植物原生质体的制备与活力检测研究进展

原生质体是进行植物遗传改良和细胞各种生理生化特性研究的平台.本文对近些年制备原生质体的材料选择、预处理、游离、纯化和活力检测等方面的研究进展进行了综述,分析了影响原生质体的分离和纯化的有关因素,并根据相关文献讨论了今后原生质体重点研究方向.     

HPLC法测定香菇中有机酸含量的检测技术研究

本实验建立了HPLC法同时测定香菇中酒石酸、苹果酸、乙酸和柠檬酸的方法。在酸提条件下,利用Agela Venusll MP C18色谱柱,以2%CH3OH-20 mM(NH4)2HPO4(pH 2.40)为流动相,流速为1.0 mL/min,柱温为35℃,检测波长为210 nm,进样量为10μL。四种有机酸均在8 min内出峰。本法准确、可靠、快速、简便,检出限较低,适合于大批量样品的分析与检测。     

蛇床子有效成分的RP—HPLC测定及其4种提取方法的比较研究

以蛇床子素、欧前胡素、佛手柑内酯为指标成分,建立了蛇床子有效成分分离测定的HPLC新方法,并对95%乙醇冷浸、超声、回流、索氏提取蛇床子所得的香豆素成分进行HPLC分析与比较。结果表明,HPLC新方法适合于蛇床子成分的质量控制,其中蛇床子不同提取液中各香豆素成分含量测定差异较大,且以超声提取为最佳。     

大黄药材、浸膏及其清胃颗粒剂质量的RP—HPLC分析

建立适合大黄药材、大黄浸膏及清胃颗粒剂过程分析与质量控制的反相高效液相色谱方法.采用YWG-ODS 10μm(200×4.0mm ID)色谱柱,流动相组成为甲醇-水-高氯酸(75250.1V/V),检测波长254nm,流速1.0ml/min,柱温控制20℃.以芦荟大黄素、大黄素、大黄酸、大黄酚和大黄素甲醚的峰面积作为定量信息,标准曲线外标法测定样品含量.结果表明,清胃颗粒剂中五种游离蒽醌成分的平均加样回收率分别为芦荟大黄素95.61%(RSD3.05%)、大黄素98.03%(RSD2.77%)、大黄酸100.1%(RSD2.21%)、大黄酚97.49%(RSD3.64%)和大黄素甲醚96.79%(RSD4.12%).本法操作简便、检测灵敏,适合于大黄药材、大黄浸膏及清胃颗粒剂过程分析与质量控制.     

甘蓝型油菜花粉超低温保存及其花粉活力的研究

研究超低温方法保存油菜花粉过程中的预冻和解冻处理方式对油菜花粉的形态、大小及其活力的影响。结果表明:采用0℃(12h)→-4℃(12h)→-20℃(12h)→-80℃(12h)变温预冻处理和用-80℃(1h)→-20℃(1h)→4℃(1h)逐步解冻后对油菜花粉形态大小和活力影响最小。而经过25℃室温解冻法和42℃快速解冻法处理后油菜花粉出现破裂,破裂率分别达到7.6%和9.1%。同时液氮保存油菜花粉的时间长短对花粉的大小及活力影响不大。     

甘蓝型油菜CMS微粉活力研究及其对杂交制种纯度的影响

通过对微粉和正常花粉在不同和同一雌蕊上的结实力比较和结实竞争研究,表明微粉虽能够授粉结实,但活力低、萌发慢、持续时间短、受精结实能力差,与正常花粉的竞争力弱,授微粉后间隔３－７２ｈ,再授恢复系花粉,其结实表现仍可达到或超过单独授恢复系花粉的结实率,最高的还超过了只授恢复系花粉结实率的９．５７％;授微粉后间隔０－４８ｈ再授恢复系花粉仍可获得７０．３５％－９１．００％的异交率,说明微粉对油菜杂交制种过     

植物乳杆菌高活力保存方法的研究及其应用

本研究旨在探讨植物乳杆菌的高活力保存方法,为植物乳杆菌饲料添加剂规模化、工业化生产奠定基础。采用4℃低温保存法、36℃烘干后常温保存法、阳离子活性载体保存法等3种方法对植物乳杆菌进行活力保存比较试验。以保存后活菌数不低于原值50%为参照标准,结果显示,对照组可保存15 d,4℃低温保存法可保存30 d,36℃烘干后常温保存法只能保存一周,而阳离子活性载体保存法则可保存2个月以上。结果表明,阳离子活性载体保存法可应用于植物乳杆菌饲料添加剂的规模化、工业化生产实践中。     

高活力胰岛素研究：B3—Lys猪胰岛素的结晶及初步晶体学分析

用化学半合成方法制备的Ｂ３—Ｌｙｓ猪胰岛素（Ｂ３ＫＰＩ）具有与天然猪胰岛素相同的体内生物活力,但其体外生物活力及受体结合活力比天然猪胰岛素高。本文报道Ｂ３ＫＰＩ的晶体生长,初步晶体学分析及衍射数据与处理。Ｂ３ＫＰＩ晶体的衍射分辨率可以达到２．１Ａ（１Ａ＝０．１ｎｍ）,空间群为Ｒ３,晶胞参数为：ａＨ＝ｂＨ＝８２．２３ＡＣＨ＝３４．１７Ａ．收集了一套２．１Ａ分辨率的衍射数据,数据完全度为８１％,Ｒｍｅｒｇｅ为２．９％。     

B27K—去B链C端三肽胰岛素的制备、生物活力与自聚合性质

液相合成五肽Gly Phe Phe Tyr Lys(Boc)Obut,与B链C端去八肽胰岛素酶促缩合得到B2 7K 去B链C端三肽胰岛素(B2 7K DTrI,B2 7K destripeptideinsulin)。用小鼠惊厥法和小鼠降血糖法测得B2 7K DTrI的整体生物活力为标准胰岛素的 80 % ,B2 7K DTrI与人胎盘细胞膜胰岛素受体结合能力为标准胰岛素的 ( 12 5± 13 ) %。用凝胶过滤法证明B2 7K DTrI自聚合性质降低 ,具有与去B链C端五肽胰岛素相同的单体性质。在B2 7K DtrI结构中 ,B2 7T被K取代 ,其优点是在酵母中表达其前体后 ,可以很方便地通过胰蛋白酶水解获得     

HPLC特征图谱结合含量测定的白芍及其炮制品的质量对比研究

建立白芍、炒白芍、酒白芍、硫熏白芍HPLC特征图谱,并结合多成分含量测定,为白芍、炒白芍、酒白芍和硫熏白芍的质量控制提供参考。采用Intersustain C₁₈(250 mm×4.6 mm,5μm)色谱柱,流动相为乙腈-0.1%醋酸水溶液,流速为每分钟1 mL,梯度洗脱,检测波长为230 nm,柱温为30℃,进样量为10μL。14批白芍、炒白芍、酒白芍和硫熏白芍的特征图谱,标定了6个共有峰,并均被指认,分别为没食子酸、儿茶素、芍药内酯苷、芍药苷、1,2,3,4,6-五没食子酰葡萄糖和苯甲酰芍药苷,而硫熏白芍标定7个共有峰,峰7为白芍硫熏后产生;且各色谱谱峰有较好的分离,但不同炮制品特征图谱存在一定差异;含量测定结果显示,白芍炒制、酒制及硫熏后,6种成分均有不同程度的变化;借助中药色谱指纹图谱相似度评价系统和SIMCA-P13.0软件对14批白芍、炒白芍、酒白芍和硫熏白芍进行相似度和正交偏最小二乘判别(OPLS-DA)分析,所建立的白芍和炮制品及硫熏品的质量评价方法稳定性、重复性好,可用于白芍、炒白芍、酒白芍和硫熏白芍的质量控制和评价。     

Pentazocine monitoring in rat hair and plasma by HPLC-fluorescence detection with DIB-Cl as a labelling reagent.

Pentazocine (PZ) in rat hair and plasma was determined by HPLC-fluorescence detection with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a labelling reagent and cyclazocine (CZ) as an internal standard (IS). PZ and IS extracted from hair or plasma sample were derivatized with DIB-Cl and the resulted solution was cleaned up with solid phase extraction. The isocratic separation of DIB-PZ and -CZ within 20 min could be achieved by a Wakopak Handy-ODS column (250 x 4.6 mm i.d.) using a mobile phase composed of 0.1 mol/L acetate buffer (pH 6.2):acetonitrile (25:75, v/v). The detection limits of PZ at a signal-to-noise ratio of 3 for rat hair and plasma were 0.18 ng/mg and 0.57 ng/mL, respectively. Reproducible and precise results could be obtained by an IS method with RSD values less than 6.6% for within- and between-day measurements. The method was successfully applied for the monitoring of PZ levels in Zucker rat hair and plasma samples after a single administration of 25 mg/kg PZ. Moreover, incorporation rates of PZ into black and white hair of Zucker rat were evaluated.     

现代教育理论在生物科技活动中的应用

以认知学习理论、建构主义理论、创新教育理论、多元智能理论、合作学习理论、服务学习理论为例,初步探讨了教育理论在生物科技活动中的应用。     

Microassay of heme oxygenase by high-performance liquid chromatography: Application to assay of needle biopsies of human liver

We developed a microassay for heme oxygenase, in which bilirubin (BR) production was measured by HPLC, and compared it to previously reported spectrophotometric methods. The microassay required as little as 5 mg wet human, rat, or chick embryo liver. Using the HPLC assay, values for heme oxygenase activity in extracts (10,000 g supernatant) of normal human liver obtained by needle biopsies were 44 +/- 7 (pmol BR.min-1.mg protein-1). Spectrophotometric assays of homogenates of human liver resulted in low values for heme oxygenase, due to unknown sources of interference. Comparative values of microsomal heme oxygenase activity were 294 +/- 25, 95 +/- 3, and 87 +/- 9 pmol BR.min-1.mg protein-1 for chick, rat, and human livers, respectively.     

农业活动对生物多样性的影响

农业生产活动如土地的农业利用、耕作、作物间套种植方式、放牧、农药化肥的使用以及农业动植物遗传改良（包括外来种引入）等是农业生产力提高的重要途径,同时也是影响生物多样性的重要因素之一。土地的不合理开发利用易导致生境破碎、生物多样性下降;大规模的机械耕作导致土壤动植物区系的变化,甚至某些物种的消失;农药（除草剂、杀虫剂等）的高度使用使非靶标动植物受到伤害;品种改良、外来种的引入以及远缘外源遗传物质的利用（如远缘杂交和ＤＮＡ导入分子育种）在丰富了遗传多样性的同时导致农作物类型和品种的简单化、一些古老的地方种和农家种等传统资源丢失等;而一些合理的农业措施（间套作、实行有机农场等）将有利于生物多样性的保持。农业活动注重的是农业生产力的提高而往往忽视其对农业系统中野生动植物（包括有害和无害）的影响以及野生动植物在维持系统稳定和平衡的作用。本文论述农业活动对生物多样性的影响以及生物多样性保护对提高农业生产力的作用,启示人们采取合理的农业活动方式,合理管理有害生物,减少农业活动对生物多样性的负面影响。     

Determination of isocitrate lyase and malate synthase activities in a marine bivalve mollusk by a new method of assay

1. A new method for the assay of isocitrate lyase (EC 4.1.3.1) was developed, based on the isolation of 14C-glyoxylate semicarbazone by co-crystallization with authentic carrier. The method was easily adapted to measure malate synthase (EC 4.1.3.2). 2. Interfering reactions were avoided with this method, and isocitrate dehydrogenase (ID) was easily distinguished from isocitrate lyase (IL). Assay of IL in germinating pumpkin seeds gave rates proportional to the amount of extract, with greater sensitivity and less variability than the spectrophotometric method. 3. Six species of marine bivalve mollusks were tested for IL activity, and two species produced glyoxylate: Crassostrea virginica at 0.10 mumol/hr/g tissue, and Petricola pholadiformis at 0.85 mumol/hr/g. The other four species, and four other marine invertebrates from other phyla, lacked detectable IL activity. 4. The rate of disappearance of glyoxylate in the malate synthase (MS) reaction indicated that Petricola had an activity of 0.60 mumol/hr/g: this is the first demonstration of activities of both IL and MS in a marine invertebrate.     

Stress degradation studies on betahistine and development of a validated stability-indicating assay method

The purpose of this work was to study the stability of betahistine (BET) at different stress conditions and to develop a sensitive stability-indicating high-performance liquid chromatographic (HPLC) assay method. The stress conditions applied were including the effect of heat, moisture, acid-base, and ultra-violet (UV) light. Betahistine and its decomposition products were derivatized by reaction with dansyl chloride (Dan-Cl) and analyzed by HPLC equipped with fluorescence detector (FL) set at 336 and 531 nm as excitation and emission wavelengths, respectively. The drug was particularly labile at UV light and oxygen rich media. Two potential degradation products could be separated and identified by spectral methods. The chromatographic method involved Zorbax Eclipse XDB-C(18) column kept at 30+/-2 degrees C and a gradient elution with mobile phase composed of acetonitrile and 0.02 mol L(-1) sodium acetate. The response factor of dansylated BET monitored by fluorescence detection was 32 times more than its UV response. The calibration curve of BET in bulk form was linear from 0.005 to 4.2 ng microL(-1). Intraday and interday precision were less than 0.04% (CV), and accuracy was between 99.2% and 100.9% over 2.0 ng microL(-1). The limit of detection was 0.002 ng microL(-1). The method was also validated for sample stability during reaction, robustness and selectivity. The method was applied for purity testing of betahistine in tablet form.     

The dual oxygenase and peroxidase activities of porphobilinogen oxygenase and horseradish peroxidase: a study using the reaction with phenylhydrazine.

Porphobilinogen oxygenase and horseradish peroxidase show dual oxygenase and peroxidase activities. By treating porphobilinogen oxygenase with phenylhydrazine in the presence of H2O2 both activities were inhibited. When horseradish peroxidase was treated in the same manner only the peroxidase activity was lost while its oxygenase activity toward porphobilinogen remained unchanged. The phenylhydrazine treatment alkylated the prosthetic heme group of porphobilinogen oxygenase and N-phenylheme as well as N-phenylprotoporphyrin IX were isolated from the treated hemoprotein. In horseradish peroxidase the modified heme was mainly 8-hydroxymethylheme. The apoproteins of the alkylated enzymes were isolated and recombined with hemin IX. The oxygenase and peroxidase activities of porphobilinogen oxygenase were entirely recovered in the reconstituted enzyme, while the reconstituted horseradish peroxidase regained 75% of its peroxidase activity.     

橙盖鹅膏多糖(AC-1)体外免疫调节活性、细胞毒性和抗肿瘤活性的研究

为研究橙盖鹅膏多糖的体外免疫调节活性、细胞毒性和抗肿瘤活性,本研究运用细胞学技术探究AC-1对T细胞、B细胞和RAW264.7三种免疫细胞的体外免疫调节活性;研究AC-1对小鼠成纤维细胞(L929)的细胞毒性以及对小鼠胃癌细胞(MFC)、小鼠肉瘤细胞(S180)的体外抗肿瘤活性。结果表明,AC-1能在体外显著刺激三种免疫细胞的增殖及RAW264.7细胞的吞噬,表明AC-1在增强机体免疫方面具有重要作用,进一步研究发现AC-1主要通过显著促进IgE和IgG的分泌来增强体液免疫。在浓度为5~20μg/mL时AC-1对L929细胞无显著的促进及抑制作用,说明AC-1对正常细胞无毒性;在浓度为10~20μg/mL时AC-1能显著抑制小鼠胃癌细胞(MFC)的生长,但对小鼠肉瘤细胞(S180)的抑制效果较弱,说明AC-1在体外能够直接抑制肿瘤细胞的生长,但对不同的肿瘤细胞具有不同的抑制效果。综上所述,橙盖鹅膏多糖(AC-1)在体外具有良好的免疫调节活性、抗肿瘤活性,但对不同的肿瘤细胞具有不同的抑制效果并且对正常细胞无毒性。