PEGylated Phospholipid Membrane on Polymer Cushion and Its Interaction with Cholesterol

By employing poly(ethylene glycol) (PEG) shielding and a polymer cushion to achieve air stability of the lipid membrane, we have analyzed PEG influence on dried membranes and the interaction with cholesterol. Small unilamellar vesicles (SUVs) formed by the mixture of 1,2-dimyristoylphosphatidylcholine (DMPC) with different molar fraction of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-2000] (DSPE-PEG(2000)) adsorb and fuse into membranes on different polymer-modified silicon dioxide surfaces, including chitosan, poly(L-lysine) (PLL), and hyaluronic acid, Dried membranes arc further examined by ellipsometer and atomic force microscopy (AFM). Only chitosan can support a visible and uniform lipid array. The thickness of dry PEGylated lipid membrane is reduced gradually as the molar fraction of PEG increases. AFM scanning confirms the lipid membrane stacking for vesicles containing low PEG, and only a proper amount of PEG can maintain a single lipid hi lover; however, the air stability of the membrane will be destroyed if overloading. PEG. Cholesterol incorporation can greatly improve the structural stability of lipid membrane, especially for those containing high molar fraction of PEG. Different amounts of cholesterol influence the thickness and surface morphology of dried membrane.

Ion channel behavior of amphotericin B in sterol-free and cholesterol- or ergosterol-containing supported phosphatidylcholine bilayer model membranes investigated by electrochemistry and spectroscopy

Amphotericin B (AmB) is a popular drug frequently applied in the treatment of systemic fungal infections. In the presence of ruthenium (II) as the maker ion, the behavior of AmB to form ion channels in sterol-free and cholesterol- or ergosterol-containing supported phosphatidylcholine bilayer model membranes were studied by cyclic votammetry, AC impedance spectroscopy, and UV/visible absorbance spectroscopy. Different concentrations of AmB ranging from a molecularly dispersed to a highly aggregated state of the drug were investigated. In a fixed cholesterol or ergosterol content (5 mol %) in glassy carbon electrode-supported model membranes, our results showed that no matter what form of AmB, monomeric or aggregated, AmB could form ion channels in supported ergosterol-containing phosphatidylcholine bilayer model membranes. However, AmB could not form ion channels in its monomeric form in sterol-free and cholesterol-containing supported model membranes. On the one hand, when AmB is present as an aggregated state, it can form ion channels in cholesterol-containing supported model membranes; on the other hand, only when AmB is present as a relatively highly aggregated state can it form ion channels in sterol-free supported phosphatidylcholine bilayer model membranes. The results showed that the state of AmB played an important role in forming ion channels in sterol-free and cholesterol-containing supported phosphatidylcholine bilayer model membranes.

Interaction of scopolamine and cholesterol with sphingomyelin bilayers by FT-Raman spectroscopy

The interaction of scopolamine and cholesterol with sphingomyelin bilayers has been investigated by FT-Raman spectroscopy in head-group region (600-1000 cm(-1)), the C-C stretching (1000-1200 cm(-1)), CH2 deformation (1400-1500 cm(-1)) and the C-H stretching (2800-3000 cm(-1)) mode regions. The results indicate that scopolamine and cholesterol do not change the conformation of O-C-C-N+ backbone in the choline group of sphingomyelin bilayers, the polar headgroup is still extending parallel to the bilayer surface and O-C-C-N+ group is still in its gauche conformer. Scopolamine and cholesterol lower the order of the interface, the interchain, CH2 crystal lattices and the lateral chain-chain packing, and increase their fluidity.

Interaction of La3+ and cholesterol with dipalmitoylphosphatidylglycerol bilayers by FT-Raman spectroscopy

The interaction of La3+ and cholesterol with the negatively charged phospholipid dipalmitoylphosphatidylglycerol bilayers was studied by Fourier transform-Raman spectroscopy. La3+ was shown to increase interchain order and intermolecular ordering of the lipid lattice, cholesterol exhibited less of an effect, the La3+-DPPG-cholesterol complex was more ordered than cholesterol=DPPG nd less ordered than La3+-DPPG complexes, cholesterol modulates the order/disorder parameters of DPPG bilayers.

CHOLESTEROL SENSOR-BASED ON ELECTRODEPOSITION OF CATALYTIC PALLADIUM PARTICLES

A layer of palladium particles was electrodeposited on a glassy carbon electrode. The dispersed Pd particles resulted in a large decrease in overvoltage for the electrochemical oxidation of H2O2 down to +0.4 V vs. Ag/AgCl, based on which a new kind of cholesterol sensor was fabricated. Cholesterol oxidase was immobilized on the Pd-dispersed electrode by cross-linking with glutaraldehyde and a layer of poly(o-phenylenediamine) (PPD) film was electropolymerized on the enzyme layer. The sensor shows a linear response in the concentration range 0.05-4.50 mmol l-1 with a rapid response of less than 20 s. The polymer film can prevent interference from uric acid and ascorbic acid and also increases the thermal stability of the sensor. The sensor can be used for 200 assays without an obvious decrease in activity.

青蒿反义鲨烯合酶基因对烟草的转化

利用反义技术研究生物代谢途径以及对其生物合成进行调控成为植物次生代谢研究领域内一个重要手段之一，并与新兴的RNAi技术一起成为本领域内重要的研究热点。在植物类异戊二烯代谢途径中存在着羟甲基戊二酰辅酶A还原酶(HMGR)、法呢基焦磷酸合酶（FPS）和鲨烯合酶（SQS）等几种关键的分支酶，他们被认为在异戊二烯类的生物合成中发挥着关键的调节作用。其中，鲨烯合酶处于HMGR和FPS的下游，并与倍半萜合酶等利用共同的前体－法呢基二磷酸（FPP），以FPP起始合成一系列的下游产物。因此，FPP成为类异戊二烯途径中的关键调节点之一。本论文基于此目的，利用反义技术研究了FPP合成鲨烯这一途径受到抑制对其他以FPP为生物合成前体的代谢支路的影响。 利用植物双元转化载体pBI121，将青蒿中鲨烯合酶基因的cDNA（约1.5kb）序列插入到pBI121中，取代原有的GUS序列，构建成植物转化载体pBIASS。以根癌农杆菌为介导，将青蒿鲨烯合酶反义基因序列导入到烟草，整合到其基因组中 ，成功获得转基因植株。对转基因烟草进行分子检测表明，外源鲨烯合酶基因的序列已经稳定整合到烟草基因组中，并对内源的烟草鲨烯合酶基因表达产生影响。转基因烟草中检测到内源鲨烯合酶基因的mRNA的水平降低。对鲨烯合酶下游产物之一的胆固醇的含量分析显示，活性减低的鲨烯合酶使胆固醇的生物合成下降约40％左右。同时，另一条以FPP为共同前体的二萜代谢途径产物之一GA3的含量得到了提高，比对照提高约30％。

水稻GA3应答基因表达谱微阵列分析与OsGAS1功能初步研究

利用反义技术研究生物代谢途径以及对其生物合成进行调控成为植物次生代谢研究领域内一个重要手段之一，并与新兴的RNAi技术一起成为本领域内重要的研究热点。在植物类异戊二烯代谢途径中存在着羟甲基戊二酰辅酶A还原酶(HMGR)、法呢基焦磷酸合酶（FPS）和鲨烯合酶（SQS）等几种关键的分支酶，他们被认为在异戊二烯类的生物合成中发挥着关键的调节作用。其中，鲨烯合酶处于HMGR和FPS的下游，并与倍半萜合酶等利用共同的前体－法呢基二磷酸（FPP），以FPP起始合成一系列的下游产物。因此，FPP成为类异戊二烯途径中的关键调节点之一。本论文基于此目的，利用反义技术研究了FPP合成鲨烯这一途径受到抑制对其他以FPP为生物合成前体的代谢支路的影响。 利用植物双元转化载体pBI121，将青蒿中鲨烯合酶基因的cDNA（约1.5kb）序列插入到pBI121中，取代原有的GUS序列，构建成植物转化载体pBIASS。以根癌农杆菌为介导，将青蒿鲨烯合酶反义基因序列导入到烟草，整合到其基因组中 ，成功获得转基因植株。对转基因烟草进行分子检测表明，外源鲨烯合酶基因的序列已经稳定整合到烟草基因组中，并对内源的烟草鲨烯合酶基因表达产生影响。转基因烟草中检测到内源鲨烯合酶基因的mRNA的水平降低。对鲨烯合酶下游产物之一的胆固醇的含量分析显示，活性减低的鲨烯合酶使胆固醇的生物合成下降约40％左右。同时，另一条以FPP为共同前体的二萜代谢途径产物之一GA3的含量得到了提高，比对照提高约30％。

青蒿素生物合成相关基因对烟草遗传转化的研究

　　青蒿素是存在于中药青蒿(Artemisia annua L.)中的一种含有过氧桥的倍半萜内酯化合物，是中国科学家研发出的当今最有潜力的抗疟药剂，较传统抗疟药很少或无毒副作用，因此青蒿素的生产备受人们关注。目前，青蒿素的生产主要以植物提取为主，但由于青蒿植株中青蒿素的含量很低（约占干重的0.01%～0.8%），从而导致青蒿素价格昂贵，使许多贫困地区的疟疾患者无法得到医治，故提高青蒿植株中青蒿素的含量或扩大青蒿素的来源，降低生产青蒿素的成本具有重要的意义。　　 　　本论文基于扩大青蒿素的来源和提高青蒿植株中青蒿素含量的目的，开展了以下两方面的工作： 一、紫穗槐二烯在烟草中组合生物合成的研究 　　紫穗槐二烯合酶（amorpha-4,11-diene synthase，ADS）是青蒿素生物合成的关键酶之一，为了能在烟草中合成青蒿素的前体，本研究将青蒿的紫穗槐二烯合酶基因置于CaMV 35S启动子控制下，通过根癌农杆菌介导转入烟草（Nicotiana tobacum L.），并获得了转ADS基因烟草植株。经PCR及Southern杂交分析表明，ADS基因已经整合到转基因烟草基因组中；RT-PCR及对转基因烟草中ADS酶活性和产物中紫穗槐二烯和植物甾醇的测定分析，进一步证明整合的ADS基因在转录、翻译水平上均已经表达。上述结果表明，利用基因工程将青蒿素生物合成途径的关键酶基因导入植物，转基因植物中能够合成青蒿素的前体，这一研究结果为利用转基因植物生产青蒿素或其前体奠定了基础。 二、青蒿鲨烯合酶双链干涉基因对烟草的遗传转化研究 　　鲨烯合酶（squalene synthase, SQS）是甾醇类生物合成分支途径的关键酶之一，利用RNA干扰技术(RNA interference，RNAi)抑制目标基因表达的技术已日趋成熟。本文根据植物中hpRNA(hairpin RNA)的原理，在与烟草SQS同源性高达80％的青蒿ASQS序列的5/端保守区选择622 bp作为构建RNAi的序列，借助中间克隆载体，经过三次亚克隆，最后形成含ASQS－RNAi表达盒的双元表达载体pART27－ASQS，并转入农杆菌EHA105。采用农杆菌介导的烟草叶盘转化法，共获得了12棵转基因植株。转基因植株经过PCR和PCR-Southern blotting 检测，证实外源ASQS基因已经导入烟草中，并已经成功整合到烟草基因组中；通过RT-PCR分析说明，转基因烟草中SQS基因的表达已被成功抑制，部分转基因植株中内源SQS的干扰效果高达90％以上。对SQS的直接产物鲨烯和最终产物植物甾醇的检测显示，转基因烟草的植物甾醇和鲨烯的含量明显低于对照。本实验的结果为下一步将此RNA干扰载体导入青蒿，抑制青蒿中ASQS基因的表达，从而提高青蒿素的含量提供了可能。

Effect of adrenalectomy and corticosterone replacement on prepulse inhibition and locomotor activity in mice

1 Stress is a risk factor in psychiatric illnesses such as schizophrenia. The aim of the present study was to investigate the effect of different circulating levels of the adrenal steroid corticosterone (CORT) on locomotor hyperactivity and prepulse inhibition of acoustic startle, two behavioural animal models of aspects of schizophrenia. 2 Male C57BL/6J mice (n = 10 per group) were anaesthetised with isoflurane and sham-operated or adrenalectomised (ADX). ADX mice were implanted with 50 mg pellets consisting of 100% cholesterol, or 2, 10 or 50 mg of CORT mixed with cholesterol. CORT pellet implantation dose dependently increased plasma CORT levels 3 weeks after surgery. Starting 1 week after surgery, mice were tested for prepulse inhibition after injection of saline or 5 mg kg(-1) of haloperidol. 3 In intact mice and in mice implanted with 10 mg of CORT, haloperidol treatment significantly increased prepulse inhibition (average values from 38 - 42 to 52%). Similar results were observed when testing the mice for amphetamine-induced locomotor hyperactivity (5 mg kg(-1)). In contrast, there was no significant effect of haloperidol in mice implanted either with cholesterol or 2 or 50 mg of CORT. 4 These results in behavioural animal models of schizophrenia suggest an important role of the stress hormone CORT in modulating dopaminergic activity in this illness.

Interaction of corticosterone and nicotine in regulation of prepulse inhibition in mice

The aim of the present Study was to investigate if different levels of circulating corticosterone (CORT) modulate the effect of nicotine on prepulse inhibition (PPI), a measure of sensorimotor gating that is disrupted in schizophrenia and other mental illnesses. Four groups of mice were investigated: sham-operated, adrenalectomized (ADX) and implanted with a cholesterol pellet, ADX and implanted with a 10 mg CORT pellet, or ADX and 50 mg, of CORT. Different CORT levels or doses of nicotine did not significantly affect startle responses. Baseline PPI was significantly reduced in mice implanted with the highest dose of CORT. In ADX mice implanted with cholesterol, nicotine treatment influenced PPI depending on the prepulse intensity. In ADX mice implanted with 50 mg of CORT, treatment with 10 mg/kg of nicotine caused a significant increase in PPI at all prepulse intensities. Binding studies showed that corticosterone treatment had significantly affected nicotinic acetylcholine receptor (nAChR) density in the mouse brain. Treatment with 50 mg CORT decreased I-125-epibatidine binding in the globus pallidus and I-125-alpha-bungarotoxin binding in the claustrum. These results suggest a possible interaction of corticosterone and nicotine at the level of the alpha4- and alpha7-type nAChR in the regulation of PPI. In situations of high circulating levels of corticosterone, nicotine may be beneficial to restore disruption of PPI. (C) 2004 Elsevier Ltd. All rights reserved.

测定乌鳢Ophiocepalus argus腦中胆固醇的初步報告

<正> 鱼脑中胆固醇(cholesterol)的含量,一般说来,还没有广泛地研究过。除鰵鱼(Rosenheim,1914a)外,共他鱼脑中胆固醇的含量都不知道,故就数种習见的鱼,把它们脑中胆固醇的含量加以分析是十分必要的。此地鸟鳢极易得到,我们现先就此鱼作了较详尽的分析,所得结果,报告如下。

Plasma biochemical responses of the omnivorous crucian carp (Carassius auratus) to crude cyanobacterial extracts

Healthy crucian carp (Carassius auratus) were treated by intraperitoneal (i.p.) injection of crude cyanobacterial extracts at two doses, 50 and 200 mu g MC-LR equiv kg(-1) BW. High mortality (100%) was observed within 60 h post injection in the high-dose group. In the treated fish, activities of four plasma enzymes, alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), all showed substantial increases, with both dose and time-dependent effects. These increases of enzyme activity indicate severe impairment occurred in the liver of crucian carp over time. Plasma concentrations of energy-related biomolecules including glucose (GLU), cholesterol (CHO), triglyceride (TG), and total protein (TP) showed marked changes in the high-dose group, possibly a nutritional imbalance correlated with the liver injury caused by intraperitoneal exposure to crude cyanobacterial extracts.

Dietary phosphorus requirement of juvenile black seabream, Sparus macrocephalus

A growth trial was conducted to estimate the optimum requirement of dietary available phosphorus (P) for black seabream (Sparus macrocephalus) in indoor net cages (1.5x1.0x1.0 m). Triplicate groups of black seabream (11.45 +/- 0.02 g) were fed diets containing graded levels (0.18, 0.36, 0.54, 0.72, 0.89 and 1.07%) of available P to satiation for 8 weeks. The basal diet (diet 1), containing 0.18% available P, was supplemented with graded levels of monosodium phosphate (NaH2PO4 2H(2)O) to formulate five experimental diets. The fish were fed twice daily (08:00 h and 16:00 h) and reared in seawater (salinity, 26-29 g l(-1)) at a temperature of 28 +/- 1 degrees C. Dissolved oxygen during the experiment was above 5 mg l(-1). The specific growth rate (SGR), weight gain (WG), feed efficiency (FE) and protein efficiency ratio (PER) were all significantly improved by dietary phosphorus up to 0.54% (P<0.05) and then leveled off beyond this level. Hepatosomatic index (HSI) was inversely correlated with dietary phosphorus levels (P< 0.05). Efficiency of P utilization stabled in fish fed diets containing 0.18%-0.54% available P and then decreased dramatically with further supplementation of dietary phosphorus. Body composition analysis showed that the whole-body lipid, ash, calcium and phosphorus contents were all significantly affected by dietary available P concentration (P<0.05), however, no significance were found in whole-body calcium/phosphorus (Ca/P) ratios among all the treatments (P>0.05). Dietary phosphorus levels also affected the mineralization of vertebrae, skin and scale (P<0.05). Ca/P ratios in vertebrae and scale were not influenced by dietary P supplementation, while skin Ca/P ratio increased statistically with dietary available P levels (quadratic effect, P<0.001). The blood chemistry analysis showed that dietary available P had distinct effects on enzyme activities of alkaline phosphatase (ALP) and plasma lysozyme (LSZ), as well as contents of triacyglycerol (TG) and total cholesterol (T-CHO) (P<0.05). Broken-line analysis showed maximum weight gain (WG) was obtained at dietary available P concentrations of 0.55%. Quadratic analysis based on P contents in whole fish, vertebrae or scale indicated that the requirements were 0.81, 0.87 and 0.88%, respectively. Signs of phosphorus deficiency were characterized by poor growth, slightly reduced mineralization and an increase in body lipid content. (C) 2008 Published by Elsevier B.V.

Effect of dietary carbohydrate-to-lipid ratios on growth and feed utilization in Chinese longsnout catfish (Leiocassis longirostris Gunther)

An 8-week growth trial was carried out in a semi-recirculation system at 26 +/- 0.5 degrees C to investigate the optimal dietary carbohydrate-to-lipid (CHO:L) ratio for carnivorous Chinese longsnout catfish (Leiocassis longirostris Gunther). Triplicate tanks of fish were assigned to each of five isocaloric and isonitrogenous diets with different carbohydrate-to-lipid ratios (0.75, 1.48, 1.98, 2.99 and 5.07). The results showed that a higher specific growth rate (SGR) and feed rate (FR) were observed in the fish fed diet ratios of 1.98 CHO:L (P < 0.05). Overloading dietary carbohydrate (5.07 CHO:L ratio) caused skeletal malformations. Apparent digestibility of dry matter (ADC(d)) significantly increased with dietary CHO:L ratio (P < 0.05), while significantly higher apparent digestibility of protein (ADC(p)) and apparent digestibility of energy (ACD(e)) was observed only in the 1.98 CHO:L group (P < 0.05). Whole body contents of dry matter, lipid and energy significantly increased as the CHO:L ratio decreased (P < 0.05). The hepatosomatic index (HSI) was highest at 1.98 CHO:L ratio (P < 0.05). Highest dietary CHO:L ratio resulted in lower liver glycogen, liver lipid, plasma glucose and plasma triacylglycerol (P < 0.05), whereas there was no significant difference in plasma total cholesterol (P > 0.05). High dietary CHO:L ratio caused pathological changes in fish morphology and liver histology. Based on maximum growth, the optimal carbohydrate-to-lipid ratio was 1.98 for Chinese longsnout catfish.

Identification of differentially expressed genes in Tetrahmena thermophila in response to dichlorodiphenyltrichloroethane (DDT) by suppression subtractive hybridization

The insecticide dichlorodiphenyltrichloroethane (DDT) is persistent in the environment, and continues to cause health problems. Tetrahymena has potential as a model organism for assaying low levels of DDT and for analysing the mechanisms of its toxicity. We constructed the suppression subtractive hybridization library of T thermophila exposed to DDT, and screened out 90 Expressed Sequence Tags whose expressions were significantly up- or downregulated with DDT treatment. From this, a series of important genes related to the DDT metabolism and detoxification were discovered, such as P450 gene, glutathione S-transferase gene and sterol carrier protein 2 gene. Furthermore, their expressions under different concentrations of DDT treatment were detected by real-time fluorescent quantitative PCR. The results show that Tetrahymena is a relevant and useful model organism for detecting DDT in the environment and for discovering biomarkers that can be used to develop specific bio-reporters at the molecular and genomic levels.