathifolia Gilib) transformants. Embryogenic callus lines DE grown on MS medium with 2.4-D and NA-3 grown on medium with NAA and adenine sulfate showed about a 20 times higher enzyme activity than the habituated non-embryogenic line Z5b/T grown on medium without hormones. A rise in peroxidase activity indicated that somatic embryogenesis was triggered in a few habituated tissue cultures. Separated globular embryoids had a manifold lower enzyme activity than the callus from which they originated. SDS-electrophoresis showed distinct polypeptide patterns between the horse-radish leaves and crown galls, but the tumor characteristic protein bands failed to be identified. In horse-radish crown galls and short bushy plants regenerated from hairy roots an enhanced peroxidase activity was registered. Due to its high peroxidase level and abundant biomass production horse-radish transformants should facilitate enzyme production.     

九连小蘖悬浮培养细胞生理生化研究 Ⅰ.细胞生长与培养液的电导率和过氧化物酶活性的变化

本文报道了九连小蘖细胞悬浮培养过程中,细胞生长与培养液的电导率、pH值、可溶性糖含量及过氨化物酶活性的变化。实验表明细胞生长曲线与培养液的电导率、可溶性糖含量变化的曲线恰成镜像关系。而细胞生长曲线与培养液的过氨化物酶活性变化的曲线相互平行。从而,可以通过监测培养液的电导率和过氧化物酶活性的变化来了解细胞生长状况,并可作为植物细胞培养过程中生物量增长的参考指标。     

九连小蘖悬浮培养细胞生理生化研究 Ⅰ.细胞生长与培养液的电导率和过氧化物酶活性的变化

本文报道了九连小蘖细胞悬浮培养过程中,细胞生长与培养液的电导率、pH值、可溶性糖含量及过氨化物酶活性的变化。实验表明细胞生长曲线与培养液的电导率、可溶性糖含量变化的曲线恰成镜像关系。而细胞生长曲线与培养液的过氨化物酶活性变化的曲线相互平行。从而,可以通过监测培养液的电导率和过氧化物酶活性的变化来了解细胞生长状况,并可作为植物细胞培养过程中生物量增长的参考指标。     

Progress and prospects in the use of peroxidase to study cell development

The need for peroxidase purification is stressed as a requirement for comparative studies on isoenzyme structure as well as for detailed investigations on biosynthesis. A single cationic protein possessing the major peroxidase activity was isolated from the medium in which peanut cells had grown. The antibodies raised against this pure protein were employed as a probe to study the site of synthesis of peroxidase in the cell as well as the proportion of total synthesized protein which was peroxidase. Structural studies on the purified isoenzymes suggest the presence of three gene loci for peroxidase in cultured peanut cells. The results are discussed together with potential assays for induction of this enzyme and the relationship to cell development.     

On Extraction and Quantitation of Plant Peroxidase Isoenzymes

Peroxidase in tobacco callus tissue differed in extract-ability depending on the subcellular distribution of the enzyme. Based on extractability it consisted of four fractions: freely soluble and less freely soluble in phosphate buffer, KCl-soluble, and insoluble. The latter two fractions were un-extractable by a phosphate buffer alone. The different fractions contained varied proportions of peroxidase isoenzymes. The extractability of indoleacetic acid oxidase was similar. A medium of high ionic strength is essential for quantitative extraction of peroxidase and indoleacetic acid oxidase isoenzymes. For quantitation of isoperoxidase activity on polyacryl-amide gel following electrophoretic separation, benzidine and o-dianisidine were better hydrogen donors than guaiacol and pyrogallol. The optimum pH was 4.5, but a citrate buffer was inhibitory. The optimum conditions included an acetate buffer at pH 4.5, a substrate concentration of 0.03 %, benzidine as the hydrogen donor, and a 3-minute treatment with 7 % acetic acid after staining. The color intensity of the bands remained unchanged for at least three days. With appropriate sample size and reaction time there was a linear relationship between enzyme concentration and activity.     

Modelling the growth kinetics of Phanerochaete chrysosporium in submerged static culture.

The potential commercial application of Phanerochaete chrysosporium requires methods for quantitatively predicting growth and substrate utilization. The growth kinetics of P. chrysosporium INA-12 (CNCM I-398) were investigated and modelled under nonlimiting nitrogen and carbon conditions in submerged static culture. This strain, unlike other strains, does not require nutrient limitation for induction of lignin peroxidase. Maximum levels of lignin peroxidase activity were reached 7 days after culture initiation, when almost 80% of the initial glycerol and 70% of the initial nitrogen were still present. Lignin peroxidase levels then decreased, while biomass levels increased until about day 14. The ratio of cell dry weight to wet weight was constant until the maximum biomass concentration was achieved, after which there was a decrease in the water content. The change in this ratio reflects cell lysis as it correlated with increased concentrations of nitrogen in the media, arising from cell leakage. The suitability of four growth models to predict growth, and in some cases glycerol consumption, was evaluated. A simple linear model and the Emerson model performed poorly for the early stages of growth, while a modified Williams model and the Monod model predicted substrate and biomass concentrations equally well. All models will predict biomass concentrations during the active growth phase, but they should not be used to predict biomass concentrations after the stationary growth phase, when cell lysis becomes significant.