药物在水产饲料中的应用现状及其生态效应

在水产养殖过程中,因为功能和作用显著,药物在水产饲料中被广泛应用;但由于药物的残留、富集及其在环境中存在迁移和转化作用,药物的使用同时也给生态环境带来了一系列的问题。对当前水产饲料中不同种类药物的应用状况进行了概述,并着重对渔用药物的使用所导致的生态效应进行以下几个方面的总结和分析:养殖动物体内的药物代谢,药物在环境中的归趋,药物对水环境中水生生物、细菌耐药性及其最终对人的影响;阐明了药物在水产饲料中的应用与生态环境和人类健康有着十分密切的关系,对其进一步深入研究有重要的理论和实际意义。     

化学发光法用于芳香酯酶抑制剂体外筛选

在前期的研究中,我们将9-(4-氯苯氧羰基)-10-甲基吖啶酯三氟甲基磺酸盐(CPOCMA)用于测定血清芳香酯酶活性,取得满意结果.在此基础上,本文以CPOCMA为底物,建立化学发光法评估药物对芳香酯活性影响的新方法.以硝酸甘油为模型药物,比较了化学发光法与UV方法的一致性.并将此法应用于评价三种抗炎药吲哚美辛、阿司匹林和乙酰氨基酚对芳香酯酶活性的影响.药物的加入使血清催化CPOCMA水解的动力学减缓,这表明这些药物均为抑制剂.吲哚美辛、阿司匹林和乙酰氨基酚表现出的IC50值分别为0.254、0.564和0.656 mmol/L,抑制常数ki分别为0.154、1.38和2.98 mmol/L.加入药物后的Lineweaver-Burk曲线表明这三种药物对PON的抑制均为竞争性抑制.根据加入药物后的动力学曲线,其IC50值、抑制常数和米氏常数的变化均表明这三种药物的抑制能力大小顺序:吲哚美辛阿司匹林乙酰氨基酚.CPOCMA可以作为PON底物体外评价药物对PON的抑制能力和抑制机理.UV法不适合评价紫外吸收峰与UV法的检测波长重叠的药物,而新建立的化学发光法对这类药物的筛选有独特优势.     

The proteomic response of Mycobacterium smegmatis to anti-tuberculosis drugs suggests targeted pathways

Mycobacterium smegmatis is a fast-growing model mycobacterial system that shares many features with the pathogenic Mycobacterium tuberculosis while allowing practical proteomics analysis. With the use of shotgun-style mass spectrometry, we provide a large-scale analysis of the M. smegmatis proteomic response to the anti-tuberculosis (TB) drugs isoniazid, ethambutol, and 5-chloropyrazinamide and elucidate the drugs' systematic effects on mycobacterial proteins. A total of 2550 proteins were identified with approximately 5% false-positive identification rate across 60 experiments, representing approximately 40% of the M. smegmatis proteome ( approximately 6500 proteins). Protein differential expression levels were estimated from the shotgun proteomics data, and 485 proteins showing altered expression levels in response to drugs were identified at a 99% confidence level. Proteomic comparison of anti-TB drug responses shows that translation, cell cycle control, and energy production are down-regulated in all three drug treatments. In contrast, systems related to the drugs' targets, such as lipid, amino acid, and nucleotide metabolism, show specific protein expression changes associated with a particular drug treatment. We identify proteins involved in target pathways for the three drugs and infer putative targets for 5-chloropyrazinamide.     

Drug affinity to immobilized target bio-polymers by high-performance liquid chromatography and capillary electrophoresis

This review addresses the use of high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) as affinity separation methods to characterise drugs or potential drugs-bio-polymer interactions. Targets for the development of new drugs such as enzymes (IMERs), receptors, and membrane proteins were immobilized on solid supports. After the insertion in the HPLC system, these immobilized bio-polymers were used for the determination of binding constants of specific ligands, substrates and inhibitors of pharmaceutical interest, by frontal analyses and zonal elution methods. The most used bio-polymer immobilization techniques and methods for assessing the amount of active immobilized protein are reported. Examples of increased stability of immobilized enzymes with reduced amount of used protein were shown and the advantages in terms of recovery for reuse, reproducibility and on-line high-throughput screening for potential ligands are evidenced. Dealing with the acquisition of relevant pharmacokinetic data, examples concerning human serum albumin binding studies are reviewed. In particular, papers are reported in which the serum carrier has been studied to monitor the enantioselective binding of chiral drugs and the mutual interaction between co-administered drugs by CE and HPLC. Finally CE, as merging techniques with very promising and interesting application of microscale analysis of drugs' binding parameters to immobilized bio-polymers is examined.     

Inhibition of the repair of neutron-induced DNA single-strand breaks in experimental tumor cells

Nicotinamide and arabinoside cytosine mixed with hydroxyurea were shown to influence the relative amount of double-stranded DNA in Ehrlich ascites tumor cells in vitro subjected to single irradiation (10-30-52 Gy) and in Guerin's carcinoma in rat lungs exposed to fractionated 6 MeV neutron-radiation (1.25 Gy X 4). The DMF values for Ehrlich ascites tumor were a function of a dose range and the duration of the drugs' effect. Guerin's carcinoma DNA was found to be affected more readily when treated with radiation and drugs than when exposed to neutron radiation alone.     

Analysis of immediate stress mechanisms upon injection of polymeric micelles and related colloidal drug carriers: implications on drug targeting

Polymeric micelles are ideal carriers for solubilization and targeting applications using hydrophobic drugs. Stability of colloidal aggregates upon injection into the bloodstream is mandatory to maintain the drugs' targeting potential and to influence pharmacokinetics. In this review we analyzed and discussed the most relevant stress mechanisms that polymeric micelles and related colloidal carriers encounter upon injection, including (1) dilution, (2) interactions with blood components, and (3) immunological responses of the body. In detail we analyzed the opsonin-dysopsonin hypothesis that points at a connection between a particles' protein-corona and its tissue accumulation by the enhanced permeability and retention (EPR) effect. In the established theory, size is seen as a necessary condition to reach nanoparticle accumulation in disease modified tissue. There is, however, mounting evidence of other sufficient conditions (e.g., particle charge, receptor recognition of proteins adsorbed onto particle surfaces) triggering nanoparticle extravasation by active mechanisms. In conclusion, the analyzed stress mechanisms are directly responsible for in vivo success or failure of the site-specific delivery with colloidal carrier systems.     

Visualization and quantitative analysis of G protein-coupled receptor-β-arrestin interaction in single cells and specific organs of living mice using split luciferase complementation

Methods used to assess the efficacy of potentially therapeutic reagents for G protein-coupled receptors (GPCRs) have been developed. Previously, we demonstrated sensitive detection of the interaction of GPCRs and β-arrestin2 (ARRB2) using 96-well microtiter plates and a bioluminescence microscope based on split click beetle luciferase complementation. Herein, using firefly luciferase emitting longer wavelength light, we demonstrate quantitative analysis of the interaction of β2-adrenergic receptor (ADRB2), a kind of GPCR, and ARRB2 in a 96-well plate assay with single-cell imaging. Additionally, we showed bioluminescence in vivo imaging of the ADRB2-ARRB2 interaction in two systems: cell implantation and hydrodynamic tail vein (HTV) methods. Specifically, in the HTV method, the luminescence signal from the liver upon stimulation of an agonist for ADRB2 was obtained in the intact systems of mice. The results demonstrate that this method enables noninvasive screening of the efficacy of chemicals at the specific organ in in vivo testing. This in vivo system can contribute to effective evaluation in pharmacokinetics and pharmacodynamics and expedite the development of new drugs for GPCRs.     

Base specific interaction of reductively activated nitroimidazoles with DNA

To exert biological activity, nitroimidazole drugs require reductive activation in vivo. Nucleic acids are susceptible to the activated drug in vitro and are presumably the major target in vivo. We carried out electrolytical reduction of several 5-nitroimidazoles at a controlled potential either in the presence or prior to the addition of DNA. Using a nucleotide sequence specific test to analyse cleavage products, specific interaction of the reduced nitroimidazole intermediate(s) towards the guanine residues is prominent. Since the strand scission depends on subsequent piperidine treatment, it can be concluded that the primary interaction between the activated drug and guanine is a covalent modification weakening the glycosidic bond.     

Concerted action of antiepileptic and antidepressant agents to depress spinal neurotransmission: Possible use in the therapy of spasticity and chronic pain

Chronic pain states and epilepsies are common therapeutic targets of voltage-gated sodium channel blockers. Inhibition of sodium channels results in central muscle relaxant activity as well. Selective serotonin reuptake inhibitors are also applied in the treatment of pain syndromes. Here, we investigate the pharmacodynamic interaction between these two types of drugs on spinal neurotransmission in vitro and in vivo. Furthermore, the ability of serotonin reuptake inhibitors to modulate the anticonvulsant and windup inhibitory actions and motor side effect of the sodium channel blocker lamotrigine was investigated. In the hemisected spinal cord model, we found that serotonin reuptake inhibitors increased the reflex inhibitory action of sodium channel blockers. The interaction was clearly more than additive. The potentiation was prevented by blocking 5-HT(2) receptors and PKC, and mimicked by activation of these targets by selective pharmacological tools, suggesting the involvement of 5-HT(2) receptors and PKC in the modulation of sodium channel function. The increase of sodium current blocking potency of lamotrigine by PKC activation was also demonstrated at cellular level, using the whole-cell patch clamp method. Similar synergism was found in vivo, in spinal reflex, windup, and maximal electroshock seizure models, but not in the rotarod test, which indicate enhanced muscle relaxant, anticonvulsant and analgesic activities with improved side effect profile. Our findings are in agreement with clinical observations suggesting that sodium channel blocking drugs, such as lamotrigine, can be advantageously combined with selective serotonin reuptake inhibitors in some therapeutic fields, and may help to understand the molecular mechanisms underlying the interaction.     

外源EGFP基因在肿瘤细胞中的稳定表达

通过将增强型绿色荧光蛋白（EGFP）基因转染到人肺腺癌细胞（A549）内,建立以EGFP为探针的体外抗癌药物细胞株。使用电穿孔法导入EGFP基因到A549细胞;然后用G418筛选以及梯度稀释法筛选出EGFP高度表达的细胞株。初步建立稳定表达绿色荧光蛋白的人肺癌细胞株。     

The interaction of daunorubicin and mitoxantrone with the red blood cells of acute myeloid leukemia patients

The effect of DNR and MIT on erythrocyte membrane structure was examined using Electron Spin Resonance spectroscopy and the fluorimetric technique. The results suggest that the in vivo interaction of the drugs with the RBCs of AML patients led to a perturbation in the structure of plasma membrane components. Differences between DNR and MIT were only noted in the interaction of the drugs with deeper regions of the lipid bilayer     

Inhibition of the DNA catenation activity of type II topoisomerase by VP16-213 and VM26

Studies suggest that the anticancer drugs VP16-213 and VM26 produce cytotoxicity by inducing protein-associated DNA breakage in vivo through interaction with a yet unknown nuclear component. The effects of these drugs and their congeners on topoisomerase activities was investigated. VP16-213, VM26, and congeners active toward inducing DNA breaks also inhibited the catenation activity of eukaryote type II topoisomerase in vitro at very low drug concentrations. A structure-activity relationship was obtained for inhibition of catenation that parallels in vivo DNA breakage and cytotoxic activities. Type I topoisomerase activity was totally unaffected by these drugs.     

Characterization of physical interaction between Casiopeina III-ia and chitosan. Toward a Cas III-ia drug delivery system

Casiopeínas® are a new generation of anticancer drugs that have shown great in vitro and in vivo antineoplastic activities. Information about interaction drug-excipient, for developing a based-nanoparticle drug delivery system, has not been investigated yet. In order to elucidate if chitosan (CS) modifies the copper complex due to its interaction with Cu²⁺ ion, different studies in aqueous media between CS and Casiopeina III-ia (Cas III-ia) were carried out. CS–Cas III-ia mixtures were characterized by viscosity curves, UV–vis, EPR, and in vivo activity against HeLa cell line. Rheological behavior showed a decrease of viscosity when the drug was present due to diminished electrostatic interactions of charged amine group. UV–vis results illustrate that Cas III-ia is not stable at low pH as a result of interaction with acetic acid. However, when chitosan is present at the acidic solution Cas III-ia is stable. These results are supported by EPR studies. Finally, activity of the drug against HeLa cell line was not modified. Therefore, the present work presents evidence that there is no breaking of copper complex due to interaction between CS and Cas III-ia in acidic media. In addition, Cas III-ia maintains both its stability and effectiveness against cancer cell line.     

Spontaneous and mitomycin-C induced sister-chromatid exchanges : Comparison of in vivo and in vitro systems

Frequencies of sister-chromatid exchanges (SCE) were measured in vitro in mouse fibroblasts and in vivo in mouse bone-marrow cells. SCE levels in these cell systems were measured in response to varying concentrations of bromodeoxyuridine (BrdU) and mitomycin-C (MMC). Although BrdU was found to induce SCE in both cellular systems, baseline SCE levels were 2- to 3-fold higher in vitro than in vivo. SCE induction was found to be a linear function of MMC concentration in vivo and in vitro; however the slope of the in vivo curve was 5-fold higher. The interaction of BrdU substituted DNA and MMC was examined by administering a fixed dose of MMC with increasing concentrations of BrdU. The induced SCE frequencies appeared to be additive. In addition to measuring drug-induced SCE, the BrdU differential staining technique allows concomitant measurement of the inhibition of cellular replication by the test drugs.     

In vivo determination of cell cycle kinetics of non-Hodgkin's lymphomas using iododeoxyuridine and bromodeoxyuridine.

Using sequential infusions of two S-phase-specific drugs, iododeoxyuridine and bromodeoxyuridine, we have developed an in vivo method for determining the labeling index (LI), the S-phase duration (Ts), and total cell cycle times (Tc) of non-Hodgkin's lymphomas. In nine non-Hodgkin's lymphomas studied, the LI ranged from 1.5% in a follicular small cleaved-cell lymphoma to 29.6% in a diffuse large-cell lymphoma. The Ts ranged from 16 hr in a large-cell lymphoma (immunoblastic type) to 117 hr in a follicular small cleaved-cell lymphoma. The Tc varied from 69 hr in a large-cell lymphoma (immunoblastic type) to over 1000 hr in all low-grade lymphomas studied. Immunohistochemical methods using anti-BrdU antibodies were used to detect cell incorporation of the two S-phase-specific drugs. In this manner, cell cycle times could be calculated while the architecture of the tumor specimen was preserved. Difficulties in using this methodology, specifically in the calculation of the growth fraction and total cell cycle times, are pointed out. This in vivo method does, however, allow for Ts calculations independent of growth fraction considerations. Correlations of cell cycle data with various biological and clinical factors await further patient follow-up.     

Multimodal action of antitumor agents on DNA: the ellipticine series

Most cytotoxic anticancer agents interact directly or indirectly with nuclear DNA, the ultimate target for this class of compounds. For a given type of drug both direct and indirect action at the DNA level usually causes various types of interference or damage. This multimodal mechanism of action is well illustrated by antitumor drugs in the ellipticine series which may bind to DNA through intercalation, may undergo covalent binding, may generate oxidizing species, and may interfere with the catalytic activity of topoisomerase II. The antitumor activity of these compounds may, therefore, result from alternative cytotoxic events. The present review summarizes information obtained with ellipticine compounds on the relation between the nature of the drugs' action on DNA and their cytotoxic and/or antitumor activity. The occurrence of topoisomerase-mediated DNA cleavage appears to be responsible for antitumor activity. The capability of the drugs to interfere with the action of topoisomerase II requires the presence of an oxidizable phenolic group on their structure. This feature (or a related one) is shared by all antitumor drugs acting on this enzyme.