Osmotic microenvironments within tissue culture well plates are an important cause of variability in plant protoplast cultures

Summary To determine if the medium osmolality of plant protoplast cultures in 24-well tissue culture plates changes sufficiently during the culture period to affect development of the protoplasts the osmolality and the division of the protoplasts was monitored within the plates over a three week period. Large increases in osmolality were measured in cell-free experiments indicating that overall evaporation from the plates was substantial. The amount of evaporation from a given culture well depended on the position of the well within the plate; three microenvironments corresponding to the corner, edge, and middle positions of the plate consistently developed. Water placed in the recessed area between each culture well moderated the desiccation of the medium but did not eliminate the formation of microenvironments. The osmolality of the medium in protoplast cultures was higher than in the cell-free experiments but similar trends in terms of plate position were recorded. After 3 weeks of incubation of plates with water added between the wells, the osmolality of the medium in the protoplast cultures had increased 209 mOsm in the corner wells, had increased 77 mOsm in the edge wells, and had decreased 39 mOsm in the middle wells. As a result, there was a three-fold higher incidence of division of the protoplasts in the middle wells than in the corner wells. The non-uniformity of medium osmolality in protoplast cultures within tissue cultures well plates is important in experimental design, in the reproducibility of procedures between different laboratories, and in the preparation of replenishment medium for protoplast cultures to minimize osmotic shock. This work was supported by the College of Agriculture and Life Sciences and the Graduate School, University of Wisconsin-Madison; by McIntire-Stennis project WIS 3082; and by the USDA-Forest Service, North Central Forest Experiment Station (B. Haissig, Project Leader).     

Applications of cytotoxicity assays and pre-lethal mechanistic assays for assessment of human hepatotoxicity potential

While drug toxicity (especially hepatotoxicity) is the most frequent reason cited for withdrawal of an approved drug, no simple solution exists to adequately predict such adverse events. Simple cytotoxicity assays in HepG2 cells are relatively insensitive to human hepatotoxic drugs in a retrospective analysis of marketed pharmaceuticals. In comparison, a panel of pre-lethal mechanistic cellular assays hold the promise to deliver a more sensitive approach to detect endpoint-specific drug toxicities. The panel of assays covered by this review includes steatosis, cholestasis, phospholipidosis, reactive intermediates, mitochondria membrane function, oxidative stress, and drug interactions. In addition, the use of metabolically competent cells or the introduction of major human hepatocytes in these in vitro studies allow a more complete picture of potential drug side effect. Since inter-individual therapeutic index (TI) may differ from patient to patient, the rational use of one or more of these cellular assay and targeted in vivo exposure data may allow pharmaceutical scientists to select drug candidates with a higher TI potential in the drug discovery phase.     

广西灵香草挥发油化学成分

灵香草(Lysimachia foenum-graecum Hance)又名零陵香,为报春花科(Primulaceae)排草属(Lysimachia L.)植物,主产广西、云南、广东、四川、贵州等地[1].灵香草性味甘、淡、平,具行气、止痛、驱蛔功能,药理实验证明具有抑制流感病毒作用[2]; 其挥发油香气浓郁且持久,是烟用香精的重要赋香剂之一.     

水稻害虫化学生态调控研究进展

自然界中,在昆虫和植物种内与种间都存在着复杂的化学联系。开发利用生态系统中这些调控生物种内种间关系的生态功能分子,可望有效降低害虫的种群密度,从而减少化学农药的使用。本文根据目前国内外在利用生态功能分子调控水稻害虫方面的最新研究成果,分别就利用昆虫性信息素、水稻挥发物、非寄主植物提取物、化学激发子以及遗传改良水稻品种等在调控水稻害虫及其天敌中作用的研究进展进行了综述,并提出了今后的研究重点与方向。希望通过本文能促进化学生态调控技术在水稻害虫治理中的应用,以减轻水稻害虫治理对化学农药的依赖。     

白簕叶挥发油的化学成分

用GCMS联用技术首次研究了云南西双版纳白簕叶挥发油的化学成分,并应用色谱峰面积归一化法计算各成分的相对含量。一共分离出108个峰,确认了其中的81种成分,所鉴定的组分占挥发油总量的96.50%,主要是萜烯类及其含氧衍生物等。     

红厚壳挥发油化学成分

红厚壳（Calophyllum inophyllum Linn．）,又名海棠果、胡桐等,为藤黄科（Guttiferae）红厚壳属（Calophyllum Linn．）植物,主要分布于中国海南岛。其果实和种子富含油脂,可制香料;植株根系发达,耐盐碱、抗风性强,可以在贫瘠干旱的海滨、山地中生长,具有良好的开发应用前景。在中国民间,红厚壳被用作治疗眼病、外伤出血、风湿骨痛、跌打损伤等。红厚壳属于半红树植物,可生长于海岸潮间带,     

乌榄叶挥发油化学成分分析

采用气相色谱-质谱联用(GC-MS)方法首次对乌榄叶挥发油成分进行了测试分析,并应用色谱峰面积归一化法计算各成分的相对含量。分离出19个峰,确认了19种化合物,所鉴定的组分占挥发油总量的100%,主要成分是石竹烯(33.47)、α-蒎烯(18.03%)、d-柠檬烯(16.82%)、α-侧柏烯(11.74%)和α-水芹烯(6.51%)。     

ACAT1 deletion in murine macrophages associated with cytotoxicity and decreased expression of collagen type 3A1

In contrast to some published studies of murine macrophages, we previously showed that ACAT inhibitors appeared to be anti-atherogenic in primary human macrophages in that they decreased foam cell formation without inducing cytotoxicity. Herein, we examined foam cell formation and cytotoxicity in murine ACAT1 knockout (KO) macrophages in an attempt to resolve the discrepancies. Elicited peritoneal macrophages from normal C57BL6 and ACAT1 KO mice were incubated with DMEM containing acetylated LDL (acLDL, 100 microg protein/ml) for 48h. Cells became cholesterol enriched and there were no differences in the total cholesterol mass. Esterified cholesterol mass was lower in ACAT1 KO foam cells compared to normal macrophages (p<0.04). Cytotoxicity, as measured by the cellular release of [(14)C]adenine from macrophages, was approximately 2-fold greater in ACAT1 KO macrophages as compared to normal macrophages (p<0.0001), and this was independent of cholesterol enrichment. cDNA microarray analysis showed that ACAT1 KO macrophages expressed substantially less collagen type 3A1 (26-fold), which was confirmed by RT-PCR. Total collagen content was also significantly reduced (57%) in lung homogenates isolated from ACAT1 KO mice (p<0.02). Thus, ACAT1 KO macrophages show biochemical changes consistent with increased cytotoxicity and also a novel association with decreased expression of collagen type 3A1.     

Assessment of environmental contamination associated with a mammalian cell transformation assay

Summary To estimate worker exposures to, and environmental contamination from, test chemicals and organic solvents used in an in vitro assay to assess the carcinogenic potential of chemicals, sodium fluorescein, a noncarcinogenic fluorescent material, was dissolved in tissue culture medium used to maintain early passage hamster embryo cells. Personal and environmental samples were taken over a 14-d period. The assay was performed according to standard procedures in a ventilated glove box or laminar flow safety cabinet. Considerably more than 99% of the chemical contamination found was recovered from the interiors of the glove box and hood and from disposable equipment. Contamination outside the containment units (less than 1 μg) resulted from intralaboratory transport of chemicals, treated cultures, and contaminated equipment. We conclude that the standard operating practices and procedures provided adequate safeguards for personnel and the environment. Research sponsored by the National Cancer Institute under Contract N01-CO-75380, with Litton Bionetics, Inc.     

马缨丹叶片水提物与挥发油的生物活性及化学成分研究

研究了马缨丹 ( Lantana camara L.)叶片水提物和挥发油水溶液的化感作用。结果表明 ,马缨丹叶片水溶物浓度在 0 .2 5 g FW m L- 1时 ,对所有受试植物的幼苗生长均有一定的抑制作用 ,浓度降至 0 .1 0 g FWm L- 1时 ,其抑制作用显著降低 ;马缨丹叶片挥发油水溶液在浓度为 1 0 0、2 0 0、30 0 μg m L- 1时 ,对受试植物均有很强的抑制作用 ,且具有明显的浓度效应。采用 GC/MS分析了马缨丹叶片挥发油的化学成分 ,鉴定了 1 9种化感物质 ,其中α-子丁香烯和β-子丁香烯为主要物质 ,分别占挥发油含量的 1 6 .2 9%、2 2 .2 9%。     

福建石荠荣挥发油化学成分分析

报道了福建省不同产地石蔼荣全草挥发油含量在不同生育期的变化,其含量变化范围为0.320%-1.020%。对五个不同产地的全草挥发油主要化学成分进行分析,其挥发油主要成分为：番荆芥酚、1,8－桉叶油素、百里香酚、β－石竹烯、侧柏酮、异胡萍荷酮等,不同产地的挥发油化学成分存在遗传相似性,但亦有一定差异,石荠荣资源在香精香料、医药、化妆品等行业具有广阔的开发利用前景。     

微囊藻毒素LR诱导细胞毒性机制研究的细胞模型建立

以18种细胞系为材料,研究微囊藻毒素LR(20μg/mL和50μg/mL)所诱导的细胞毒性。形态观察表明,在经过30h以上的微囊藻毒素处理后,PC-3,J82,786-0,5637,VERO-E6等5种细胞出现了明显的细胞形态改变,毒素浓度越高,形态改变越厉害。微囊藻毒素LR的细胞毒性用LDH泄漏来表示。结果显示,5种毒素处理细胞的LDH泄漏呈剂量依赖性增加,其中5637和PC-3的LDH泄漏在同样的处理后较为厉害;同对照比较,SOD活力在20μg/mL MCLR处理下呈增加趋势,但在50μg/mL浓度下则下降;GSH含量在两种浓度处理下呈总体下降趋势。鉴于对微囊藻毒素敏感性差异分析,作者选择以5637细胞为基础,建立微囊藻毒素的毒理机制研究模型。       

Increased resistance to nitric oxide cytotoxicity associated with differentiation of neuroblastoma-glioma hybrid (NG108-15) cells

Nitric oxide (NO), synthesized by the enzyme nitric oxide synthase (NOS), acts as an intercellular messenger associated with various physiological and pathological events. In this study, we investigated whether there exits a difference in the vulnerability to NO-induced cytotoxicity between undifferentiated and differentiated NG108-15 cells, and if so, the mechanisms responsible for the difference. Following a 7- to 8-day exposure to dibutyryl cAMP (dbcAMP), NG108-15 cells exhibited a neuron-like morphology associated with the expression of the neuronal protein, synaptophysin, and with increased NADPH-d activity. Neuron-like differentiated NG108-15 cells acquired resistance to exogenously applied NO. This increased resistance to NO toxicity in differentiated cells was almost completely cancelled out by inhibiting the activity of superoxide dismutase (SOD), but not by inhibiting the activity of NOS. The present study suggested that the activity of SOD increased in parallel with the activity of NOS associated with differentiation and was crucial for the acquired resistance to NO toxicity in differentiated cells.     

Capture of volatile metabolites for tracking the evolution of gibberellic acid in a solid-state culture of Gibberella fujikuroi

The evolution of volatile compounds produced during solid substrate cultivation (SSC) of Gibberella fujikuroi on wheat bran was tracked looking for volatile metabolites related with GA₃ production. Ethyl acetate and isoamyl acetate gave identical profiles and sharp increases that abated as the production of GA₃ began, while ent-kaurene displayed a profile matching that of the development of GA₃. ent-Kaurene is a precursor in the synthesis of gibberellins and was the most abundant compound found.     

Ophiostomatoid fungi can emit the bark beetle pheromone verbenone and other semiochemicals in media amended with various pine chemicals and beetle-released compounds

Fungal volatile compounds can mediate fungal-insect interactions. Whether fungi can emit insect pheromones and how volatile chemicals change in response to chemicals the fungi naturally encounter is poorly understood. We analyzed volatiles emitted by Grosmannia clavigera (symbiont of the mountain pine beetle) and Ophiostoma ips (symbiont of the pine engraver beetle) growing in liquid media amended with compounds that the fungi naturally encounter: (−)-α-pinene, (+)-α-pinene, (−)-trans-verbenol, verbenone, or ipsdienol. Nine volatile compounds were identified among the fungal and amendment treatments. Volatiles qualitatively and quantitatively differed between fungal species and among amendment treatments. The bark beetle anti-aggregation pheromone (−)-verbenone was detected from both fungi growing in (−)-trans-verbenol-amended media. G. clavigera and O. ips can emit beetle pheromones and other beetle semiochemicals, suggesting that ophiostomatoid fungi could contribute to the chemical ecology of bark beetles. However, such contributions could be modulated by the presence of other environmental chemicals.     

不同提取方法对黔产水香薷挥发性成分的影响

比较不同提取方法对黔产水香薷挥发性成分影响。采用固相微萃取法、水蒸气蒸馏法提取水香薷挥发性成分,用GC-MS联用仪进行测定,结合NIST05和WILEY275谱库鉴定各化合物,峰面积归一化法测定各成分相对含量。利用两种提取方法供从水香薷中鉴别出67个化合物。从固相微萃取法提取物中检测并鉴定了45个化合物,而从水蒸气蒸馏法提取物种检测并鉴定了55个化合物。其中挥发性相同的化合物有33种,两种不同提取方法得到的挥发性成分虽有差异,但主要成分变化不大。体外抗菌试验表明,水香薷挥发性成分具有抑菌作用。这些研究结果为香料植物水香薷的开发提供了科学依据。     

Predictive models for estimating cytotoxicity on the basis of chemical structures

Cytotoxicity is a critical property in determining the fate of a small molecule in the drug discovery pipeline. Cytotoxic compounds are identified and triaged in both target-based and cell-based phenotypic approaches due to their off-target toxicity or on-target and on-mechanism toxicity for oncology and neurodegenerative targets. It is critical that chemical-induced cytotoxicity be reliably predicted before drug candidates advance to the late stage of development, or more ideally, before compounds are synthesized. In this study, we assessed the cell-based cytotoxicity of nearly 10,000 compounds in NCATS annotated libraries against four ‘normal’ cell lines (HEK 293, NIH 3T3, CRL-7250 and HaCat) using CellTiter-Glo (CTG) technology and constructed highly predictive models to estimate cytotoxicity from chemical structures. There are 5,241 non-redundant compounds having unambiguous activities in the four different cell lines, among which 11.8% compounds exhibited cytotoxicity in two or more cell lines and are thus labelled cytotoxic. The support vector classification (SVC) models trained with 80% randomly selected molecules achieved the area under the receiver operating characteristic curve (AUC-ROC) of 0.88 on average for the remaining 20% compounds in the test sets in 10 repeating experiments. Application of under-sampling rebalancing method further improved the averaged AUC-ROC to 0.90. Analysis of structural features shared by cytotoxic compounds may offer medicinal chemists heuristic design ideas to eliminate undesirable cytotoxicity. The profiling of cytotoxicity of drug-like molecules with annotated primary mechanism of action (MOA) will inform on the roles played by different targets or pathways in cellular viability. The predictive models for cytotoxicity (accessible at https://tripod.nih.gov/web_adme/cytotox.html) provide the scientific community a fast yet reliable way to prioritize molecules with little or no cytotoxicity for downstream development.     

Evaluation of the proposed interaction of nucleic acid with Clostridium difficile toxins A and B and the effects of nucleases on cytotoxicity

Both DNA and RNA were found to co-purify with Clostridium difficile toxin B but not toxin A. DNAase treatment greatly reduced the cytotoxicity of toxin B but not of toxin A. RNAase had no effect on either toxin. The effects on toxin B were shown to be due to a contaminating protease and could be inhibited by the serine protease inhibitor phenylmethylsulphonyl fluoride.