In vitro reassembly of nuclear envelopes and organelles in Xenopus egg extracts

We reconstituted bilayer nuclear membranes, multilayer membranes, and organelles from mixtures ofXenopus laevis egg extracts and demembranatedXenopus sperm nuclei. Varying proportions of the cytosolic and vesicular fractions from the eggs were used in the reconstitution mixtures. A cytosol：vesicle ratio of 10：1 promoted reassembly of the normal bilayer nuclear membrane with inserted nuclear pore complexes around the decondensed Xenopus sperm chromatin. A cytosol： vesicle ratio of 5：1 caused decondensed and dispersed sperm chromatin to be either surrounded by or divided by unusual multilayer membrane structures with inlaid pore complexes. A cytosol：vesicle ratio of 2.5：1 promoted reconstitution of mitochondria, endoplasmic reticulum networks, and Golgi apparatus. During reassembly of the endoplasmic reticulum and Golgi apparatus, vesicular fragments of the corresponding organelles fused together and changed their shape to form flattened cistemae, which were then stacked one on top of another.

Association of DNA with nuclear matrix in in vitro assembled nuclei induced by rDNA from Tetrahymena shanghaiensis in Xenopus egg extracts

The nuclei assembled from exogenous DNA or chromatin in egg extracts resemble their in vivo counterparts in many aspects. However, the distribution pattern of DNA in these nuclei remains unknown. We introduced rDNA from the macronuclei of Tetrabymena into Xenopus cellfree extracts to examine the association of specific DNA sequences with nuclear matrix (NM) in the nuclei assembled in vitro. Our previous works showed the 5’NTS (nontranscription sequences) of the rDNA specifically bind to the NM system in the macronuclei. We show now the rDNA could induce chromatin assembly and nuclear formation in Xenopus cell-free system. When we extracted the NM system and compared the binding affinity of different regions of rDNA with the NM system, we found that the 5’NTS still hold their binding affinity with insoluble structure of the assembled nuclei in the extracts of Xenopus eggs.

Nuclear assembly of purified Crythecodinium cohnii chromosomes in cell-free extracts of Xenopus laevis eggs

Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation, nuclear envelope assembly, and nuclear reconstitution.Dinoflagellate Crythecodinium cohnii is a kind of primitive eukaryote which possesses numerous permanently condensed chromosomes and discontinuous double-layered nuclear membrane throughout the cell cycle. The assembled nuclei, being surrounded by a continuous double membrane containing nuclear pores and the uniformly dispersed chromatin fibers are morphologically distinguishable from that of Dinoflagellate Crythecodinium cohnii. However, incubation of dinoflagellate Crythecodinium cohnii chromosomes in the extracts from dinoflagellate Crythecodinillm cohnii cells does not induce nuclear reconstitution.

The microtubule aster formation and its role in nuclear envelope assembly around the sperm chromatin in Xenopus egg extracts

Nuclear envelope is a dynamic structure in the cell cycle. At the beginning of mitosis, nuclear envelope breaks down and its components disperse into the cytoplasm. At the end of mitosis, nuclear envelope reassembles using the dispersed components. Searching for the mechanisms of the nuclear disassembly and reassembly has for a long time been one of the key projects for cell biologists. In this report we show that microtubules take a role in the nuclear envelope assembly around the sperm chromatin in Xenopus egg ex-tracts. Microtubule cytoskeleton has been demonstrated to take roles in the transport of intracellular membranes such as Golgi and ER vesicles. We found that the nuclear envelope assembly needs functional microtubules. At the beginning of the nuclear assembly, microtubules nucleated to form a microtubule aster around the centrosome at the base of the sperm head. Using the microtubule drug colchicine to dis-rupt the microtubule nucleation, nuclear envelope reassem-bly was seriously inhibited. If the microtubules were stabi-lized by taxol, another microtubule drug, the nuclear enve-lope reassembly was also interfered, although a significantly large aster formed around the chromatin. Based on these observations, we propose that microtubules play an impor-tant role in the nuclear envelope reassembly maybe by transporting the nuclear envelope precursors to the chroma-tin surfaces.

Specific degradation of keratin in Xenopus laevis egg extracts undergoing apoptosis

Cytochrome c activates apoptosis specific protease XCPP32 when being added to Xenopus laevis egg extracts, and induces apoptosis in this cell-free system. During apoptosis, cyto-skeleton proteins in egg extracts are degraded. Western blot assay indicates that 42-ku acidic keratin in egg extracts has been degraded by XCPP32. The degradation of 42-ku keratin may be crucial in apoptosis.

NUCLEAR RECONSTITUTION IN THE Xenopus laevis EGG EXTRACTS CELL-FREE SYSTEM

Ⅰ. INTRODUCTIONNuclear reconstitution (nuclear assembly )was reported by several laboratories in recent years and regarded as an important advance in cell biology. In vitro nuclear assembly system provides a good model to study the organization and function of nucleus, chromatin, nuclear envelope and nuclear matrix. Although it is too early to evaluate its significance in theory and in bioengineering, there is no doubt that the prospect in this field is inspiring.

Nuclear reconstitution of demembranated Orychophragmus violaceus sperm in Xenopus laevis egg extracts

The cell-free extracts from animal Xenopus laevis egg could induce chromatin decon- densation and pronuclear formation from demembranated plant (Orychophragmus violaceus)sperm. The demembranated Orychophragmus violaceus sperm began to swell in 30 min incubation, and then were gradually decondensed. The reassembly of nuclear envelope in the reconstituted nuclei had been visualized by means of electron microscopy and fluorescent microscopy. Membrane vesicles fused to form the double envelope around the periphery of the decondensed chromatin. The morphology of the newly formed nucleus, with a double membrane, was similar to those nuclei after fertilization. Transmission electron microscope micrograph of the whole mount prepared nuclear matrix-lamina showed the reconstituted nucleus to be filled with a dense network.

Extraction and identification of membrane proteins from black widow spider eggs

The eggs of oviparous animals are storehouses of maternal proteins required for embryonic development. Identification and molecular characterization of such proteins will provide much insight into the regulation of embryonic development. We previously analyzed soluble proteins in the eggs of the black widow spider （Latrodectus tredecimguttatus）, and report here on the extraction and mass spectrometric identification of the egg membrane proteins. Comparison of different lysis solutions indicated that the highest extraction of the membrane proteins was achieved with 3%-4% sodium laurate in 40 mmol/L Tris-HCI buffer containing 4% CHAPS and 2% DTT （pH 7.4）. SDS-PAGE combined with nLC- MS/MS identified 39 proteins with membranelocalization annotation, including those with structural, catalytic, and regulatory activities. Nearly half of the identified membrane proteins were metabolic enzymes involved in various cellular processes, particularly energy metabolism and biosynthesis, suggesting that relevant metabolic processes were active during the embryonic development of the eggs. Several identified cell membrane proteins were involved in the special structure formation and function of the egg cell membranes. The present proteomic analysis of the egg membrane proteins provides new insight into the molecular mechanisms of spider embryonic development.

Identification of a DNase activated in Xenopus egg extracts undergoing apoptosis

A cell-free apoptosis system was established by adding dATP and cytochrome c to Xenopus laevis egg extracts S-150. Accompanied by an incubation process, an apoptosis-specific DNase was activated in egg extracts which depended on Mg 2+ and inhibited by Zn 2+. Two nucleases existing in egg extracts were revealed by in-gel nuclease assay. Further experiments showed that 27 ku nuclease which was different from other Ca 2+/Mg 2+-dependent nucleases was a possible candidate involved in apoptosis.

Effects of Mango Skin or Mango Skin Combined with Paprika Extracts on Production Performance, Egg Quality and Egg Yolk Polyphenols

The phenc, lic compounds found in mango fruit are antioxidants, and contribute to a reduction in the risk of cardiovascular diseases. Mango carotenoids are synthesized in mango fruit during ripening. A major by-product of mango fruit is the skin, which is available after the fruits is consumed or used, particularly in the preparation of jams and fruit juices by the fruit canning industry. The objective of this study was to investigate the effects of dietary mango skin （MS） or mango skin combined with paprika extracts （PE） on production performance, egg quality, and egg yolk polyphenols. Total of 60 44-week-old Boris Brown hens was assigned, based on egg production rate and body weight, to 6 groups （10 birds in each group）. The laying hens were fed a basal diet （control group, 18 CP, 2,800 kcal/kg ME）, a basal diet supplemented with 0.1% PE, 1% or 5% MS, or with a mixture of 0.1% PE with 1% or 5% MS respectively. During the three-week experimental feeding period, the birds had free access to feed and water. Feed consumption was measured weekly and egg production was recorded daily. The results for initial body weight, feed consumption, hen-day production, egg mass, mortality, and final body weight did not indicate any effects of the different treatments （P 〉 0.05）. There were no significant differences （P 〉 0.05） in shell-breaking strength, shell thickness, shell ratio, yolk ratio or Haugh units, except in the case of egg yolk color. Roche yolk color fan scores were better in all experimental groups than in the control （P 〈 0.0001）. The yolk color, yellow index, and ratio of redness to yellowness were greater （P 〈 0.0001） in the 0.1% PE, the 0.1% PE ＋ 1% MS, and the 0.1% PE ＋ 5% MS group than in the 1%, 5% MS, and control groups. Compared with the control, lightness was decreased significantly in the 0.1% PE, the 0.1% PE ＋ 1% MS, and the 0.1% PE ＋ 5% MS groups （P 〈 0.05）, whereas redness was increased significantly in the 0.1% PE, 0.1% PE ＋ 1% MS, and 0.1% PE ＋ 5% MS groups （P 〈 0.0001）. No significant differences among the treatments were observed in either yellowness or egg yolk polyphenols. The results of the present experiment indicate that dietary mango skin or mango skin combined with paprika extracts did not show adverse effects on production performance, egg quality or egg yolk polyphenols. Moreover, supplementation with only mango skin did not enhance egg yolk color.

Does the African garden egg offer protection against experimentally induced ulcers?

Objective:To evaluate the possible antiulcer effect of the African garden egg,Solanum aethiopicum(S.aethiopicum)(a domestic vegetable) experimentally in rats.Methods:A methanol extract of the plant fruit was prepared by maceration.Twenty five overnight fasted rats for each model were divided randomly into five groups of five rats.Groups 1,2,3,4 and 5 received normal saline,extract dose levels of 100,200 and 400 mg/kg and 100mg/kg of ranitidine respectively.All administrations were given orally.For the indomethacin and aspirin models, ulcerogenic agents(indomethacin,50 mg/kg and aspirin 200 mg/kg) were given thirty minutes after extract treatments and animals sacrificed 8 h later.The acidified ethanol model(ethanol 60%+0.1 mol/L HC1) was given 1hr after extract treatment and animals sacrificed 1 h later. Ulcer index was checked and analysed with appropriate statistical tools.Results:Extract of S. aethiopicum showed positive effect on all the models used.It produced higher ulcer inhibition than ranitidine in the indomethacin and acid-ethanol models.All the anti-ulcer effects of the extract at different doses were dose dependent but only in indomethacin model did it produce statistically significant(P【0.05) ulcer reduction in all doses compared to control.Conclusions: Garden egg,a readily cultivated crop vegetable possesses ulcer protective properties against ulcers induced experimentally making it a cheap source of natural anti-ulcer remedy.

不同C_(18)固相萃取柱对鹌鹑蛋中6类兽药残留测定的影响研究

使用不同C_(18)固相萃取柱对鹌鹑蛋进行处理,通过对比基质效应、回收率、精密度的结果,评价不同C_(18)固相萃取柱对6类兽药残留测定情况的影响。结果表明,使用不同C_(18)固相萃取柱后,6类目标物质保留时间出峰一致,且目标物质峰形良好,净化效果基本一致。对同一种化合物的基质效应标准偏差均小于10%。3种不同浓度下,6类兽药的平均回收率为71.4%~116.1%,精密度为2.6%~15.5%,均满足使用效果。

黄芩提取物对热应激蛋鸡产蛋性能、蛋品质和免疫功能的影响

文章旨在探讨黄芩提取物对热应激蛋鸡产蛋性能、蛋品质和免疫功能的影响。试验将600只产蛋性能相近、70周龄左右的健康罗曼粉蛋鸡随机分为5组,每组8个重复,每个重复15只。其中对照组和热应激组分别饲喂基础日粮。试验Ⅰ、Ⅱ和Ⅲ组分别在基础日粮中添加15、30和60 mg/kg黄芩苷提取物的,试验为期9周。结果表明:1~3周,试验Ⅱ和Ⅲ组蛋鸡产蛋率较热应激组分别显著提高8.27%和11.92%(P<0.05),料蛋比显著降低7.21%和9.34%(P<0.05),软破蛋率分别显著降低7.38%和26.57%(P<0.05);4~6周,试验Ⅱ和Ⅲ组蛋鸡产蛋率较热应激组分别显著提高11.35%和10.98%(P<0.05),料蛋比显著降低10.50%和10.45%(P<0.05),软破蛋率分别显著降低15.33%和16.11%(P<0.05);7~9周,试验Ⅱ和Ⅲ组蛋鸡产蛋率较热应激组分别显著提高4.08%和7.71%(P<0.05),料蛋比显著降低2.95%和6.01%(P<0.05),软破蛋率分别显著降低14.09%和15.28%(P<0.05)。试验Ⅱ和Ⅲ组蛋壳强度较热应激组显著提高(P<0.05),试验Ⅲ组哈氏单位较热应激组显著提高(P<0.05),试验Ⅱ和Ⅲ组蛋粗蛋白质和钙含量较热应激组显著提高(P<0.05),试验Ⅱ和Ⅲ组蛋鸡血清白细胞介素-10(IL-10)和肿瘤坏死因子α(TNF-α)含量显著降低(P<0.05),免疫球蛋白A(IgA)和免疫球蛋白G(IgG)含量显著提高(P<0.05);总之,试验数据显示,补充黄芩提取物(60 mg/kg)可通过增强热应激蛋鸡免疫力提高蛋鸡生产性能,改善蛋品质。

西番莲茎叶提取物对热应激下蛋鸡产蛋性能、蛋品质和抗氧化性能的影响研究

试验旨在了解西番莲茎叶提取物对夏季热应激下蛋鸡产蛋性能、蛋品质和抗氧化性能的影响。试验于夏季选取210日龄海兰褐蛋鸡240只,随机分为对照组、低剂量组、中剂量组和高剂量组4组,对照组饲喂基础日粮,低、中、高剂量组分别在基础日粮中添加250、500、1000 mg/kg的西番莲茎叶提取物,试验期42 d。结果显示,低、中、高剂量组蛋重均显著高于对照组(P<0.05),高剂量组产蛋率显著高于对照组(P<0.05),高、中剂量组平均日采食量显著高于对照组(P<0.05),高剂量组料蛋比显著低于对照组(P<0.05)。高、中剂量组鸡蛋的蛋白高度和哈夫单位显著高于对照组(P<0.05),高剂量组鸡蛋的蛋壳厚度显著高于对照组(P<0.05);各组蛋黄重、蛋黄比例、蛋形指数和蛋黄颜色无显著差异(P>0.05)。高、中剂量组血清中谷胱甘肽过氧化物酶活性显著高于对照组(P<0.05);低、中、高剂量组血清中丙二醛(MDA)含量显著低于对照组(P<0.05);各组间血清中超氧化物歧化酶、总抗氧化能力和过氧化氢酶活性差异不显著(P>0.05)。说明西番莲茎叶提取物可提高夏季热应激条件下海兰褐蛋鸡产蛋性能、蛋品质和抗氧化性能。

通过式固相萃取结合高效液相色谱-四极杆飞行时间质谱测定鸡蛋中62种兽药残留

建立了固相萃取结合高效液相色谱-四极杆飞行时间质谱(HPLC-QTOF)同时测定鸡蛋中62种兽药残留的方法。鸡蛋均质后经90%甲酸乙腈水溶液提取后,PRiME HLB固相萃取小柱净化,氮吹后复溶,用(HPLC-QTOF)进行检测。62种兽药在0.5~500 ng/mL浓度范围内线性关系良好(r^(2)≥0.99),回收率在71.2%~113%之间,日内变异系数范围为1.3%~14.3%,日间变异系数范围为4.2%~18.3%,检测限为1.0~2.0μg/kg,定量限为2.0~5.0μg/kg。本方法简便快捷、准确可靠,适用于鸡蛋中62种兽药的同时测定。

5种植物提取物对小菜蛾的生物活性

植物源杀虫剂相较于化学农药,具有源于自然易分解、生物活性多样、害虫不易产生抗药性等优点。为研制高效低毒的植物源杀虫剂,为开发研究新型植物杀虫剂提供数据基础,从已报道的具有杀虫活性的5个科中选取丁香、甘草、独活、赤芍、八角5种植物,并在室内测定5种植物水醇提取物对小菜蛾3种虫态的生物活性。采用浸卵法、浸叶法和非选择叶盘法测定5种植物提取物对小菜蛾卵触杀、幼虫胃毒、成虫产卵忌避效果;并通过高倍显微镜观察5种植物提取物处理后小菜蛾卵的外部形态。结果表明,5种植物提取物对小菜蛾卵和幼虫均有显著的杀虫活性,对成虫具有产卵忌避活性,生物活性与剂量基本呈正相关;在植物提取物质量浓度为100 mg/mL时,对小菜蛾卵和幼虫的校正死亡率以及成虫产卵忌避率均在85%以上。丁香、甘草、独活、赤芍、八角提取物对小菜蛾卵的毒力LC50分别为9.57、16.16、20.42、9.00、7.57 mg/mL,对小菜蛾幼虫的毒力LC50分别为7.46、13.49、21.31、54.67、9.87 mg/mL。其中,丁香提取物对小菜蛾卵和幼虫的毒力最高;5种植物提取物处理后的小菜蛾卵发育受到影响,卵内组织器官发育受阻或虫体畸形。丁香、甘草、独活、赤芍、八角提取物具有作为植物源杀虫剂材料来源用于防控小菜蛾的潜在价值。

高效液相色谱-串联质谱法检测鸡组织及鸡蛋中新型兽药氟雷拉纳残留

基于高效液相色谱-串联质谱法建立鸡组织(鸡肉、鸡肝、鸡皮和鸡肾)及鸡蛋中新型兽药氟雷拉纳残留的检测方法。将低(0.10 mg/kg)、中(0.25 mg/kg)、高(0.50 mg/kg)剂量氟雷拉纳分2次灌胃(间隔7 d)白羽母鸡后获得阳性动物样品。样品经乙腈提取、PRiME HLB固相萃取小柱净化后,以甲醇和5 mmol/L甲酸铵溶液为流动相,Eclipse Plus C18色谱柱为分离柱进行梯度洗脱,电喷雾负离子模式电离,在多反应监测模式下检测,绘制基质匹配校准曲线,外标法定量。结果表明:氟雷拉纳在0~200 ng/mL质量浓度范围内具有良好的线性关系,相关系数(R^(2))>0.99,方法检出限为2.0μg/kg,定量限为5.0μg/kg;在5、15、50μg/kg加标水平下,鸡组织(鸡肉、鸡肝、鸡皮、鸡肾)和鸡蛋中氟雷拉纳的平均加标回收率为81.2%~99.1%,相对标准偏差为1.29%~7.86%(n=6)。该方法成功应用于阳性动物样品中氟雷拉纳残留量的检测。综上,该方法简便、快速、灵敏,适用于鸡组织及鸡蛋中氟雷拉纳兽药残留的检测。

固相萃取结合高效液相色谱-串联质谱法测定禽蛋中5种硝基咪唑类药物残留

目的:建立一种固相萃取结合高效液相色谱-串联质谱快速测定禽蛋中甲硝唑、洛硝哒唑、替硝唑、奥硝唑和二甲硝唑残留的分析方法。方法:禽蛋样品经5%三氯乙酸溶液涡旋提取、MCX固相萃取小柱净化,采用ACQUITYUPLC^(TM)BEH C_(18)色谱柱作为分析柱,采用0.1%甲酸水-甲醇作为流动相体系,采用正离子喷雾模式电离、多反应离子监测方式进行分析。结果:在0.5~50.0 ng/mL的线性范围内,5种硝基咪唑类药物的线性方程均呈良好线性关系,相关系数r^(2)>0.9984。5种硝基咪唑类药物的检出限低至0.25μg/kg,在低、中、高三组质量浓度水平下,平均加标回收率在70.8%~111.3%范围内,相对标准偏差在1.4%~6.2%范围内。结论:该方法有机试剂消耗少、低毒环保、前处理简单易操作、分析速度快、灵敏度和准确度较高,重现性好,适用于禽蛋中硝基咪唑类药物残留的检测分析。

食品及饲料脂肪检测方法的优化开发

对食品、饲料、蛋及蛋制品中脂肪检测方法进行探讨,并提出一种改进后新的脂肪测定方法,以酸水解-索氏抽提法为基础,对实验条件进行改进后,新方法能够同时满足食品、饲料、蛋及蛋制品中脂肪的测定。通过数据比较,新方法的相对标准偏差为0.81%~3.76%。与不同产品脂肪检测国家标准方法相比,相对误差平均值在0.77%~1.75%。新方法的提出,为实验室及日常检验检测工作提供了一个实验更加便捷、准确度更好、污染更小、试剂回收率更好的检测方法。

金银花黄芩桑叶粗提物对产蛋后期蛋鸡生产性能、蛋品质及血清生化指标的影响

试验旨在探究金银花黄芩桑叶粗提物对产蛋后期蛋鸡生产性能、蛋品质、屠宰性能及肉品质的影响。选取4320只生产性能相近、健康状况良好的670日龄大午金凤蛋鸡,随机分成2组,每组6个重复,每个重复360只。对照组饲喂基础饲粮,正常饮水,试验组在饮水中添加0.3%的金银花黄芩桑叶粗提物。试验期30 d。结果表明,与对照组相比,试验组正常蛋数量、产蛋总数、正常蛋总重量、正常蛋均重、产蛋率、小黄色卵泡数量显著升高(P<0.05);异常蛋比例、平均斑点蛋数、平均钙盐沉积蛋数、平均波纹蛋数、斑点蛋比、钙盐沉积蛋比显著降低(P<0.05)。与对照组相比,试验组胸肌pH值、腿肌pH值、腿肌系水力、腿肌红度(a*)值和黄度(b*)值均显著升高(P<0.05);试验组胸肌亮度(L*)值和腿肌L*值均显著降低(P<0.05)。研究表明,产蛋后期蛋鸡饮水中添加金银花黄芩桑叶粗提物可有效提高产蛋性能,改善肉品质和血清生化指标。