Cytochrome P450-catalysed reactive oxygen species production mediates the (-)schisandrin B-induced glutathione and heat shock responses in AML12 hepatocytes

Sch B (schisandrin B), the most abundant dibenzocyclooctadiene lignan in Fructus schisandrae, can induce glutathione antioxidant and heat shock responses, as well as protect against oxidant-induced injury in various tissues, including the liver in rodents and AML12 (alpha mouse liver 12) hepatocytes. (-)Sch B is the most potent stereoisomer of Sch B in its cytoprotective action on AML12 hepatocytes. To define the role of ROS (reactive oxygen species) arising from CYP (cytochrome P450)-catalysed metabolism of (-)Sch B in triggering glutathione antioxidant and heat shock responses, the effects of a CYP inhibitor [ABT (aminobenzotriazole)] and antioxidants [DMTU (dimethylthiouracil) and TRX (trolox)] on (-)Sch B-induced ROS production and associated increases in cellular GSH level, as well as Hsp25/70 (heat-shock protein 25/70) production, were investigated in AML12 hepatocytes. The results indicated that (-)Sch B causes a dose dependent and sustained increase in ROS production over 6 h in AML12 hepatocytes, which was completely suppressed by pre-/co-treatment with ABT or DTMU/TRX. Incubation with (-)Sch B for 6 h caused optimal and dose-dependent increases in cellular GSH level and Hsp25/70 production at 16 h post-drug exposure in AML12 hepatocytes. These cellular responses were associated with protection against menadione-induced apoptosis. Pre-/co-treatment with ABT or antioxidants completely abrogated the (-)Sch B-induced glutathione antioxidant and heat shock responses, as well as protection against menadione-induced apoptosis. Experimental evidence obtained thus far supports the causal role of ROS arising from the CYP-catalysed metabolism of (-)Sch B in eliciting glutathione antioxidant and heat shock responses in AML12 hepatocytes.     

Differential effect of schisandrin B and dimethyl diphenyl bicarboxylate (DDB) on hepatic mitochondrial glutathione redox status in carbon tetrachloride intoxicated mice

The effects of schisandrin B (Sch B), a dibenzocyclooctadiene derivative isolated from the fruit of Schisandra chinensis, and dimethyl diphenyl bicarboxylate (DDB), a synthetic intermediate of schisandrin C (also a dibenzocyclooctadiene derivative), on hepatic mitochondrial glutathione redox status in control and carbon tetrachloride (CCl₄)-intoxicated mice were examined. Treating mice with Sch B or DDB at a daily oral dose of 1 mmol/kg for 3 d did not produce any significant alterations in plasma alanine aminotransferase (ALT) and sorbital dehydrogenase (SDH) activities. CCl₄ treatment caused drastic increases in both plasma ALT and SDH activities in mice. Pretreating mice with Sch B or DDB at the same dosage regimen significantly suppressed the CCl₄-induced increase in plasma ALT activity, with the inhibitory effect of Sch B being much more potent. Sch B, but not DDB, pretreatment could also decrease the plasma SDH activity in CCl₄-intoxicated mice. The lowering of plasma SDH activity, indicative of hepatoprotection against CCl₄ toxicity, by Sch B pretreatment was associated with an enhancement in hepatic mitochondrial glutathione redox status as well as an increase in mitochondrial glutathione reductase (mtGRD) activity in both non-CCl₄ and CCl₄-treated mice. DDB pretreatment, though enhancing both hepatic mitochondrial glutathione redox status and mtGRD activity in control animals, did not produce any beneficial effect in CCl₄-treated mice. The difference in hepatoprotective action against CCl₄ toxicity between Sch B and DDB may therefore be related to their ability to maintain hepatic mitochondrial glutathione redox status under oxidative stress condition.     

Cloning,characterization, hypoxia and heat shock response of hypoxia inducible factor-1 (HIF-1) from the small abalone Haliotis diversicolor

In this study, hypoxia inducible factor-1α (HIF-1α) and hypoxia inducible factor-1β (HIF-1β) from small abalone Haliotis diversicolor were cloned. The cDNA of H. diversicolor HIF-1α (HdHIF-1α) is 2833 bp encoding a protein of 711aa and H. diversicolor HIF-1β (HdHIF-1β) is 1919 bp encoding a protein of 590aa. Similar to other species' HIF-1, HdHIF-1 has one basic helix–loop–helix (bHLH) domain and two Per-Arnt-Sim (PAS) domains, and HdHIF-1α has a oxygen-dependent degradation domain (ODDD) with two proline hydroxylation motifs and a C-terminal transactivation domain (C-TAD) with an asparagine hydroxylation motif. Under normoxic conditions, HdHIF-1α and HdHIF-1β mRNAs were constitutively present in all examined tissues. Under hypoxia (2.0 mg/L DO at 25 °C) stress, HdHIF-1α expression was up-regulated in gills at 4 h, 24 h and 96 h, and in hemocytes at 24 h and 96 h, while HdHIF-1β remained relatively constant. Under thermal stress (31 °C), HdHIF-1α expression was significantly increased in gills at 4 h, and hemocytes at 0 h and 4 h, while HdHIF-1β expression still remained relatively constant. These results suggested that HIF-1α may play an important role in adaption to poor environment in H. diversicolor.     

The effect of neuronal expression of heat shock proteins 26 and 27 on lifespan, neurodegeneration, and apoptosis in Drosophila

Heat shock proteins (Hsps) are chaperones thought to increase lifespan, enhance stress resistance, and prevent apoptosis and neurodegenerative diseases. Our previous study reported that ubiquitous expression of hsp26 or hsp27 extended Drosophila lifespan. The effect of neuronal expression of hsp26 and hsp27 in Drosophila on the above-mentioned functions has not yet been investigated. Here, we show that neuronal expression of hsp26 or hsp27 improved lifespan and increased resistance to oxidative stress. However, only neuronal expression of hsp27 ameliorated Parkinsonism climbing disorder and attenuated mild polyglutamine-induced toxicity. Additionally, neuronal expression of hsp27 specifically partially rescued hid-induced lethality, but was not able to rescue reaper/grim-induced lethality. However, unlike hsp27, neuronal expression of hsp26 did not rescue hid-induced or reaper/grim-induced lethality. In summary, we demonstrate the functional similarities and differences of neuronal expression of hsp26 and hsp27 in adult Drosophila.     

Effects of artificially enhanced levels of ascorbate and glutathione on the enzymes monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase in spinach (Spinacia oleracea)

Effect of high intracellular concentrations of the antioxidants ascorbate and glutathione on the extractable activity of the reducting enzymes dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase were investigated with spinach cells ( Spinacia oleracea ). An elevated ascorbate concentration was obtained by treatment with the ascorbate biosynthesis precursor L-galactono-1,4-lactone (GAL). To increase the intracellular level of glutathione, cells were treated with the 5-oxo-L-proline analog L-2-oxothiazolidin-4-carboxylate (OTC), or with the peroxidative herbicide acifluorfen (sodium 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid). Extractable monodehydroascorbate reductase activity increased in the presence of a high level of ascorbate or glutathione, and enzyme activity was at maximum when cells were treated with acifluorfen + OTC, or acifluorfen + GAL. Extractable dehydroascorbate reductase activity decreased when the intracellular concentration of glutathione was high and non-enzymatic reduction of dehydroascorbate by glutathione was the dominant reaction. Maximal decrease of enzyme activity was found in cells treated with acifluorfen + OTC. Extractable activity of glutathione reductase (GR) increased after treatment of cells with acifluorfen alone, or acifluorfen + OTC, but enzyme activity was unaffected by a high intracellular concentration of glutathione obtained by treatment of cells with OTC alone, or by treatment with acifluorfen + GAL. The degree of GR activation seemed to be controlled by several factors including inhibition by a high concentration of glutathione and possibly oxidative damage to the enzyme. Overall, the enzymes tested in this study, which provide the reduced forms of ascorbate and glutathione, were differently affected by high antioxidant levels.     

Variation in heat shock protein expression at the latitudinal range limits of a widely‐distributed species,the Glanville fritillary butterfly (Melitaea cinxia)

Studies of heat shock response show a correlation with local climate, although this is more often across altitudinal than latitudinal gradients. In the present study, differences in constitutive but not inducible components of heat shock response are detected among populations of the Glanville fritillary butterfly Melitaea cinxia L. that exist at the species' latitudinal range limits (Finland and Spain). The study demonstrates that macroclimatic differences between these sites should cause greater exposure of the Spanish population to higher temperatures. Thermal stress treatments are used to estimate differences in the expression of four genes potentially relevant for tolerating these temperatures. For the analysis, three heat‐shock proteins and glyceraldehyde‐3‐phosphate dehydrogenase (G3PDH), a glycolysis enzyme that also modulates cell growth based on metabolic state, are chosen. Two constitutive differences are found between the sites. First, insects from Spain have higher levels of Hsp 21.4 than those from Finland regardless of thermal stress treatment; this protein is not inducible. Second, insects from Finland have higher levels of G3PDH. The two remaining Hsps, Hsp20.4 and Hsp90, show dramatic up‐regulation at higher temperatures, although there are no significant differences between insects from the different populations in either constitutive levels or inducibility. In nature, differences between the study populations likely occur in the expression of all four genes that were studied, although these differences would be directly climate‐induced in Hsp20.4 and Hsp90 and constitutive in Hsp21.4 and G3PDH. Inducibility may mitigate the need for constitutive variation in traits that adapt insects to local climate.     

水稻IR36及光温敏核雄性不育系培矮64S小孢子母细胞减数分裂期间微管骨架的变化

对水稻（Oryza sativa L.)IR36及光温敏核雄性不育系培矮64S小孢子母细胞减数分裂期间微管骨架的变化的研究显示,微管在小孢子母细胞正常发育的情况下,每一个发育阶段（即造孢细胞时期、细线期、偶线期、粗线期、双线期、终变期、中期Ⅰ、后期Ⅰ）都出现不同的组织结构形态和分布。在IR36中,还发现了一些新的和特殊的 管组织结构和分布。主要包括：（1）在细线期,从核膜向胞质射的微管呈螺旋状,显示核具旋转功能;（2）在偶线期,微管的分布呈极性分布现象;（3）在终变期,核被一组微管形成的宽带围绕着（perinuclear broad band）。而培矮64S小孢子母细胞分裂期间,各发育阶段的细胞内的;管管骨架结构呈现许多不正常现象。如：（1）在偶线期具极性分布的微管分布不存在;（2）在小孢子母细胞减数分裂期间的细胞内出现许多特别粗的微管束;（3）在终变期围绕核的微管宽带结构松散及内含微管密度低。由于微管骨架的分布变化可以使人们较早地在雄性不育细胞内看到败育现象,故此微管的分布和构型变化应可作为一个早期确立雄性不育现象的视觉标记(visual marker）或形态指标予以利用。     

Inhibitory effect of SJSZ glycoprotein (38?kDa) on expression of heat shock protein 27 and 70 in chromium (VI)-treated hepatocytes

Chromium (VI) is as an extremely toxic chemical substance, and is also an internationally recognized human carcinogen. The principal objective of this study was to determine whether or not Styrax japonica Siebold et al. Zuccarini (SJSZ) glycoprotein prevents hepatocarcinogenesis in chromium-treated BNL CL.2 cells and ICR mice. Firstly, it was evaluated that SJSZ glycoprotein has strong antioxidant character and scavenges radicals. In an effort to assess the chemopreventive effects of SJSZ glycoprotein on hepatocarcinogenesis, ICR mice were intraperitoneally injected with chromium (10?mg/kg, BW) for 8?weeks. After sacrifice, we evaluated indicators of liver tissue damage [the activities of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT), and thiobarbituric acid-reactive substances (TBARS)], antioxidative enzymes [activities of superoxide dismutase (SOD), catalase (CAT) and gluthathione peroxidase (GPx)], and initiating hepatocarcinogenic indicator [heat shock protein (HSP) 27 and 70] and protein kinase C (PKC), p38 MAPK and PCNA via biochemical methods and immunoblot analysis. The results obtained from this study demonstrated that the SJSZ glycoprotein (50?μg/ml) inhibited the production of intracellular ROS in BNL CL.2 cells. In addition, the SJSZ glycoprotein (10?mg/kg, BW) attenuated the levels of LDH, ALT, and TBARS, whereas it increased antioxidative enzymes in mouse serum. SJSZ glycoprotein attenuated the activity of HSP27, HSP70, PKC, MAPKs, and PCNA in BNL CL.2 cells and liver tissue. Taken together, our results indicate that SJSZ glycoprotein might be have a potent preventive effect against hepatocarcinogenesis induced by oxidative stress.     

Purification,regulation and cloning of a glutathione transferase (GST) from maize resembling the auxin-inducible type-III GSTs

The glutathione transferases (GSTs) from maize (Zea mays L.) with activities toward the chloroacetanilide herbicide metolachlor and the diphenyl ether herbicide fluorodifen were fractionated into two pools based on binding to affinity columns. Pool 1 GSTs were retained on Orange A agarose and were identified as isoenzymes Zea mays (Zm) GST I-I, Zm GST I-II and Zm GST I-III, which have been described previously. Pool 2 GSTs selectively bound to S-hexyl-glutathione-Sepharose and were distinct from the pool 1 GSTs, being composed of a homodimer of 28.5 kDa subunits, termed Zm GST V-V, and a heterodimer of the 28.5 kDa polypeptide and a 27.5 kDa subunit, termed Zm GST V-VI. Using an antibody raised to Zm GST V-VI, a cDNA expression library was screened and a Zm GST V clone identified showing sequence similarity to the type-III auxin-inducible GSTs previously identified in tobacco and other dicotyledenous species. Recombinant Zm GST V-V showed high GST activity towards the diphenyl ether herbicide fluorodifen, detoxified toxic alkenal derivatives and reduced organic hydroperoxides. Antibodies raised to Zm GST I-II and Zm GST V-VI were used to monitor the expression of GST subunits in maize seedlings. Over a 24 h period the Zm GST I subunit was unresponsive to chemical treatment, while expression of Zm GST II was enhanced by auxins, herbicides, the herbicide safener dichlormid and glutathione. The Zm GST V subunit was more selective in its induction, only accumulating significantly in response to dichlormid treatment. During development Zm GST I and Zm GST V were expressed more in roots than in shoots, with Zm GST II expression limited to the roots.     

Heat shock protein 70 promotes coxsackievirus B3 translation initiation and elongation via Akt‐mTORC1 pathway depending on activation of p70S6K and Cdc2

We previously demonstrated that coxsackievirus B3 (CVB3) infection upregulated heat shock protein 70 (Hsp70) and promoted CVB3 multiplication. Here, we report the underlying mechanism by which Hsp70 enhances viral RNA translation. By using an Hsp70‐overexpressing cell line infected with CVB3, we found that Hsp70 enhanced CVB3 VP1 translation at two stages. First, Hsp70 induced upregulation of VP1 translation at the initiation stage via upregulation of internal ribosome entry site trans‐acting factor lupus autoantigen protein and activation of eIF4E binding protein 1, a cap‐dependent translation suppressor. Second, we found that Hsp70 increased CVB3 VP1 translation by enhancing translation elongation. This was mediated by the Akt‐mammalian target of rapamycin complex 1 signal cascade, which led to the activation of eukaryotic elongation factor 2 via p70S6K‐ and cell division cycle protein 2 homolog (Cdc2)‐mediated phosphorylation and inactivation of eukaryotic elongation factor 2 kinase. We also determined the position of Cdc2 in this signal pathway, indicating that Cdc2 is regulated by mammalian target of rapamycin complex 1. This signal transduction pathway was validated using a number of specific pharmacological inhibitors, short interfering RNAs (siRNAs) and a dominant negative Akt plasmid. Because Hsp70 is a central component of the cellular network of molecular chaperones enhancing viral replication, these data may provide new strategies to limit this viral infection.     

Pharmacokinetic data of propranolol enantiomers in a comparative human study with (S)- and (R,S)-propranolol

The pharmacokinetics of (S)-propranolol were compared after the oral administration of a 40 mg dose of the pure enantiomer and an 80 mg dose of a racemic mixture of (R,S)-propranolol. The results of this study indicate that the bioavailability of (S)-propranolol, as expressed by the mean area under the concentration-time curve (AUC) and maximum serum concentration, is lower after 40 mg of the optically pure drug than after the racemic drug.     

柑橘全爪螨热激蛋白基因PcHsp90的克隆及表达模式分析

【目的】研究热激蛋白基因Hsp90在柑橘全爪螨Panonychus citri生长发育及响应高温和低温胁迫方面的作用。【方法】采用RT-PCR和RACE技术克隆柑橘全爪螨Hsp90基因cDNA全长序列;利用生物信息学软件分析该基因的序列特性;运用荧光Real-time PCR技术,分析Hsp90基因mRNA在该螨各发育阶段、高温及低温胁迫条件下的表达模式。【结果】克隆鉴定出柑橘全爪螨一条Hsp90基因的c DNA全长序列,命名为PcHsp90(Gen Bank登录号:GQ495086),全长为2 763 bp,包含2 193 bp的开放阅读框,编码730个氨基酸,编码蛋白质的理论分子量和等电点分别为83.85kDa和4.99,氨基酸序列包括Hsp90家族的5个特征基序及细胞质型Hsp90的特征序列"MEEVD"。系统进化分析表明,PcHsp90与朱砂叶螨Tetranychus cinnabarinus的Hsp90首先聚为一支,然后再与肩突硬蜱Ixodes scapularis的Hsp90聚合,说明它们较近的亲缘关系。PcHsp90在柑橘全爪螨的各发育阶段均有所表达,其中在幼螨期表达量较低,且显著低于若螨和成螨期的表达水平(P=0.015)。0~10℃低温胁迫下,Pc Hsp90的mRNA相对表达量无显著变化(P=0.492);但在35~41℃高温胁迫下,Pc Hsp90的mRNA相对表达量随胁迫温度的升高而上调,尤其是当温度升高到41℃时,mRNA相对表达量达到对照(25℃)的6.75倍,且差异达显著水平(P=0.007)。【结论】柑橘全爪螨PcHsp90不仅对该螨的生长发育具有重要作用,而且是其响应高温胁迫的重要机制之一。     

Effect of N,N-diallyl-2,2-dichloroacetamide (R-25788) on efflux and synthesis of glutathione in tobacco suspension cultures

The low level of glutathione synthesis observed in tobacco suspension cultures grown under heterotrophic conditions was stimulated by the addition of t     

Phylogeny of coral-inhabiting barnacles (Cirripedia; Thoracica; Pyrgomatidae) based on 12S, 16S and 18S rDNA analysis

The traditional phylogeny of the coral-inhabiting barnacles, the Pyrgomatidae, is based on morphological characteristics, mainly of the hard parts. It has been difficult to establish the phylogenetic relationships among Pyrgomatidae because of the apparent convergence of morphological characteristics, and due to the use of non-cladistic systematics, which emphasize ancestor-descendant relationships rather than sister-clade relationships. We used partial sequences of two mithochondrial genes, 12S rDNA and 16S rDNA, and a nuclear gene, 18S rDNA, to infer the molecular phylogeny of the pyrgomatids. Our phylogenetic results allowed us to reject previous classifications of Pyrgomatidae based on morphological characteristics. Our results also suggested the possibility of paraphyly of the Pyrgomatidae. The hydrocoral barnacle Wanella is not found on the same clade as the other pyrgomatids, but rather, with the free-living balanids. The basal position of Megatrema and Ceratoconcha is supported. The archeaobalanid Armatobalanus is grouped with Cantellius at the base of the Indo-Pacific pyrgomatines. Fusion of the shell plate and modification of the opercular valves are homoplasious features that occurred more than three times on different clades. The monophyly of the "Savignium" group, comprising four nominal genera, is also not supported, and the different taxa are placed on different clades.     

烟夜蛾谷胱甘肽S-转移酶基因的克隆、序列分析与表达

为探明谷胱甘肽S-转移酶（GSTs）在昆虫嗅觉识别中的作用, 本研究采用RT-PCR和RACE方法, 从烟夜蛾Helicoverpa assulta（Guenée）雄虫触角中克隆获得了1个GSTs基因的全长cDNA序列（GenBank登录号为EU289223）。将该基因推导的氨基酸序列与其他物种的GSTs进行同源性比对和系统发育分析, 发现该蛋白属于昆虫特异性Epsilon家族成员, 因此将该基因命名为HaGSTe1。同时从烟夜蛾基因组DNA中克隆获得了该基因序列, 发现序列中含有5个内含子, 长度分别为415,513,296,333和269 bp。利用半定量RT-PCR和实时荧光定量PCR方法对HaGSTe1在雌、 雄虫不同组织的表达进行了定性和定量分析, 结果显示, 该基因在雌、 雄虫的头部（去掉触角和喙）、触角、喙、胸、足、翅以及雌虫的腹部均有表达, 并且在雄虫触角中的表达量最高, 且显著高于雌虫触角, 这种表达情况提示其可能与触角中性信息素及其他外源物质的分解有关。     

Effect of hypoxia on in vivo thromboxane and prostacyclin levels in arterial blood of rainbow trout, Oncorhynchus mykiss (Walbaum)

Endogenous levels of TXB₂ and 6-keto PGF_1a are reported in dorsal aortic blood from rainbow trout. Acute hypoxia induced an increase in TXB₂ levels whereas 6-keto PGF_1a was unaffected. We suggest that enhanced thromboxane synthesis might have a role in microcirculation of various organs in fish hypoxia.