Microwave-Accelerated and Metal-Enhanced Fluorescence Myoglobin Detection on Silvered Surfaces: Potential Application to Myocardial Infarction Diagnosis

In this short paper, we describe a novel approach to both significantly accelerate and optically amplify fluorescence-based immunoassays. Our approach utilizes metal-enhanced fluorescence (MEF) to intrinsically optically amplify fluorescence signatures, which, when combined with the use of low-power microwaves to kinetically accelerate assays, provides for both ultrafast and ultrabright immunoassays. Surprisingly, the use of low-power microwaves and silver nanostructures provides for localized heating, concentrating the effect to the particles themselves as compared to the generic heating of the high dielectric assay fluid. We have subsequently applied our microwave-accelerated MEF approach to the detection of myoglobin, where its rapid quantification is paramount for the clinical assessment of an acute myocardial infarction.     

Using budding yeast to screen for anti-prion drugs

Prions are misfolded proteins capable of propagating their altered conformation which are commonly considered as the causative agent of transmissible spongiform encephalopathies, a class of fatal neurodegenerative diseases. Currently, no treatment for prion-based diseases is available. Recently we have developed a rapid, yeast-based, two-step assay to screen for anti-prion drugs [1]. This new method allowed us to identify several compounds that are effective in vivo against budding yeast [PSI+] and [URE3] prions but also able to promote mammalian prion clearance in three different cell culture-based assays. Taken together, these results validate our method as an economic and efficient high-throughput screening approach to identify novel prion inhibitors or to carry on comprehensive structure-activity studies for already isolated anti-mammalian prion drugs. These results suggest furthermore that biochemical pathways controlling prion formation and/or maintenance are conserved from yeast to human and thus amenable to pharmacological and genetic analysis. Finally, it would be very interesting to test active drugs isolated using the yeast-based assay in models for other diseases (neurodegenerative or not) involving amyloid fibers like Huntington's, Parkinson's or Alzheimer's diseases.     

Trypanosoma evansi: Pharmacological evidence of a nicotinic acetylcholine receptor

The role of calcium and its relevance have been deeply revised with respect to trypanosomatids, as the mechanism by which calcium enters trypanosomes was, until now, not well understood. There is evidence supporting the presence of a nAChR in another member of the trypanosomatidae family, Trypanosoma cruzi, these receptors being one entry path to calcium ions. The aims of this work were to determine if there was a nicotinic acetylcholine receptor (nAChR) in Trypanosoma evansi, and to subsequently perform a partial pharmacological characterization of this receptor.After being loaded with FURA-2AM, individual cells of T. evansi, were exposed to cholinergic compounds, and the cells displayed a dose-dependent response to carbachol. This observation indicated that a cholinergic receptor may be present in T. evansi. Although a dose-dependent response to muscarine could not be demonstrated, nicotine could promote an incremental dose-dependent response. The relative potency of this specific agonist of nAChR is in agreement with previous reports. The estimated affinity values were a K_d1 value of 29.6 ± 5.72 nM and a K_d2 value of 315.9 ± 26.6 nM, which is similar to the K_d value reported for the α4 nicotinic receptor. The Hill coefficients were determined to be an n₁ of 1.2 ± 0.3 and an n₂ of 4.2 ± 1.3. Finally, our calculations indicated that there are about 1020 receptors in each T. evansi parasite, which is approximately 15-fold lower than the number reported in Torpedo californica electric cells. These results suggest the presence of a nAChR in T. evansi, which is able to bind nicotinic ligands and induce calcium signals.     

Prostaglandin-H-synthase (PGHS)-1 and -2 microtiter assays for the testing of herbal drugs and in vitro inhibition of PGHS-isoenzyms by polyunsaturated fatty acids from Platycodi radix

In order to test inhibition of prostaglandin-H-synthase-1 and -2 (PGHS-1 and -2) by plant extracts, we have established two enzyme based in vitro assays with enzyme immunoassay (EIA) evaluation. The assays have been evaluated with known synthetic inhibitors and with plant extracts. In a screening of traditionally used Chinese herbs for anti-inflammatory activity, a series of n-hexane and dichloromethane extracts showed significant inhibitory effect in comparison with the known specific PGHS-2 inhibitors NS-398 (IC(50) = 2.6 microM) and nimesulide (IC(50) = 36 microM). The lipophilic extracts of the Chinese drug Jiengeng, the dried roots of Platycodon grandiflorum (Jacq.) A. DC. (Campanulaceae), showed good inhibitory activity against both PGHS isoenzymes. The directly prepared DCM-extract exhibited better activity against PGHS-2 (IC(50) = 4.0 microg/ml) than against PGHS-1 (IC(50) = 17.6 microg/ml). We identified fatty acids as main active constituents and quantified them. Linoleic acid showed the highest content (ca. 20% of the dried extract) and a high and preferential PGHS-2 inhibitory activity (IC(50) (PGHS-1) = 20 microM; IC(50) (PGHS-2) = 2 microM). The comparison of the concentration of linoleic acid and the inhibitory activity of the direct DCM-extract showed, that linoleic acid is mainly responsible for the in vitro activity of the extract on PGHS-2.     

A modified Vibrio harveyi mutagenicity assay based on bioluminescence induction

AIMS: Mutagenic pollution of natural environment is currently one of the most serious ecological problems. Therefore, rapid detection of the presence of mutagens is a very important issue. Although many mutagenicity assays have already been described, only a few are suitable for testing samples from natural environment. One of such assays is a microbiological mutagenicity test based on genetically modified Vibrio harveyi strains. The aim of this work was to modify and improve the V. harveyi assay. METHODS AND RESULTS: A series of V. harveyi dark and dim mutants were tested for reversion of their phenotype towards efficient light emission in response to incubation with known mutagens. Luminescence of the A16 strain (luxE mutant) increased significantly after a few hours of such a treatment with various mutagenic agents, revealing a dose-response correlation. Sensitivity of the assay has been determined for different mutagens. CONCLUSIONS: The luminescence-based V. harveyi mutagenicity assay is rapid, sensitive and reveals a dose-response correlation for various mutagens. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay developed in this study is a potentially useful tool in studies on mutagenic pollution of environment, especially marine water.     

Detection of competitive enzyme inhibition with end point progress curve data

A model for a dimensionless factor, the inhibition detection limit (IDL), which describes the limit of detection of competitive inhibition for end point assays as a function of the proportion of substrate converted into product, has been developed. For a given end point enzymatic assay, the IDL function has a maximum that is dependent on the error structure parameters (four parameters) of the assay, the value of [S]o/K(ms), and the extent of product inhibition (K(ms)/K(mp)). Accordingly, the substrate conversion level that maximized the ability to detect samples with high Ki/[I] ratios was predicted for each member of a population of simulated assays. Furthermore, we identified a consensus substrate conversion level where the probability of a near-optimal robustness and detection limit for all the members of the assay population is maximal. Unlike the optimal substrate conversion level for individual assays, this consensus substrate conversion level was dependent only on [S]o/K(m), K(ms)/K(mp), and whether the signal increases or decreases during the course of the reaction. Consensus substrate conversion levels were beyond the initial velocity range for almost all the analyzed assay populations. It was shown that the IDL factor was a more informative indicator of assay quality than the popular Z' factor.     

Correlation between cell survival,clonogenic activity and micronuclei induction in DMBA-OC-1R cells treated with immunospecific albumin microspheres containing cisplatin and 5-fluorouracil

An ideal chemotherapeutic strategy would be to deliver a high concentration of drug that would be released in sustained small amounts from targeted microspheres to effectively kill only the tumour cells and thus reduce toxicity to normal tissue. Clonogenic and cell survival growth curve assays, as well as the micronucleus assay, were used to determine the feasibility of employing targeted immunomicrospheres in the treatment of cancer. Cells of a rodent ovarian carcinoma cell line, were exposed to cisplatin and 5-fluorouracil, either as free drug or encapsulated in albumin microspheres that were either conjugated to monoclonal antibodies or not. In cell survival growth curve assays, cell survival was reduced to 1.2% of the control when cells were treated with drug-containing immunomicrospheres. 3.2-fold more micronuclei were found in those cells that had been exposed to the drugs in immunomicrospheres than in those subjected to untargeted microspheres. All three assays demonstrated that the targeted immunomicrospheres were more effective in delivering cisplatin and 5-fluorouracil directly to the cells than the unconjugated microspheres, thus suggesting that targeted chemotherapy might be a more effective option in the treatment of cancer.     

我国西南元江大理茶的挥发性成分及其抗氧化活性(英文)

大理茶 ( Camellia taliensis) 为山茶科山茶属茶组植物,主要分布于云南横断山脉澜沧江至伊洛瓦底江流域,即从云南的西部及西南部至缅甸北部。在其分布区,大理茶亦被称为野生大茶树,常用于加工制作茶叶。采用水蒸气蒸馏法、GC 及 GC/MS 联用技术,首次对大理茶的鲜幼叶和鲜幼叶及老叶分别制成的绿茶中的挥发性成分进行提取和分析,共鉴定出 91 个化合物。研究结果表明,大理茶鲜幼叶的主要香气成分为棕榈酸 ( 30. 52%) ,亚油酸 ( 19. 82%) ,植醇 ( 8. 75%) 和亚麻酸乙酯 ( 2. 54%) 等有机酸及其酯和二萜类,而制成绿茶后,其主要香气成分则为芳樟醇 ( 28. 43%) ,脱氢芳樟醇 ( 1. 13%) ,α-松油醇( 11. 68%) ,橙花醇 ( 4. 92%) 和香叶醇 ( 12. 34%) 等单萜醇类成分。从大理茶鲜叶到由其制成的绿茶,香气成分发生了较大变化,形成了 28 种原鲜叶中未检测到的香气成分,其中,( Z,Z,Z) -9,12,15-十八烷三烯-1-醇的含量分别达到 1. 21% ( 幼叶绿茶) 和 11. 2% ( 老叶绿茶) ,是大理茶制作的绿茶的特征香气成分。DPPH 和 ABTS+自由基清除实验结果显示大理茶鲜叶及其制成的绿茶的挥发性成分均具有一定的抗氧化活性,但均弱于茶多酚的抗氧化活性。