Anodic voltammetric behavior of inosine on a glassy carbon electrode

The anodic voltammetric behavior of inosine (I) was investigated by linar-sweep voltammetry, differential-pulse voltammetry and cyclic voltammetry at a glassy carbon electrode. In a medium of 0.1 mol/L N2HPO4, inosine showed a well defined anodic peak. The peak potential was about 1.42 V (vs. Ag/AgCl). A linear relationship held between the peak current and the concentration of inosine in the rang of 5 x 10(-4) similar to 8 x 10(-2) g/L. The peak potential decreased with the decrease of the acidity of the solution. The four anodic peaks of inosine with hypoxanthine, xanthine and uric acid were obtained. Their peak potentials were about at 1.42, 1.07, 0.72 and 0.26 Vt vs. Ag/AgCl). The method has been used for the direct determination of inosine in injections. Recoveries of inosine in urine samples were about 85%. Experimental result proved that the electrode reaction was diffusion-controlled and irreversible.

In situ infrared spectroelectrochemical studies on adsorption and oxidation of nucleic acids at glassy carbon electrode

The adsorption and oxidation of yeast RNA and herring sperm DNA (HS DNA) at glass carbon (GC) electrode are studied by differential pulse voltammetry (DPV) and in situ FTIR spectroelectrochemistry. Two oxidation peaks of yeast RNA are obtained by DPV, whose peak potentials shift negatively with increasing pH. The peak currents decrease gradually in successive scans and no corresponding reduction peaks occur, thus indicating that the oxidation process of yeast RNA is completely irreversible. The IR bands in the 1200-1800 cm-l range, attributed to the stretching and ring vibrations of nucleic acid bases, show the main spectral changes when the potential is shifted positively, which gives evidence that the oxidation process takes place in the base residues. The oxidation process of HS DNA is similar to that of yeast RNA. The results both from DPV and in situ FTIR spectroelectrochemistry confirm that the guanine and adenine residues can be oxidized at the electrode surface, which is consistent with the oxidation mechanism of nucleic acids proposed previously. (C) 2001 Elsevier Science B.V. All rights reserved.

End-column amperometric detection of aesculin and aesculetin by capillary electrophoresis

Determination of aesculin (AL) and aesculetin (AT) by capillary electrophoresis with end-column amperometric detection using a 33 mu m microdisk carbon fiber electrode is described. The HDVs, the effect of pH, buffer concentration, injection voltage, injection time and separation voltage on the peak current response (i(p)) of the analytes and the number of theoretical plates (N) were studied. The method has high sensitivity and good reproducibility. Under the optimum condition - 10 mM, pH 9.00 phosphate buffer, 4 s at 9 kV injection, separation at 15 kV and +1.0 V as the detection potential - low detection limits (S/N = 3) of 0.06 and 0.3 mu M were obtained for AL and AT, respectively. The calibration curve was linear over three orders of magnitude. The relative standard deviations (n = 15) of peak current and migration time were 3.9% and 4.6%, and 0.96% and 0.75% for 15 consecutive injections of 5 mu M AL and AT, respectively. The use of this method for the separation and detection of the two compounds present in the traditional Chinese medicine and human urine samples is also reported. (C) 1999 Elsevier Science B.V. All rights reserved.

Voltammetric behavior of tetracine on glassy garbon electrode and its application

The anodic voltammetric behavior of anaesthetic tetracine and its application were studied. In 0.1 mol/L HClO4 solution, the potential of anodic peak for tetracine is 1.04 V(vs. Ag/AgCl) at a glassy carbon electrode. A linear relationship between the peak height and the concentration of tetracine in the range of 5 x 10(-4) similar to 1 x 10(-1) g/L was obtained. The peak current decreases with the decreasing acidity of the solution. the mehtod has been used for the direct determination of tetracine in injections. The average recoveries of tetracine in urine samples were 98.5%. The mechanism of the electrode reaction was also discussed.

ELECTROCATALYTIC OXIDATION AND FLOW DETECTION OF CYSTEINE AT AN AQUOCOBALAMIN ADSORBED GLASSY-CARBON ELECTRODE

A chemically modified electrode (CME) constructed by adsorption of aquocobalamin (VB12a) onto a glassy carbon electrode surface was demonstrated to catalyze the electro-oxidation of cysteine, a sulfhydryl-containing compound. The sulfhydryl oxidation occured at 0.54-0.88 V vs. Ag/AgCl depending on pH value (3.0-10.0). The electrocatalytic behavior of cysteine is elucidated with respect to solution pH, operating potential and other variables as well as the CME preparation conditions. When used as the sensing electrode in flow injection amperometric detection, the CME permitted detection of the compound at 0.8 V. The detection limit was 1.7 pmol. The linear response range went up to 1.16 nmol. The stability of the CME was shown by RSD (4.2%) over 10 repeated injections.

LIQUID-CHROMATOGRAPHY WITH ELECTROCATALYTIC DETECTION OF CYSTEINE, N-ACETYLCYSTEINE AND GLUTATHIONE BY A PRUSSIAN BLUE FILM-MODIFIED ELECTRODE

Chemically modified electrodes prepared by adsorbing prussian blue on a glassy carbon electrode are shown to catalyse the electro-oxidation of cysteine, N-acetylcysteine and glutathione in acidic media. The catalytic response is evaluated with respect to the potential scan rate, the solution pH, the concentration dependence, and other variables. Covering the electrode with Nafion(R) film improved the stability and reproducibility in liquid chromatography with electrochemical detection to the extent that repetitive sample injections produced relative standard deviations of less than 5% over several hours of operation. The limit of detection was 4 pmol for cysteine, 33 pmol for glutathione and 61 pmol for N-acetylcysteine.

Hybrids between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus: karyotype, allozyme and RAPD analyses

The hybrid between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus was produced by artificial insemination of olive flounder eggs with stone flounder sperm. Sinistral and dextral are two types of hybrid progeny after metamorphosis. Karyotypes of both hybrid flounders are the same as those of the two parental species. Of the 22 loci examined from 12 allozymes, 12 confirmed hybridization of the paternal and maternal loci in hybrids and no difference was found in allozyme patterns of sinistral and dextral hybrid fishes. RAPD patterns of these specimens were also studied with 38 primers selected from 104 tested. Among them, the PCR products of 30 primers showed hybridization of the paternal and maternal bands. Genetic variation between hybrids and their parental stocks was analyzed by RAPD using 10 of the above 38 primers. The average heterozygosity and genetic distance were calculated. The results suggested that the filial generation could inherit a little more genetic materials from paternal fish than that from maternal fish.

Gonad development characteristics and sex ratio in triploid chinese shrimp (Fenneropenaeus chinensis)

This paper details for the first time the gonad development characteristics and sex ratio of triploid shrimp (Fenneropenaeus chinensis). In triploid shrimp the development of gonad is apparently impaired, especially in females. In the ovary of triploids, germ cells mainly remain at oogonia stage during September through December. From January to February of the next year, partial primary oocytes developed in the ovary lobes. Spermatocytes and spermatids could be observed in the testes of triploids, and a few sperm were observed in the vas deferens and spermatophores. The morphology of sperms in triploid shrimp was abnormal. Flow cytometry was used to detect the ploidy of sperm in the vas deferens. The data showed that triploidy could affect the sex ratio in Chinese shrimp. The female-to-male ratio in triploids of about 4:1 will favor triploid shrimp aquaculture.

Microsatellite-centromere distances and microsatellite diversity in different ploidy classes of Chinese shrimp (Fenneropenaeus Chinensis)

This is the first report of microsatellite-centromere mapping in this commercial species Fenneropenaeus Chinensis, and will be important for providing fixed points in the linkage groups of genetic maps. Triploid Chinese shrimp was induced by heat shock. The fertilized eggs were treated either by retention of the first polar body or the second polar body to produce Meiosis I (MI) or Meiosis II (MII) triploid. The triploidy status in each Chinese shrimp could be confirmed by nine polymorphic microsatellite loci, in which the parents with different alleles and the female parents were each heterozygous. The nine loci were mapped in relation to their centromeres in three MII triploid families, which were induced by retention of the second polar bodies after fertilization with sperm. Microsatellite-centromere (M-C) distances ranged from 9.6 cM to 37 cM under the assumption of complete interference. Information on the positions of centromeres in relation to the microsatellite loci will represent a contribution towards assembly of genetic maps in F. chinensis. Twelve polymorphic microsatellites were used to assess the heterozygosity and allelic diversity in different ploidy classes. As expected, triploids were significantly more polymorphic than diploids. The diploids had an average heterozygosity and allelic diversity value of 0.86, whereas the triploids heterozygosity averaged 0.93 and had allelic diversity value of 1.29. However, MI triploids were not significantly more polymorphic than MII in the microsatellite loci.

Synaptonemal complex analysis in spermatocytes of diploid and triploid Chinese shrimp Fenneropenaeus chinensis

A modified surface spreading technique for synaptonemal complex (SC) analysis was tested to assess the process of chromosome synapsis in spermatocytes of diploid and induced triploid Fenneropenaeus chinensis. Spermatocytes of diploid shrimp showed typical morphological characteristics of eukaryote SC, with complete synapsis of bivalents. No recognizable bivalent associated with sex chromosomes was observed in spermatocytes of diploid shrimp. However, differences in morphology of SC, including unsynapsed univalents, bivalents, totally paired trivalents with non-homologous synapsis, partnerswitches and triple synapsis were identified at early pachytene stage of triploid spermatocytes. Triple synapsis was especially common at late pachytene stage in spermatocytes of triploid shrimp. The observed abnormal synapsis behavior of chromosomes in spermatocytes indicated that triploid male shrimp may find it difficult to develop normal haploid sperm. (C) 2008 Elsevier Ltd. All rights reserved.

Cultivation of the brown alga Hizikia fusiformis (Harvey) Okamura: controlled fertilization and early development of seedlings in raceway tanks in ambient light and temperature

Commercial cultivation of the dioecious brown macroalga Hizikia fusiformis (Harvey) Okamura in East Asia depends on the supply of young seedlings from regenerated holdfasts or from wild population. Recent development of synchronized release of male and female gametes in tumble culture provides a possibility of mass production of young seedlings via sexual reproduction. In this paper, we demonstrate that controlled fertilization can be efficiently realized in ambient light and temperature in a specially designed raceway tank in which the sperm-containing water has been recirculated. The effective fertilization time of eggs by sperm was found to be within six hours. Fast growth and development of the young seedlings relied on the presence of water currents. Velocity tests demonstrated that young seedlings of 2-3 mm in length could withstand a water current of 190 cm s(-1) stop without detachment. Culture experiments at 24 h postfertilization showed that elongation of both the seedlings and their rhizoids were not hampered by high irradiance up to 600 mu mol photons m(-2) stop s(-1) stop. However, growth was slightly retarded if cultured at a temperature of 16 degrees C compared to other culture temperatures of 22, 25 and 29 degrees C. No seedling detachment was observed after transfer of the young seedlings to raft cultivation in the sea after one and 1.5 months post-fertilization, indicating the feasibility of obtaining large quantity of seedlings in such a system.

大西洋庸鲽Hippoglossus hippoglossus和大西洋鲑Salmo salar配子质量研究

种质问题是养殖健康发展的基础。在鱼类养殖中，卵子和精子的质量直接关系到受精、胚胎发育，仔稚鱼发育以及幼鱼生长等一系列过程。本论文针对大西洋庸鲽和大西洋鲑的配子质量进行研究。研究内容涉及大西洋庸鲽精子冷冻保存方法；促性腺激素释放激素类似物（GnRHa）使用对其精子冷冻保存效果、以及脂肪酸组成的影响；野生和驯养大西洋鲑卵子在脂肪酸、类胡萝卜素、矿物盐方面的差异比较。 精子冷冻保存通过提高对精子的利用效率，进而对于种质改良，推进鱼类养殖科研和生产具有重要意义。本实验建立了大西洋庸鲽精子大容量冷冻保存方法。八种抗冻剂冷冻保存实验结果表明：10% 及15% DMSO配以 HBSS 或KS 的抗冻剂组合冷冻保存效果最佳，4 mL体积冷冻保存可获得与1.6 mL同样的保存效果。 在繁殖季节后期注射GnRHa激素缓释剂，可获得质量稳定的大西洋庸鲽精液，将激素注射方法与精子冷冻保存方法相结合对于提高雄鱼利用率，扩大生产规模具有重要实用价值。本项研究分三个时间采集注射GnRHa激素后的雄鱼精子以及同期未注射激素的雄鱼精子，对所有精子样品使用同样的方法进行冷冻保存，检测冷冻保存后解冻精子的受精率与活力。结果表明，激素注射与否对于冷冻保存后精子的受精率和活力无显著影响，两类冷冻精液均达到鲜精水平。实验结果还表明，注射激素14天后的精子的密度显著的降低。说明GnRHa激素的使用可以显著降低精子密度，但不会影响精子的冷冻保存效果。 本相研究同时对注射GnRHa 缓释激素和未注射GnRHa 缓释激素的大西洋庸鲽精液脂肪酸成分进行分析，以检测该激素使用对精子生化组分的影响。结果表明激素的使用对在DHA (22:6n-3,二十二碳六烯酸)、EPA(20:5n-3,二十碳五烯酸)、AA(20:4n-6，花生四烯酸)等重要脂肪酸，不饱和脂肪酸、饱和脂肪酸以及n-3、n-6等重要种类的脂肪酸总量及其比例没有显著影响。精液脂肪酸中DHA含量最高，约占25%；PUFA约为44%。 作为世界性的重要养殖品种，野生和驯养大西洋鲑在形态、生化组成以及遗传 等方面表现出的差异被广泛关注。本论文，对野生和驯养大西洋鲑受精卵关键生化成分进行分析，通过与野生受精卵比较阐明驯养受精卵的质量状况，为亲鱼营养需求提供指导依据。本实验中野生配子和驯养配子的受精率没有显著差异，但重要脂肪酸组成、类胡萝卜素以及矿物盐含量都存在多方面显著差异。两类受精卵脂肪酸中含量最高的依次为18:1n-9（油酸）、DHA（二十二碳六烯酸）、16:0（棕榈酸）、EPA（二十碳五烯酸）。野生受精卵的单不饱和脂肪酸总量显著高于驯养受精卵，而多不饱和脂肪酸（PUFA）比例显著低于驯养的受精卵。在主要必需不饱和脂肪酸（EFA）中，DHA和EPA在野生受精卵中的比例高于驯养受精卵，AA（花生四烯酸）低于驯养受精卵。野生受精卵虾青素（Ax）的含量低于驯养受精卵而鸡油菌素（Cx）含量高于驯养受精卵。野生受精卵中多种矿物盐的含量（铝、铜、铁、硒和锌）含量显著高于驯养的受精卵。差别最大的为铜。诸多方面的差异表明，野生亲鱼与驯养亲鱼产出的卵子确实存在显著差异，因此关注亲鱼的营养极为重要。

真鲷(Pagrosomus major)胚胎冷冻保存损伤机理及冷冻精子遗传物质稳定性研究

本文对真鲷心跳期胚胎对5种常用渗透性抗冻剂（DMSO、甘油、甲醇、丙二醇、乙二醇）和3种非渗透性抗冻剂（PVP、PEG-8000、蔗糖）的耐受性进行了研究。渗透性抗冻剂分6个浓度梯度（5%；10%；15%；20%；25%；30%）和3个时间组（10min；30min；1h）。非渗透性抗冻剂中，PVP、PEG-8000分3个浓度梯度（5%、10%、15%）和2个时间组（10min、30min），蔗糖为4个浓度梯度（5%、10%、15%、20%）和2个时间组（10min、30min）。实验结果表明，在渗透性抗冻剂组中，浓度为5%的处理组的孵化率（>90%）与对照组差异均不显著，随着抗冻剂浓度增大及处理时间的延长，真鲷心跳期胚胎的孵化率显著下降（P<0.05），在最高浓度的最长处理时间中胚胎孵化率均降到了0。总体上，真鲷心跳期胚胎对五种渗透性抗冻剂的耐受性从小到大依次为：甲醇 < 甘油 < 乙二醇 < DMSO < 丙二醇。对影响胚胎孵化率的三个因素（抗冻剂、浓度、时间）进行的因素效应分析结果表明，三种因素对孵化率的影响显著（P<0.05），并且浓度效应 > 时间效应 > 抗冻剂效应。在非渗透性抗冻剂组中，蔗糖组胚胎孵化率未呈显著变化；PVP组随着浓度及时间的增大，孵化率显著下降（P<0.05）；PEG-8000组随着浓度增大孵化率显著下降（P<0.05），但在两个时间组间差异不显著。相同处理情况下PEG-8000对真鲷心跳期胚胎的毒性要小于PVP。因素效应分析比较结果表明仅时间效应不显著，且抗冻剂效应 > 浓度效应 > 时间效应。 对所用各种抗冻剂进行了渗透压测量，实验中使用的渗透性抗冻剂（5%-30%）的渗透压值在959-7980mOsm/kg之间，均高于使用海水的渗透压值（919mOsm/kg）；使用的非渗透性抗冻剂的渗透压值在316-1040mOsm/kg之间，除20%蔗糖渗透压值（1040mOsm/kg）高于海水外，其他非渗透性抗冻剂的渗透压值均要低于海水。对孵化率与相应的溶液渗透压值进行相关回归分析结果表明，渗透性抗冻剂的渗透压与孵化率呈显著的负相关（P<0.05），而非渗透性抗冻剂的渗透压与孵化率相关不显著。渗透性抗冻剂组的回归分析结果表明，二次方程的曲线拟合度最高，得到的回归方程分别为：Y10min = -2×10-8X2 10min - 6×10-5 X 10min + 1.5635 （R2 = 0.713），Y30min= 5×10-8X2 30min－0.0007 X 30min + 2.097（R2 = 0.681），Y1h = 7×10-8X2 1h－0.0008 X 1h+ 2.0397（R2= 0.725）。 在真鲷胚胎对抗冻剂耐受性实验的基础上，挑选五种抗冻剂--10%DMSO、5%甘油、10%甲醇、20%丙二醇、10%乙二醇，浸泡真鲷心跳期胚胎30min后，分别以超速（130℃/min）、快速（20℃/min）、慢速（3℃/min）的速度降温并使用低温显微镜进行观察，依次记录Toif（油球结冰）、Teif（胚胎外部结冰）、Tiif（胚胎内部结冰）等结冰点，Toif值在-9~-23℃之间；Teif值在-21~-35℃之间；Tiif值在-21~-52℃之间。结冰顺序为先油球结冰，然后胚胎外部结冰随之内部马上瞬间变黑形成内部冰晶。随着降温速度的提高，各结冰温度值显著下降。各抗冻剂之间的Teif及Tiif值不同，Toif值之间没有显著差异。对两种玻璃化冷冻方法进行模拟观察，发现胚胎冰晶形成的顺序与非玻璃化过程不同--先内部结冰然后逐渐蔓延至外部形成外部冰晶，而且模拟玻璃化的内部结冰温度Tiif值（-52.56℃）显著（P<0.05）低于使用低浓度的同种抗冻剂超速降温组的Tiif值（-40.11℃）。在快速及慢速降温组中，20%丙二醇组的Tiif要显著的低于其他组（P<0.05）；在超速降温中，甲醇组的Tiif值要显著的低于其他组（P<0.05）。在Tiif小于30℃的实验组中获得形态完整胚胎的比例平均仅有30.77%；在Tiif大于30℃的实验组中获得形态完整胚胎的平均比例高达70.37%，模拟玻璃化组达到100%。各抗冻剂之间，复温后胚胎形态完整率10%甲醇组最高（77.78%）；其次依次为10%乙二醇（66.67%）、20%丙二醇（55.56%）和10%DMSO（55.56%）；5%甘油组最低（11.11%）；推测甲醇的对胚胎的渗透效果要好于其他组。综上推测：使用丙二醇、甲醇作为抗冻剂以及玻璃化冷冻保存方法对真鲷心跳期胚胎超低温保存也许较为合适。 我们对低温保存的真鲷精子核DNA损伤进行了研究以期为下一步胚胎遗传物质稳定性研究提供参考依据。研究方法为单细胞凝胶电泳（SCGE），针对研究对象，在实验过程中对传统的碱性单细胞凝胶电泳在铺胶方法、电泳条件等进行了改进。对精子细胞进行预处理，在碱性电泳液中使核DNA双链解链变性后电泳，EB染色lOmin后，在荧光显微镜下观察，每次随机观察50个左右的核DNA。结果表明，对荧光显微镜下观察到的精子核按彗尾长度及荧光强度划分等级，出现损伤的精子核DNA的损伤程度主要为轻度损伤和中度损伤，很少见有完全损伤的真鲷精子核。经5%、10%、18%、20%、25%、30%DMSO冷冻保存后的精子彗星率分别为33.47％ ± 8.95％; 35.91％ ± 19.44％; 48.95％ ± 8.90％; 43.33％ ± 11.19％; 55.80％ ± 38.94％。鲜精彗星率为31.43 ％ ± 2.68％。对比真鲷冷冻精液与新鲜精液的精子DNA的损伤状况，表明仅用30％ DMSO冷冻精子DNA损伤状况与鲜精差异显著(P<0.05)。 综上所述，渗透性抗冻剂对胚胎的毒性与其渗透压值呈显著的负相关关系。丙二醇对真鲷心跳期胚胎毒性最小，甲醇较其他抗冻剂能更好的渗透入胚胎；玻璃化方法能显著降低Tiif值并能更好的保持超低温保存后胚胎的形态完整性，因此，使用丙二醇、甲醇作为抗冻剂以及玻璃化冷冻保存方法对真鲷心跳期胚胎超低温保存也许较为合适。常规使用的用于超低温保存真鲷精子的DMSO（浓度<15%）不会对精子核物质稳定性造成明显影响。由于胚胎较精子结构要复杂许多，对于真鲷胚胎损伤机理的研究还有大量工作可以开展。

Osmoregulatory actions of growth hormone in juvenile tilapia (Oreochromis niloticus)

Growth hormone (GH) effectively promotes seawater (SW) adaptation in salmonids, but little is known of its effect in tilapias. Experiments were performed to investigate the effects of recombinant eel GH (reGH) on osmoregulatory actions and ultrastructural features of gill chloride cells in juvenile tilapia, Oreochromis niloticus. Tilapia showed a markedly improved SW survival, when directly transferred from freshwater (FW) to 62.5% SW 24h after a single reGH injection (0.25 or 2.5 mu g g(-1)) or 3 reGH injections (0.25 mu g g(-1) every other day). Plasma Na+ and Mg2+ levels were significantly reduced by reGH (0.25 and 2.5 mu g g(-1)) compared with saline injections; Ca2+ concentrations were reduced significantly by high dose of reGH (2.5 mu g g(-1)) after SW transfer. However, fish failed to survive more than 24h when directly transferred to 70 % SW, although the fish treated with reGH could survive longer than the controls. When examined by electron microscopy, the chloride cells were identified as mitochondrion-rich and an extensive tubular system was induced by GH treatment. The results of the present study suggest that, similar to its effect on salmonids, GH also exerts acute osmoregulatory actions and enhances SW adaptation in juvenile tilapia. GH also stimulates the differentiation of chloride cells toward SW adaptation.