金银忍冬花粉离体萌发初探

以金银忍冬[Loniceramaackü(Rupr.)Maxim.]为试材,研究了不同种类糖、硼酸、钙离子、pH值、光照等单因子对花粉萌发的影响.结果表明,培养基中分别含有25%的蔗糖、0.01%的硼离子、0.02%～0.05%的钙离子以及pH6.7时较适合金银忍冬的花粉萌发,萌发率分别为85.63%、59.23%、78.12%和75.27%;培养基中不同种类的糖对花粉萌发的影响不同,以乳糖最适宜,其次是葡萄糖、蔗糖和果糖;在光照(0.5625 μmol m-2s-1)与黑暗下培养,金银忍冬花粉的萌发率没有明显的变化,但黑暗条件更利于花粉管的生长.金银忍冬花粉在含有25%蔗糖、0.01%硼离子、0.02%钙离子、pH为6.7的培养基上光照培养3 d,其花粉萌发率均达到80%以上,最高的萌发率可达88.1%.     

A highly sensitive ultra performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) technique for quantitation of protein free and bound efavirenz (EFV) in human seminal and blood plasma

A combined UPLC-tandem mass spectrometric (UPLC-MS/MS) technique has been validated for quantitation of protein free efavirenz (EFV) as well as total concentrations of EFV in human blood and seminal plasma. The analytical method possesses capabilities for concentration measurements of EFV ranging from 0.5 to 10,000ng/ml with an accuracy (%dev) of -5.2-8.0% and precision (%CV) of <8%. Standard curves were linear with coefficients of variation (r(2)) >0.98. The method employs a racemic fluorinated analog of EFV (F-EFV) as the internal standard. EFV and F-EFV were eluted from a reverse-phase UPLC column via gradient elution with detection via negative ion multiple reaction monitoring (MRM). EFV and F-EFV, respectively, were detected via the following MRM transitions: m/z 314.0>244.1 and m/z 298.0>227.9. The time required for the analysis of each sample was 8.0min. The analytical technique is capable of a reliable detection limit of ～15-20fmol of EFV injected on column.     

Determination of ethyl glucuronide in hair samples of Chinese people by protein precipitation (PPT) and large volume injection-gas chromatography-tandem mass spectrometry (LVI-GC/MS/MS)

Ethyl glucuronide (EtG) has been shown to be a suitable marker of excessive alcohol consumption. Determination of EtG in hair samples may help to differentiate social drinkers from alcoholics, and this testing can be widely used in forensic science, treatment programs, workplaces, military bases as well as driving ability test to provide legal proof of drinking. A method for determination of EtG in hair samples using large volume injection-gas chromatography-tandem mass spectrometry (LVI-GC/MS/MS) was developed and validated. Hair samples (in 1 mL deionized water) were ultrasonicated for 1h and incubated overnight; these samples were then deproteinated to remove impurities and derivatisated with 15 μL of pyridine and 30 μL of BSTFA. EtG was detected using GC/MS/MS in multiple-reaction monitoring mode. This method exhibited good linearity: y=0.0036 x+0.0437, R2=0.9993, the limit of detection and the limit of quantification were 5 pg/mg and 10 pg/mg, respectively. The extraction recoveries were more than 60%, and the inter-day and intra-day relative standard deviations (RSD) were less than 15%. This method has been applied to the analysis of EtG in hair samples from 21 Chinese subjects. The results for samples obtained from all of those who were teetotallers were negative, and the results for the other 15 samples ranged from 10 to 78 pg/mg, except for one negative sample. These data are the basis for interpretation of alcohol abuse.     

HPLC-MS/MS法测定黄山贡菊花、叶和茎中酚类物质

采用HPLC-MS/MS检测技术分析了黄山贡菊花、叶、茎中酚类物质。结果表明,黄山贡菊花、叶、茎中酚类物质主要为黄酮及酚酸类化合物,其中贡菊花中含有13种黄酮类化合物,9种酚酸类化合物;叶中含有11种黄酮类化合物,6种酚酸类化合物;茎中含有8种黄酮类化合物,5种酚酸类化合物。贡菊花、叶、茎中共有的酚性成分包括:绿原酸、1,5-二咖啡酰奎尼酸、3,5-二咖啡酰奎尼酸、4,5-二咖啡酰奎尼酸、芹菜素-6-C-木糖-8-C-葡萄糖、芹菜素-6-C-葡萄糖-8-C-阿拉伯糖、芹菜素-6-C-阿拉伯糖-8-C-葡萄糖、木犀草苷、木犀草素-7-O-葡萄糖醛酸苷、香叶木素-7-O-6"-丙二酰-葡萄糖苷、金合欢素-7-O-6"-丙二酰-葡萄糖苷。金合欢素-7-O-半乳糖苷存在于贡菊茎、叶中,而在贡菊花中未发现;芹菜素-7-O-葡萄糖醛酸苷只存在于贡菊叶中,在贡菊花和茎中均未发现。     

东北天然针阔混交林凋落物磷素空间异质性及其影响因素

以天然云冷杉(Picea jezoensis var.microsperma(Lindl.) W.C.ChengL.K.Fu and Abies nephrolepis(Trautv.) Maxim.)针阔混交林为研究对象,基于4块1 hm~2固定样地林分调查数据和等距离网格布点取样的400个半分解层凋落物样品的养分测定数据,采用地统计学方法,分析凋落物磷浓度、归还量及利用效率的空间格局特征及其影响因素。结果表明:4块样地凋落物磷浓度均值为1.26 g/kg,归还量均值为24.57 kg/hm~2,利用效率均值为841.74,均表现为中等强度变异,具有明显的空间异质性,且同一样地,变异程度呈现出磷归还量磷利用效率磷浓度。凋落物磷浓度、归还量(样地III和IV结构比大于75%,其空间异质性主要由随机效应引起,不适合进行空间插值)和利用效率主要受结构性因素影响,具有较高的空间自相关性。同一样地磷利用效率的空间异质性较磷浓度及归还量低,各样地磷浓度、归还量和利用效率空间自相关范围分别为9.9—40.5 m、11.9—52.9 m和8.1—39.3 m。同一样地磷利用效率的分形维数高于磷浓度,空间格局较磷浓度复杂,磷浓度空间依赖性更强,具有更好的结构性。凋落物磷浓度、归还量(除样地III和IV)和利用效率呈现条带状和斑块状梯度性分布,且磷浓度和利用效率的空间分布格局相似。凋落物磷浓度、归还量和利用效率受到郁闭度、物种数和植物多样性等多种因子的影响。     

灰斑古毒蛾口腔反吐物诱导沙冬青细胞Ca2+内流及H2O2积累

植物对昆虫取食活动进行成功防御的关键,取决于对昆虫口腔反吐物的激发子的快速识别。实验利用无损伤微测系统及激光共聚焦显微镜,研究了沙冬青细胞经灰斑古毒蛾口腔反吐物诱导后Ca2+流及H2O2的变化。结果发现:灰斑古毒蛾口腔反吐物诱导沙冬青细胞Ca2+内流及H2O2的积累,表明Ca2+内流及H2O2的积累是沙冬青细胞对口腔反吐物产生应答的早期响应事件;Ca2+钙通道阻断剂仅部分抑制Ca2+内流,说明Ca2+内流除经过质膜上的Ca2+通道进入细胞外,尚存在其他的内流途径;灰斑古毒蛾口腔反吐物中的某些成分与沙冬青细胞的质膜结合后,诱导质膜上形成允许Ca2+通过的孔道,而GdCl3不能抑制这类孔道的活性。胞外Ca2+螯合剂EGTA完全抑制H2O2的积累,GdCl3预处理仅部分抑制了H2O2的积累,说明灰斑古毒蛾诱导的沙冬青细胞内H2O2的积累依赖于Ca2+内流;抑制剂实验表明,H2O2的积累主要来源于质膜上NADPH氧化酶的作用。     

A high-throughput platform for the rapid screening of vitamin D status by direct infusion-MS/MS

Vitamin D is an important fat-soluble prohormone with pleiotropic effects on human health, such as immunomodulation of the innate and adaptive immune system. There is an unmet clinical need for a rapid screening platform for 25-hydroxyvitamin D (25OH-D) determination without chromatographic separation that offers better precision and accuracy than immunoassays. Here, we introduce a high-throughput method for assessing vitamin D status from blood specimens based on direct infusion-MS/MS (DI-MS/MS) following click derivatization using 2-nitrosopyridine. We developed an optimized liquid-phase extraction protocol to minimize ion suppression when directly infusing serum or plasma extracts via a capillary electrophoresis system for quantitative determination of 25OH-D. Acceptable reproducibility (mean coefficient of variation = 10.9%, n = 412), recovery (mean = 102% at 15, 30, and 45 nmol/l), and linearity (R² > 0.998) were achieved for 25OH-D with lower detection limits (limit of detection ～1.2 nmol/l, S/N ～ 3), greater throughput (～3 min/sample), and less bias than a commercial chemiluminescence immunoassay prone to batch effects. There was mutual agreement in 25OH-D concentrations from reference blood samples measured by DI-MS/MS as compared with LC-MS/MS (mean bias = 7.8%, n = 18). We also demonstrate that this method could reduce immunoassay misclassification of vitamin D deficiency in a cohort of critically ill children (n = 30). In conclusion, DI-MS/MS offers a viable alternative to LC-MS/MS for assessment of vitamin D status in support of large-scale studies in nutritional epidemiology as well as clinical trials to rapidly screen individual patients who may benefit from vitamin D supplementation.     

Determination of protein binding for novel 2-(2-hydroxypropanamido)-5-trifluoromethyl benzoic acid enantiomers to rats,dogs, and humans plasma by UPLC-MS/MS

R-/S-2-(2-hydroxypropanamido)-5-trifluoromethyl benzoic acid (R-/S-HFBA) is a novel COX inhibitor with remarkable anti-inflammatory and antiplatelet aggregation activities, but no gastrointestinal toxicity. In our previous study, the different pharmacokinetic profiles of the two enantiomers in rats were observed after administration of R-HFBA and S-HFBA. Stereoselective protein binding of the two enantiomers may be a reason for the different pharmacokinetic behaviors. In this study, we developed and validated an UPLC-MS/MS method for determining stereoselective binding of HFBA enantiomers to rat, dog, and human plasma in vitro. Chromatographic separation was achieved by gradient elution with a flow rate of 0.4 mL/min. MS/MS detection was operated in positive electrospray using multiple reaction monitoring (MRM) mode. The method was proved to be linear over the concentration range of 0.005 to 10 μg/mL with a lower limit of quantification of 0.005 μg/mL. The developed method was successfully employed to the plasma protein binding study of HFBA enantiomers. Equilibrium dialysis method was applied to assess drug-plasma protein interactions. The results showed that the enantiomers were both extensively bound to three species plasma and protein binding of R-/S-HFBA was concentration dependent. R-HFBA and S-HFBA showed significant species difference among rat, dog, and human plasma and stereoselective plasma protein binding.     

A practical vector for efficient knockdown of gene expression in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

In the last decade, RNA interferences (RNAi) has proven to be an effective strategy to knock out homologous genes in a wide range of species. Based on its principle, a new generation of vectors containing an inverted target sequence separated by an intron as a loop, developing simplifications to the procedure of RNAi construction are required to improve the efficiency of gene inactivation techniques. Here, a novel polymerase chain reaction (PCR)—based RNAi vector pTCK303 with a maize ubiquitin promoter, 2 specific multiple enzyme sites, and a rice intron was constructed for monocot gene silencing. With this vector, only 1 PCR product amplified by a single pair of primers and 2 ligation reactions were needed to create an RNAi construct, which shortened the time span before being transformed into the plant. To test the efficiency of vector pTCK303, a rice geneOsGAS1 was used, and its RNAi construct was introduced into rice calli. Southern blot analysis of the transgenic rice confirmed the presence of theOsGAS1 RNAi structure. The decrease inOsGAS1 level in the transgenic rice was detected by Northern blot probed with anOsGAS1-specific sequence. Moreover, the rate of inhibition of the RNA expression level in RNAi transgenic rice was approximately 85% according to our real-time PCR. Therefore, the RNAi vector pTCK303 based on the homology-dependent gene-silencing mechanisms facilitated the inhibition of endogenous genes in a monocot and was proven to be a practical and efficient platform for silencing a rice gene. These authors contributed equally to this work.     

Clinical validation of an immunoaffinity LC-MS/MS assay for the quantification of a collagen type II neoepitope peptide: A biomarker of matrix metalloproteinase activity and osteoarthritis in human urine

The degradation of type II collagen has been associated with the pathology of osteoarthritis (OA). Matrix metalloproteinases (MMPs) are enzymes that are responsible for catalyzing the degradation of collagen and, therefore, are pursued as potential targets for the treatment of OA. Collagen-derived peptides identified as a reflection of in vivo MMP activity have been investigated as target biomarkers of MMP activity as well as potential biomarkers of OA disease state and/or progression. An immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay developed for the quantification of the most abundant urinary type II collagen neoepitope (uTIINE) peptide, a 45-mer with 5 HO-proline residues resulting from MMP-13-catalyzed degradation, was validated for clinical use. Validation experiments were designed with attention to specific challenges related to quantification of endogenous analytes. The validated method is sensitive, selective, accurate (<15% relative error) and precise (<15% coefficient of variation) over a linear range of 0.156-7.50 ng/ml. Sample stability and inter- and intrasubject variability were evaluated in the urine of normal and OA populations. The method was applied to analyze human urine samples from clinical studies investigating the utility of uTIINE as a potential biomarker for OA.     

The antitumor effect of hinesol,extract from Atractylodes lancea (Thunb.) DC. by proliferation,inhibition, and apoptosis induction via MEK/ERK and NF-κB pathway in non–small cell lung cancer cell lines A549 and NCI-H1299

Lung cancer (especially, non–small cell lung cancer [NSCLC]) is one of the most malignant cancers in the world. Hinesol is the major component of the essential oil of Atractylodes lancea (Thunb.) DC and possesses the most promising anticancer function. However, the effects and molecular mechanism of hinesol on antiproliferation in NSCLC cells has not been well understood. In this study, we found that hinesol effectively inhibited the A549 and NCI-H1299 cells in a dose- and time-dependent manner by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay. In addition, hinesol induced cell cycle arrest at G0/G1 phase and apoptosis assessed by flow cytometry in A549 cells. Furthermore, Western blot analysis showed that hinesol decreased phosphorylation of mitogen-activated protein kinase, extracellular signal-regulated kinase, IκBα, and p65 inhibited the expressions of Bcl-2, cyclin D1 and upregulated the expression of Bax. Based on these results, hinesol might be a potential drug candidate of anti-NSCLC for therapy.