Characterization of the Rana grylio virus 3 beta-hydroxysteroid dehydrogenase and its novel role in suppressing virus-induced cytopathic effect

The 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) isoenzymes play a key role in cellular steroid hormone synthesis. Here, a 3 beta-HSD gene homolog,was cloned from Rana grylio virus (RGV), a member of family Iridoviridae. RGV 3 beta-HSD gene has 1068 bp, encoding a 355 aa predicted protein. Transcription analyses showed that RGV 3 beta-HSD gene was transcribed immediate-early during infection from an initiation site 19 nucleotides upstream of the translation start site. Confocal microscopy revealed that the 3 beta-HSD-EGFP fusion protein was exclusively colocalized with the mitochondria marker (pDsRed2-Mito) in EPC cells. Upon morphological observation and MTT assay, it was revealed that overexpression of RGV 3 beta-HSD in EPC cells could apparently suppress RGV-induced cytopathic effect (CPE). The present studies indicate that the RGV immediate-early 3 beta-HSD gene encodes a mitochondria-localized protein, which has a novel role in suppressing virus-induced CPE. All these suggest that RGV 3 beta-HSD might be a protein involved in host-virus interaction. @ 2006 Elsevier Inc. All rights reserved.

First report of aphantoxins in China - waterblooms of toxigenic Aphanizomenon flos-aquae in Lake Dianchi

The oligohaline cyanobacterium Aphanizomenon flos-aquae (L.) Ralfs (A. flos-aquae) has been reported in several countries to produce paralytic shellfish poisons (PSPs) or protracted toxic effects. In the past years, A. flos-aquae blooms have occurred annually in the eutrophic Lake Dianchi (300 km(2) in area, located in southwestern China). Material from natural blooms dominated by A. flosaquae was collected and lyophilized. Acute toxicity testing was performed by mouse bioassay using extracts from the lyophilized material. Clear symptoms of PSPs, intoxications were observed. To confirm the production of PSPs, a strain of A. flos-aquae (DC-1) was isolated and maintained in culture. Histopathological effects were studied by examining the organ damages using transmission electron microscopy (TEM). Slight hepatocytic damage with swollen mitochondria was found. The ultrastructural pulmonary lesions were characterized by distortied nuclei and indenting of karyotheca, together with degeneration and tumefaction of mitochondria and endoplasmic reticulum. Control animals injected with acetic acid did not exhibit histopathological damage in any organ. Toxic effects of cultured algal cells on enzymatic systems in the mouse were studied using sublethal doses of extracts. Significant glutathione-S-transferase (GST) and lactate dehydrogenase (LDH) increases, together with decrease of the glutathione (GSH) level, were measured. These results indicated a potential role of PSPs intoxicating and metabolizing in the test animals. HPLC-FLD and LC/MS analysis of extracts from cultured material demonstrated the PSP toxins produced by A. flos-aquae bloom. To the best of our knowledge, this is the first study reporting chemically and toxicologically confirmed PSP toxins related to A. flosaquae in China. (c) 2005 Elsevier Inc. All rights reserved.

Electron microscopic examination of the viromatrix of Rana grylio virus in a fish cell line

Rana grylio virus (RGV), a Ranavirus belonging to the family Iridoviridae, assembles in the viromatrix which is a factory for viral genome replication and particle assembly. Ultrastructural studies of the viromatrix will clarify the pathway of assembly. The viromatrix and quantitative changes in RGV infected epithelipma papulosum cyprini (EPC) cells, one of fish cell lines, were studied by electron microscopy. It was shown that viromatrices were adjacent to the nucleus, and the electron density was lower than that of the surrounding cytoplasm. The viromatrix contained virus particles with different forms, electron-dense materials and amorphous structures which included tubules and membranous materials. Tubules were often observed in direct continuity with empty capsids. Several bundles of intermediate filaments were seen alongside the viromatrix and crystalline aggregates. Large clusters of mitochondria occurred in proximity to viromatrix. A total of 990 cells profiles were examined. The results showed that 394 cells contained viromatrix: 89.3% contained one, and 10.7% contained two to four viromatrices. The number of viromatrices increased gradually and reached a peak at 16 h p.i. The viromatrix area at 24 h p.i. increased up to 7.4 +/- 0.69 mu m(2) which was three-times lower than that at 6 h p.i. The number of empty capsids within viromatrix was generally more than that of "full" particles at different time points, and there was a strong positive correlation between them. (c) 2005 Elsevier B.V. All rights reserved.

Cultured gill epithelial cells from tilapia (Oreochromis niloticus): a new in vitro assay for toxicants

A culture gill epithelium from seawater-adapted tilapia (Oreochromis niloticus) was developed for testing PAHs and dioxin-like contaminants in seawater. The epithelia consists two to three layers of epithelial cells incorporating both pavement cells and mitochondria-rich cells (MRCs). Polarity and a stable transepithelial resistance (TER) were maintained. and closely resembled those in fish gills in vivo. The tightness (integrity) of the epithelia remained unchanged upon exposure to benzo[a]pyrene (B[a]P). 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3,3',4,4',5-pentachlorobiphenyl (PCB#126), while a concentration-dependent response of EROD activity in the epithelia was induced within 18-24 h when the apical side was exposed to these toxicants. The 24 h EC50 of EROD activity was 2.77 x 10(-7) M for PCB#126, 1.85 x 10(-7) M for B[a]P and 7.38 x 10(-10) M for TCDD. showing: that the preparation was not only sensitive to PAHs and dioxin-like compounds, but also able to produce inductive potency of AhR agonists that generally agreed with those derived from other established in vitro and in vivo systems. The results suggest, that the cultured gill epithelia from seawater-adapted tilapia may serve as a simple. rapid and cost-effective tool for assessing exposure and potential effects of toxicants in marine waters. (C) 2004 Elsevier B.V. All rights reserved.

Total and subcellular distribution of trace elements in the liver of a mother-fetus pair of Dall's porpoises (Phocoenoides dalli)

Total and subcellular hepatic Zn, Cu, Se, Mn, V, Hg, Cd, and Ag were determined in a mother-fetus pair of Dall's porpoises (Phocoenoides dalli). Except for higher fetal Cu concentration, all maternal elements were higher. Elements existed mostly in the cytosol of both animals except in the case of maternal Ag in the microsome and fetal Cu and Ag in the nuclei and mitochondria. In the maternal cytosol, Zn, Mn, Hg, and Ag were present in the high-molecular-weight substances (HMW); Se and V were present in the low-molecular-weight substances (LMW); Cu and Cd were mostly sequestered by metallothionein (MT). In the fetal cytosol, Zn, Se, Mn, Hg, Cd, and Ag were present in the HMW and V in the LMW, while Cu and Ag were mostly associated with MT. MT isoforms were characterized using the HPLC/ICP-MS. Two and four obvious peaks appeared in the maternal and fetal MT fractions, respectively. The highest elemental ion intensities were at a retention time of 7.8 min for the mother, and for the fetus the peak elemental ion intensities occurred at a retention time of 4.3 min, suggesting that different MT isoforms may be involved in elemental accumulation in maternal and fetal hepatocytosols. (C) 2003 Elsevier Ltd. All rights reserved.

Long-term toxic impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the reproduction, sexual differentiation, and development of different life stages of Gobiocypris rarus and Daphnia magna

The sexual ratio of Gobiocypris rarus exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 17 beta -estradiol from embryo to sexually mature revealed feminization and overdevelopment of connective tissue in male fish gonad in 2-30 pg/L TCDD concentration range. Daphnia magna was not sensitive to the high dose of TCDD (0.1-1000 ng/ml), but the reproduction of D. magna treated with TCDD decreased after the 8th day. 7-Ethoxyresorufin-O-deethylase (EROD) activities in newly fertilized eggs of G. rarus exposed to TCDD dosage groups (1000-100,000 pg/L) were significantly induced and increased with TCDD concentrations at the early life stage, while no difference was found between low TCDD dosage groups (<100 pg/L), but a good relationship between the EROD activity and the TCDD concentration was observed during a long-term developmental stage. There was a pericardial edema formed in a 2-week yolk-sac at the concentration of 1000 pg/L TCDD. But in the exposure group (2 pg/L TCDD for 120 days), the cell nuclei of hepatocytes was far from the center and packed toward the cell membrane; the cristae of most mitochondria in the cell dropped and collapsed; the rough endoplasmic reticulum broke into fragments; and numerous lipid droplets formed in the cell. (C) 2001 Academic Press.

Studies on morphogenesis and cellular interactions of Rana grylio virus in an infected fish cell line

A pathogenic virus (RGV), isolated from diseased pig frog Rana grylio with lethal syndrome, was investigated with regard to morphogenesis and cellular interactions in EPC cells, a cell Line from fish. Different stages of virus amplification, maturation and assembly were observed at nucleus, cytoplasm and cellular membranes. The matured virus particles, were not only distributed diffusely in nucleus, cytoplasm and cellular surface, but also aggregated as pseudocrystalline arrays in the cytoplasm. Virions were released by budding from the plasma membranes, or following cell lysis. Various types of cell damage, such as small vacuoles, spherical inclusions, and swollen and empty mitochondria, were also found. Some typical characteristics of RGV, such as the symmetrical shape of the virions, replication process involving both nuclear and cytoplasmic phases, budding release from cellular membrane and intracellular membrane, viromatrix and paracrystalline aggregation in cytoplasm, and its acute pathogenic effects, were observed to be similar to that of other iridoviruses. Therefore, the RGV appears to be a member of the Iridoviridae based on these studies. (C) 1999 Elsevier Science B.V. All rights reserved.

Dynamics of morphological changes for mitochondrial fission and fusion

Mitochondria experience continuous fusion and fission in a living cell, but their dynamics remains poorly quantified. Here a theoretical model was developed, upon a simplified population balance equation (PBE), to predict the morphological changes induced by mitochondrial fission and fusion. Assuming that both fission and fusion events are statistically independent, the survival probability of mitochondria staying in the fission or fusion state was formulated as an exponentially-decayed function with time, which depended on the time-dependent distribution of the mitochondrial volume and the fission and fusion rates. Parametric analysis was done for two typical volume distributions. One was Gamma distribution and the other was Gaussian distribution, derived from the measurements of volume distribution for individual mitochondria in a living cell and purified mitochondria in vitro. The predictions indicated that the survival probability strongly depended on morphological changes of individual mitochondria and was inversely correlated to the fission and fusion rates. This work provided a new insight into quantifying the mitochondrial dynamics via monitoring the evolution of the mitochondrial volume.

青杨(Populus cathayana Rehd)雌雄植株对锈病和干旱胁迫的生理响应及差异蛋白质组比较

雌雄异株植物对环境的不同响应一直是一个有趣而新颖的研究领域，由于雌雄个体不同的繁殖成本及不同的生存策略，使得雌雄植株在生长、存活、生殖格局、空间分布、资源配置等方面已经表现出明显的不同，在生理和分子水平上也表现出明显的性别间差异。干旱是制约农林业发展的环境因子之一，叶锈病是对杨树危害最严重的病害之一，由于长期进化的结果，不同性别的植物必然对生物和非生物胁迫有着不同的响应。本文以雌雄异株的青杨为模式植物，研究雌雄间在生理、生化、亚细胞结构和蛋白质水平上对生物和非生物胁迫的差异响应。主要研究结果如下： （1） 青杨雌雄植株对锈病胁迫的生理生化差异响应 在正常的对照组中，雄株叶片比雌株叶片有着较高的活性氧自由基产生速率、较高的SOD、POD、PPO 和较低的CAT 活性；在锈病感染的早期阶段， SOD、POD、CAT 活性、活性氧自由基产生速率、H2O2 含量、膜脂过氧化程度和细胞膜的电渗率在雌雄株中都增加，而PPO 仅在雄株中增加明显，APX 仅在雌株中增加明显，并且雌株比雄株有着更严重的锈病感染程度、细胞膜的伤害程度和光合系统II 的破坏程度，雌株有更多的净光合速率、气孔导度和叶绿素a 含量的降低，在同工酶变化上，雌雄间对锈病也显示出不同的表达模式。结果显示，雄株比雌株对锈病有着更好的抗性和更有效的ROS 清除系统。 （2） 青杨雌雄植株对干旱胁迫的生理生化及亚细胞结构的差异响应 与较好水分条件相比，干旱下雄株比雌株有着更高的A-Ci 响应参数，如Rubisco 最大羧化速率、光呼吸速率、暗呼吸速率和最大电子传递速率等。干旱显著地增加了膜脂过氧化程度和游离脯氨酸含量，并且雄株比雌株表现出较低的膜脂过氧化程度，较高的总蛋白和游离脯氨酸含量。无论是中度干旱还是极度干旱，除了CAT 外，雄株比雌株表现为较强的抗氧化酶活性，在同工酶谱带上，雌雄间表现出不同的变化模式，并且有些条带是干旱影响应的，而有些条带是性别特异性的，这些性别特异性条带能够作为鉴定性别快速而准确的标记。干旱显著地影响了线粒体、叶绿体和细胞壁的结构，尤其在中度干旱胁迫下，雄株线粒体和叶绿体比雌株呈现出较好的完整性，并且雄株细胞壁要比雌株更厚。因此， 雄株比雌株表现出更强的干旱忍耐性和更高效的抗氧化酶系统。 （3） 青杨雌雄植株对干旱胁迫的蛋白质组差异响应 用双相电泳检测到雌雄间近1000 个蛋白点，通过对比发现对照组雌雄间有54 个差异蛋白点，干旱下雌雄间有108 个差异点，其中102 个被质谱成功鉴定。对照组雌雄间的差异蛋白主要集中在与光合作用相关蛋白、抗氧化酶、胁迫防御蛋白和一些调节基因表达的蛋白；干旱胁迫下雌雄间差异蛋白明显增多，主要有参与信号转导、调节基因表达、蛋白质加工、转录产物的转录翻译后修饰的调节性蛋白蛋白和参与氧化还原平衡、抗胁迫、细胞壁合成、光合作用、能量代谢、氨基酸代谢和脂肪酸代谢等的功能性蛋白。干旱下这些蛋白的表达量在雌雄中有的表现出相同的表达模式，如干旱下雌雄株中Rubisco 激活酶、小热激蛋白等表达都增加，而有的表现出相反的表达模式，如Rubisco 大亚基的降解片段、羰酸酯酶等在雄株中表达量上调而在雌株中却是下调。因此，雌雄间在蛋白质水平上对干旱胁迫响应的差异是显著的，也是复杂的。 It is an interesting and novel topic that dioecious plants possess different responses to environmental stress. As for the different productive cost and different survive strategy, different sexual plants have shown obviously morphological, physiological and molecular differences. Drought is one of the most worldwidely important environmental stress factors that limit plant growth and ecosystem productivity. Rust disease is one of the economically important diseases in many trees. As a result of the long evolutionary process, male and female plants should show different responses to abiotic and biotic stress. In this paper, using a dioeious tree of Populus cathayana Rehd as a model, we study the sexual differences to drought and rust disease stress in physiological, biochemical, sub-cellular and proteomics levels. The main results are follows: (1) The sexual differences in physiology and biochemistry of poplar to rust disease In controls, males showed higher production of superoxide radicals, higher activities of SOD, POD, PPO and lower CAT activity. Under rust disease, the activities of antioxidant, the content of ROS and the degree of cellular member destroyed were increased in both sexes, except for PPO in diseased males and APX in diseased females. However, females showed more seriously disease severity and cellular member and PS II destroyed degrees. Net photosynthesis rate, transpiration rate and chlorophyll a content were decreased more in diseased females than in males. There were also some different changes inantioxidant isozymes under rust disease. The results suggested that male poplar possessed a more effectively antioxidant system and were more resistant to rut disease than females. (2) The sexual differences in physiology and biochemistry of poplar to drought stress Under drought stress, there were higher rates of RuBP-saturated CO2 assimilation, dark respiration, photorespiratory release of oxygen, the max electron transportrate in CO2-saturated and carboxylation efficiency in males than in females. And males showed lower TBARS and higher proline content. Except for CAT, the activities of other antioxidants were higher in males than in females. Meanwhile, there were obviously differences in isozyme changes between teo sexes. Drought stress obviously destroyed the integralities of chloroplasts and mitochondria and the sexual differences in sub-cellular level were obviously under the moderate water stress. Male cell walls were more sensitive to drought stress than did female. The results suggested males were more resistant to drought stress. (3) The sexual differences in proteomics of poplar to drought stress By 2-D and MS analysis, we identified 102 different protein spots between males and females. Under control conditions, the different proteins were mainly in photosynthesis related proteins, antioxidants, stress response proteins and some gene expression related proteins. Under drought stress, the different proteins were focused on (i) regulated proteins such as signaling conduction, kinase, HSP, gene expressional regulation and protein modification, (ii) functional proteins such as photosynthesis, energy metabolism, antioxidant, redox, stress response, lipid metabolism and amino acid metabolism. Some protein showed the same expressional pattern, while some showed contrary expressional pattern. Thus, the results suggested that sexual differences in proteomics were significant and complex.

棘蛙族(Tribe Paini)精子形态结构及精巢年周期研究

棘蛙族(Tribe Paini)隶两栖纲(Amphibia)、无尾目(Anura)、蛙科(Ranidae)、叉舌蛙亚科(Dicroglossinae)，由棘蛙属(Paa)、倭蛙属(Nanorana) 和沙巴蛙属(Chaparana)构成（Dubois,1992）。由于特殊的形态特征和染色体核型，棘蛙族受到国内外学者的广泛重视和研究，但是到目前为止，棘蛙族的系统发育关系尚未明晰，族下属种的分类和归属问题还有待进一步研究和新的证据出现。本文通过光学显微镜、电子显微镜和石蜡切片对棘蛙族10 物种的精子和精巢进行研究，旨在了解棘蛙族精子的形态、量度、超微结构特征及不同季节精巢结构的变化规律，同时为棘蛙族的系统研究提供新的依据，也为棘蛙族濒危物种的保护和经济物种的繁殖提供基础资料。研究结果表明：棘蛙族各属物种精子的形态基本相似，精子整体呈线形，由头部、中片和尾部构成。精子头部呈长条状，顶体呈锥状，位于头部顶端并向前伸出，中片较长，尾部波动弯曲。棘蛙族各属物种精子量度差异较大，将各属物种精子头部、中片、尾部、头宽、尾宽的量度数据进行聚类分析，结果表明棘蛙族10 物种可分为三类：第一类包括棘侧蛙、合江棘蛙、小棘蛙、棘腹蛙和棘胸蛙，特点是精子较短，全长在72.6～103.35µm 之间；第二类包括倭蛙、高山倭蛙、腹斑倭蛙，特点是精子较长，全长在107.74～129.75µm 之间；第三类包括隆肛蛙和双团棘胸蛙，特点是精子最长，全长在145.89～165.84µm 之间。棘蛙族各属精子超微结构基本相似：精子头部由顶体、细胞核构成；中片由中心粒、线粒体构成；尾部由单根轴丝构成。精子顶体横切呈圆环状，细胞核电子密度高；线粒体为卵圆形，呈环状围绕轴丝排列，线粒体数目较多，约30层；尾部轴丝为典型的9＋2结构，即由2根中央微管和9对外周微管组成。不同季节的倭蛙精巢结构变化表明倭蛙精巢每年只有一个生精周期，生精周期始于7 月，繁殖季节从5 月到6 月，生精高峰期为9 月；根据倭蛙不同季节精巢结构的变化，可将生精周期分为3 个阶段：第一阶段从7 月到9 月，为精子形成期；第二阶段从10 月到翌年4 月，为精子的贮存阶段，也即倭蛙的冬眠期；第三阶段从5 月到6 月，为精子的排放阶段，即倭蛙的繁殖期。不同季节的隆肛蛙精巢结构变化表明5 月为隆肛蛙的繁殖高峰期。根据棘蛙族各属精子的形态、量度和超微结构特征，结合已有的棘蛙族形态学、生态学、染色体核型及系统学研究成果，本文认为：1．基于精子数据对棘蛙族的划分和基于形态学及分子系统学数据对棘蛙族的划分均有相同之处，精子形态结构可为棘蛙族的系统研究提供新的证据。2． 棘蛙族各属精子的形态、量度及超微结构不仅与蛙科其他属种有明显差异，而且在无尾类中也较为特殊，精子学研究结果支持将棘蛙族从蛙科中分离出来，归隶于叉舌蛙科的叉舌蛙亚科的系统学修正。3. 精子的顶体、细胞核、中片的形态结构及量度可作为蛙科的分类指标。On the base of unique morphological and kyrotype characters, Dubois(1992)recognized three genera Paa, Narnorana, Chaparana as tribe Paini, which is amember of Dicroglossinae, Ranidae. In present study, the sperm shape, size andultrastructure of 10 paini species were investigated through the light and electronmicroscope, and testis structure of N. pleskei and F. quadrana was also studied. Wesuppose this study could offer some spermatological evidence to phylogeny andreproduction study of tribe Paini. The results were as follows:The sperm shape of tribe paini is homologically similar, the spermatozoa arefiliform, composed of elongate head, long mid-piece and waved tail. The acrosome isapically associated with the nucleus and extend anteriorly.The sperm length of tribe paini differ remarkably among genera. Cluster for thelength of sperm head, mid-piece, tail, total length, head-width, tail-width of ten painifrogs indicated the 10 species could be separated into three groups: GroupⅠcontainsP. shini, P. robertingeri, P. spinosa, P. exilispinosa, P. boulengeri, the spermatozoa ischaracterized with short in total length, ranging from 72.6µm to 103.35µm; GroupⅡcontains N. pleskei, N. parkeri, N. ventripunctata, the spermatozoa ischaracterized with relatively long in total length, ranging from 107.74µm to129.75µm; Group Ⅲ contains F. quadrana and P. yunnanensis, the spermatozoa is characterized with longest in total length, ranging from 145.89µm to 165.84µm. thethree groups based on spermatological data is partially match the classification basedon morphological and molecular data.The ultrastructure of spermatozoa in tribe paini is also basic similar, includingacrosome vescile, nuleus of the head proper, centriole, mitochondriol of themid-pieces, axoneme of the tail. The acrosome vescle is circle in TEM transversesection, the density of nucleus is high; The mitochondrions is oval, surrounding theaxial filament with about 30 layers of mitochondria; The axoneme has the typical 9+2pattern of microtubules.The seasonal changes in testis of N. pleskei indicates it has only onespermatogenesis circle, which begin in July, the reproduction season is from May toJune, the spermatogenesis is active in September. On the base of seasonal changes intestis, the spermatogenesis circle can be separated into three stages: In stageⅠfromJuly to September, spermatids are formed; In stage Ⅱ from October to April next year,the spermatozoa are stored in testis,which is the hibernated period; In stage Ⅲ fromMay to June, mature spermatozoa were released from the testis, which is thereproduction season of N. pleskei. As to F. quadrana, reproduction is active in May.With the previous study of morphology, ecology, karyotypes and phylogenyresearch of tribe Paini, the spermatological data in present study suggests:1. The spermatological classification of tribe paini is partially consistant with themorphological and molecular classification respectively.2.The sperm morphology and ultrustructure of tribe paini is unique not only inthe family Ranida but also in Anura, which suggest the tribe paini is monophyletic andmight be transfered from the family Ranida to the family Dicroglossidae based onmolecular evidence.3. The acrosome, nuleus, shape, length and ultrastructure of mid-piece can beused as an alternative taxonomic character in Anura.

Evaluation of bioeffects of a long-term administering ChangLe on rat by proton NMR method and biochemical examination

High resolution H-1 nuclear magnetic resonance ( NMR) spectroscopy has been employed to assess long-term toxicological effects of ChangLe (a kind of rare earth complex applied in agriculture). Male Wistar rats were administrated orally with ChangLe at doses of 0, 0.1, 0.2, 2.0, 10 and 20 mg/kg body weight daily, respectively, for 6 months. Urine was collected at-day 30, 60, go and serum samples were taken after 6 months. Many low-molecular weight metabolites were identified by H-1 NMR spectra of rat urine. A decrease in citrate and an increase in ketone bodies, creatinine, DMA, DMG, TMAO, and taurine in the urine of the rats. receiving high doses were found by H-1 NMR spectra. These may mean that high-dosage of ChangLe impairs the specific region of liver and kidney, such as renal tubule and mitochondria. The decrease in citrate and the increase in succinate and alpha-ketoglutarate were attributed to a combination of the inhibition of certain citric acid enzymes, renal tubular acidosis and the abnormal fatty acid catabolism. The information of the renal capillary necrosis could be derived from the increase in DMIA, DMG and TMAO. The increase in taurine was due to hepatic mitochondria dysfunction. The conclusions were supported by the results of biochemical measure. merits and enzymatic assay.

Electrochemical probe of hydroxylation of 4-nitrophenol by cytochrome c with hydrogen peroxide

The reaction of hydrogen peroxide with cytochrome c makes them coupled to lead to the hydroxylation of 4-nitrophenol. In situ electrochemical probe was used to detect the hydroxylation of 4-nitrophenol, which can avoid the tedious extraction procedure, the loss of the active species and the interference of some colored substances in the detection of 4-nitrocatechol by spectroscopic method. The hydroxyl radical scavengers mannitol and sodium benzoate did not eliminate hydroxylation, but the inhibitory effect of uric acid on the hydroxylation lead to the formation of the ferryl species of the protein during the reaction. These studies suggest that the electrochemical probe might efficiently detect the trace 4-nitrocatechol from the onset of the hydroxylation reaction and thus provides a more sensitive tool.

Biochemical characterization of selenium-containing catalytic antibody as a cytosolic glutathione peroxidase mimic

A selenium-containing catalytic antibody (Se-4A4), prepared by converting reactive serine residues of a monoclonal antibody (4A4) raised against a GSH derivative into selenocysteines, acts as a mimic of cytosolic glutathione peroxidase (cGPX). To clarify the mechanism of action of this catalytic antibody, detailed studies on kinetic behaviour and biological activity were carried out. A rate of acceleration (k(cat)/K-m/k(uncat)) 10(7)-fold that of the uncatalytic reaction is observed. Under similar conditions, the turnover number (k(cat)) of Se-4A4 is 42% of that of the natural rabbit liver cGPX. The Se-4A4 reaction involves a Ping Pong mechanism, which is the same as that of the natural cGPX. The selenocysteine residue is located in the binding site of the antibody and is shown to be crucial for this activity. Of the thiol compounds tested, only GSH is able to serve as substrate for Se-4A4. It was demonstrated, using the free-radical-damage system (hypoxanthine/xanthine oxidase) of cardiac mitochondria, that Se-4A4 can protect mitochondria from free-radical damage at least 10(4)-fold more effectively than the natural cGPX.

Assembly of the subcellular parts of Bryopsis hypnoides Lamouroux into new protoplasts

The chloroplasts, mitochondria, and protoplasm devoid of mature chloroplasts (PMC) of Bryopsis hypnoides Lamouroux were isolated by low-speed and sucrose density centrifugation. The PMC aggregated in artificial seawater, and then protoplasts without mature chloroplasts (PtMCs) were formed. Transmission electron microscopy and cytochemical studies indicated that there were mitochondria, nuclei, vesicles, and other small cell organelles in the PtMCs. Scanning electron microscopy showed that there were holes on the surface of 1-h PtMCs and then fewer holes on the surface of 24-h PtMCs, suggesting that a healing process occurred. The plasma membrane was formed over the surface of the PtMCs. However, the cell wall was not regenerated, and the newly formed PtMCs were ruptured and died in 3 days. Light intensity during alga maintenance before use influenced significantly (one-way ANOVA, P < 0.0001) on the number of PtMCs formed; the highest number of PtMCs was formed at 20A mu mol/(m(2) s). When isolated chloroplasts were transferred into seawater, there were only two or three chloroplasts aggregated together. However, isolated mitochondria and the mixed six layers of cell organelles (separated by sucrose density centrifugation) could not aggregate in the artificial seawater. This indicates that the conjunction of cell organelles is important for their aggregation.

Inhibition of mouse liver lipid peroxidation by high molecular weight phlorotannins from Sargassum kjellmanianum

The inhibitory effects of high molecular weight phlorotannins (HMP) from Sargassum kjellmanianum on mouse liver lipid peroxidation were investigated by spectrophotometric methods. The content of malondialdehyde (MDA) in liver samples was measured by TBA (thiobarbituric acid) assay. It showed that HMP significantly inhibited the generation of MDA in vivo and in situations induced by CCl4 and Fe2+-Vc ( ascorbic acid), and significantly decreased membrane swelling of mouse liver mitochondria, compared with controls ( p < 0.01). HMP were found to have strong anti-oxidative activity in inhibiting mouse liver lipid peroxidation.