Two cathelicidin genes are present in both rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

Further to the previous finding of the rainbow trout rtCATH_1 gene, this paper describes three more cathelicidin genes found in salmonids: two in Atlantic salmon, named asCATH_1 and asCATH_2, and one in rainbow trout, named rtCATH_2. All the three new salmonid cathelicidin genes share the common characteristics of mammalian cathelicidin genes, such as consisting of four exons and possessing a highly conserved preproregion and four invariant cysteines clustered in the C-terminal region of the cathelin-like domain. The asCATH_1 gene is homologous to the rainbow trout rtCATH_1 gene, in that it possesses three repeat motifs of TGGGGGTGGC in exon IV and two cysteine residues in the predicted mature peptide, while the asCATH_2 gene and rtCATH_2 gene are homologues of each other, with 96% nucleotide identity. Salmonid cathelicidins possess the same elastase-sensitive residue, threonine, as hagfish cathelicidins and the rabbit CAP18 molecule. The cleavage site of the four salmonid cathelicidins is within a conserved amino acid motif of QKIRTRR, which is at the beginning of the sequence encoded by exon W. Two 36-residue peptides corresponding to the core part of rtCATH_1 and rtCATH_2 were chemically synthesized and shown to exhibit potent antimicrobial activity. rtCATH_2 was expressed constitutively in gill, head kidney, intestine, skin and spleen, while the expression of rtCATH_1 was inducible in gill, head kidney, and spleen after bacterial challenge. Four cathelicidin genes have now been characterized in salmonids and two were identified in hagfish, confirming that cathelicidin genes evolved early and are likely present in all vertebrates.

Inhibition of progesterone receptor activity in recombinant yeast by soot from fossil fuel combustion emissions and air particulate materials

Numerous environmental pollutants have been detected for estrogenic activity by interacting with the estrogen receptor, but little information is available about their interactions with the progesterone receptor. In this study, emission samples generated by fossil fuel combustion (FFC) and air particulate material (APM) collected from an urban location near a traffic line in a big city of China were evaluated to interact with the human progesterone receptor (hPR) signaling pathway by examining their ability to interact with the activity of hPR expressed in yeast. The results showed that the soot of a petroleum-fired vehicle possessed the most potent anti-progesteronic activity, that of coal-fired stove and diesel fired agrimotor emissions took the second place, and soot samples of coal-fired heating work and electric power station had lesser progesterone inhibition activity. The anti-progesteronic activity of APM was between that of soot from petroleum-fired vehicle and soot from coal-fired establishments and diesel fired agrimotor. Since there was no other large pollution source near the APM sampling sites, the endocrine disrupters were most likely from vehicle emissions, tire attrition and house heating sources. The correlation analysis showed that a strong relationship existed between estrogenic activity and anti-progesteronic activity in emissions of fossil fuel combustion. The discoveries that some environmental pollutants with estrogenic activity can also inhibit OR activity indicate that further studies are required to investigate potential mechanisms for the reported estrogenic activities of these pollutants. (c) 2005 Elsevier B.V. All rights reserved.

Identification of a novel cathelicidin gene in the rainbow trout, Oncorhynchus mykiss

We report the cloning of a novel antimicrobial peptide gene, termed rtCATH_1, found in the rainbow trout, Oncorhynchus mykiss. The predicted 216-residue rtCATH_1 prepropeptide consists of three domains: a 22-residue signal peptide, a 128-residue cathelin-like region containing two identifiable cathelicidin family signatures, and a predicted 66-residue C-terminal cationic antimicrobial peptide. This predicted mature peptide was unique in possessing features of different known (mammalian) cathelicidin subgroups, such as the cysteine-bridged family and the specific amino-acid-rich family. The rtCATH_1 gene comprises four exons, as seen in all known mammalian cathelicidin genes, and several transcription factor binding sites known to be of relevance to host defenses were identified in the 5' flanking region. By Northern blot analysis, the expression of rtCATH_1 was detected in gill, head kidney, and spleen of bacterially challenged fish. Primary cultures of head kidney leukocytes from rainbow trout stimulated with lipopolysaccharide or poly(I (.) C) also expressed riCATH_1. A 36-residue peptide corresponding to the core part of the fish cathelicidin was chemically synthesized and shown to exhibit potent antimicrobial activity and a low hemolytic effect. Thus, rtCATH_1 represents a novel antimicrobial peptide gene belonging to the cathelicidin family and may play an important role in the innate immunity of rainbow trout.

Molecular cloning and characterisation of a fish PKR-like gene from cultured CAB cells induced by UV-inactivated virus

The double-stranded-RNA-dependent protein kinase (PKR) is an important component in an antiviral defence pathway that is mediated by interferon (IFN) in vertebrates. Previously, some important IFN system genes had been identified from an IFN-producing CAB (crucian carp Carassius auratus blastulae embryonic) cells after treatment with UV-inactivated GCHV (grass carp haemorrhage virus). Here, a fish PKR-like gene, named CaPKR-like, is cloned and sequenced from the same virally infected CAB cells. It has 2192 base pairs in length with a largest open reading frame (ORF) encoding a protein of 513 amino acid residues. BLAST search reveals that the putative CaPKR-like protein is most homologous to human PKR and also has a high-level homology with all members of a family of eIF2alpha kinases. Structurally, CaPKR-like possesses a conserved C-terminal catalytic domain of eIF2alpha kinase family and the most similarity to mammalian PKRs. Within its N-terminus, there are no dsRNA-binding domains conserved in mammalian PKRs instead of two putative Z-DNA binding domains (Zalpha). Like mammalian PKRs, CaPKR-like had a very low level of constitutive expression in normal CAB cells but was up-regulated in response to active GCHV, UV-inactivated GCHV and CAB IFN, implying that the transcriptional activation of CaPKR-like by viral infection is mediated possibly by newly produced CAB IFN, which was further supported by using cycloheximide, a potent inhibitor of protein synthesis. The results together suggested that CaPKR-like was the first identified fish gene most similar to mammalian PKRs. (C) 2004 Elsevier Ltd. All rights reserved.

Identification and expression analysis of two IFN-inducible genes in crucian carp (Carassius auratus L.)

Interferon (IFN) exerts its antiviral effects mainly through activation of a subset of IFN-stimulated genes (ISG), but relatively few of fish ISGs have been isolated and characterized so far. Here, we report two fish ISGs, termed CaIF158 and CaIF156, cloned from a subtractive cDNA library constructed with mRNAs obtained from crucian carp (Carassius auratus L.) blastulae embryonic (CAB) cells infected by UV-inactivated GCHV and mock-infected cells. Database search revealed that both ISGs had a high-level homology with all members of a well conserved gene family with multiple tetratricopeptide repeat (TPR) motifs, including human IF160, IF158, IF156, IFI54 and their homologues in some other mammalian species. The transcripts of CaIF158 and CaIF156 were undetectable in CAB cells but could be induced by active GCHV, UV-inactivated GCHV or CAB IFN. Analysis of expression difference between them and IFN signal factors, CaSTAT1 and CaIRF7, indicated that their transcriptions were mediated possibly through JAK-STAT signal pathway, which was further supported by the induction analysis in UV-inactivated GCHV infected, IFN-treated and untreated cells in the presence or absence of cycloheximide (CHX), a potent inhibitor of protein synthesis. In addition, a pufferfish (Fugu rubrides) DNA sequence representing putative FrIFI56 was also revealed when CalF158 and CalF156 were used to search the pufferfish genome database. Phylogenetic analysis showed that these fish ISGs form a unique clad independent of mammalian homologues, reflecting a distant evolutionary relationship from mammals. These studies identified the first teleost IFI56 and IFI58 orthologues. (C) 2003 Elsevier B.V All rights reserved.

Estrogenic effects from household stoves

With the application of a genetically modified yeast, estrogen receptor-activating compounds were detected in the soot and emission gas of a wood-burning household stove. The EC50 value of 17beta-estradiol was divided by the EC50 value of soot, and the obtained relative estrogenic value for raw soot was 2.37E-5, indicating that soot was about 100,000 times less estrogenic than 17beta-estradiol. Chemical analysis revealed that alkyl phenol, benzonic acid, and PAHs represented the major constituents in the most potent fractions of the soot. Along with PAHs, other constituents might also contribute to the estrogenicity of soot. (C) 2002 Elsevier Science (USA).

The toxic effects of microcystin-LR on embryo-larval and juvenile development of loach, Misguruns mizolepis Gunthe

Microcystin-LR, a specific and potent hepatotoxin, was tested for its effects oil loach embryo-larval and juvenile development, The results of this study showed that loach embryos were more sensitive when exposed to microcystin-LR at a later than at an earlier stage of development, Juveniles were far less sensitive to MC-LR than were embryos and larvae. Mortality and developmental abnormality were proven to be dose-dependent and to be stage-specific sensitive. Among the abnormal changes noted were: pericardial edema and tubular heart, bradycardia, homeostasis, poor yolk resumption. small head, curved body and tail, and abnormal hatching, Liver and heart were the main targets of microcystin-LR toxicity. Ultrastructural analysis documented a complex set of sublethal effects of microcystin-LR on loach hepatocytes, chiefly including morphological alteration in nuclear and RER of loach liver cells. fit addition, microcystin-LR was lethal to loach juvenile in the subacute (7 days) exposure (LC50) = 593.3 mug/l). (C) 2002 Elsevier Science Ltd. All rights reserved.

Comparative study on in vitro inhibition of grass carp (Ctenopharyngodon idellus) and mouse protein phosphatases by microcystins

Microcystins isolated from toxic cyanobacteria are potent inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A). The inhibitory effects of three structural variants of microcystins (microcystin-LR, -YR, and -RR) on protein phosphatases isolated and purified from the liver and kidney of grass carp (Ctenopharyngodon idellus) were investigated using the P-32 radiometric assay. The relationships between percentage inhibition of protein phosphatase activity and microcystin levels followed a typical dose-dependent sigmoid curve. These results were compared to those obtained from mouse PP2A. The degree and pattern of inhibition of both fish and mouse protein phosphatases by microcystins were similar. Protein phosphatases in crude fish tissue homogenates showed similar inhibition patterns as purified fish PP2A toward microcystins. (C) 2000 by John Wiley & Sons, Inc.

Effects and numerical analysis of argon gas flow on the oxygen concentration in Czochralski silicon single crystal growth

Argon gas, as a protective environment and carrier of latent heat, has an important effect on the temperature distribution in crystals and melts. Numeric simulation is a potent tool for solving engineering problems. In this paper, the relationship between argon gas flow and oxygen concentration in silicon crystals was studied systematically. A flowing stream of argon gas is described by numeric simulation for the first time. Therefore, the results of experiments can be explained, and the optimum argon flow with the lowest oxygen concentration can be achieved. (C) 2002 Elsevier Science B.V. All rights reserved.

A switch-on mechanism to activate maize ribosome-inactivating protein for targeting HIV-infected cells

Maize ribosome-inactivating protein (RIP) is a plant toxin that inactivates eukaryotic ribosomes by depurinating a specific adenine residue at the a-sarcin/ricin loop of 28S rRNA. Maize RIP is first produced as a proenzyme with a 25-amino acid internal inactivation region on the protein surface. During germination, proteolytic removal of this internal inactivation region generates the active heterodimeric maize RIP with full N-glycosidase activity. This naturally occurring switch-on mechanism provides an opportunity for targeting the cytotoxin to pathogen-infected cells. Here, we report the addition of HIV-1 protease recognition sequences to the internal inactivation region and the activation of the maize RIP variants by HIV-1 protease in vitro and in HIV-infected cells. Among the variants generated, two were cleaved efficiently by HIV-1 protease. The HIV-1 protease-activated variants showed enhanced N-glycosidase activity in vivo as compared to their un-activated counterparts. They also possessed potent inhibitory effect on p24 antigen production in human T cells infected by two HIV-1 strains. This switch-on strategy for activating the enzymatic activity of maize RIP in target cells provides a platform for combating pathogens with a specific protease.

A colorimetric assay for screening microcystin class compounds in aquatic systems

Secondary metabolites produced by water-blooming cyanobacteria in eutrophic waters include some potent hepatotoxins, These compounds also have tumour-promoting properties, attributable to their inhibition and activation of protein phosphatases and kinases respectively. The inhibitory effect of these toxins on protein phosphatases have been employed in a commonly used radiometric assay, involving the use of a P-32-labeled substrate, for the detection and quantitation of these compounds. This paper investigates and describes a colorimetric method in which the activity of protein phosphatase 2A is determined by measuring the rate of colour production from the release of yellow p-nitrophenol using p-nitrophenyl phosphate as the substrate. Results of this study suggest that the colorimetric protein phosphatase inhibition assay is a simple, inexpensive tool for screening substances that may have tumour-promoting characteristics in aquatic systems. The detection limit of the colorimetric method is comparable to the radiometric assay. (C) 1998 Elsevier Science Ltd. All rights reserved.

Effects and numerical analysis of argon gas flow on the oxygen concentration in Czochralski silicon single crystal growth

Argon gas, as a protective environment and carrier of latent heat, has an important effect on the temperature distribution in crystals and melts. Numeric simulation is a potent tool for solving engineering problems. In this paper, the relationship between argon gas flow and oxygen concentration in silicon crystals was studied systematically. A flowing stream of argon gas is described by numeric simulation for the first time. Therefore, the results of experiments can be explained, and the optimum argon flow with the lowest oxygen concentration can be achieved. (C) 2002 Elsevier Science B.V. All rights reserved.

乙草胺对玉米根际微生物和作物抗氧化酶活性的影响

乙草胺作为我国应用最多的三大除草剂之一，被广泛地应用于玉米、花生、大豆等农田中。作为外源有机污染物，乙草胺在施入土壤后，对作物构成一定的逆境胁迫。作物在逆境胁迫下，自身会发生相应的改变抵御迫害，其中根际、活性氧清除系统、除草剂的代谢酶等在作物抵御乙草胺等除草剂的迫害中起着十分重要的作用。 本文以我国重要的粮食作物-玉米作为供试作物，在田间实验条件下研究了低（田间用量）、中（田间用量的2倍）、高（田间用量的5倍）三个浓度乙草胺处理下，乙草胺在玉米根际和非根际环境中的残留特征，并探讨了玉米根际微生物和玉米超氧化物歧化酶、过氧化物酶、还原性谷胱甘肽、谷胱甘肽-S-转移酶对乙草胺施用的响应特征。 研究表明：（1）乙草胺在土壤中的降解迅速，并且在根际的降解得到强化，随着乙草胺的施用浓度增加，其在土壤中的最终残留量增大。（2）由于根际效应的存在，根际微生物量、真菌和细菌数量都显著高于非根际土壤，且根际和非根际土壤、高浓度处理和低、中浓度处理土壤间微生物群落结构明显不同。乙草胺各处理对根际微生物量有先抑制后刺激的作用，对根际细菌尤为显著，抑制作用随乙草胺浓度的增加而加强。乙草胺对本体土壤的微生物量影响较复杂，高浓度对本体土壤微生物量主要表现为抑制作用，而低、中浓度则是抑制和刺激作用交替出现，低浓度乙草胺对非根际土壤微生物的抑制作用未达到显著水平。乙草胺对对非根际真菌的抑制程度大于根际真菌。虽然各处理的微生物量都能恢复到正常水平，但非根际的恢复比根际需要更长的时间。土壤中细菌和真菌对乙草胺响应存在非同步性，并且群落变化具有一定的补偿作用。（3）除草剂乙草胺对玉米过氧化物酶（POD）的活性影响较小，POD不能作为乙草胺污染的生物标记物；超氧化物歧化酶（SOD）对乙草胺的污染有一定的指示作用，但其对乙草胺的敏感性不如谷胱甘肽-S-转移酶（GST）和还原性谷胱甘肽（GSH）高。低、中浓度乙草胺处理使玉米GST活性和GSH含量先升高后降低，高浓度抑制GST活性，降低GSH的含量。GST和GSH对乙草胺的响应非常敏感，可以作为评价除草剂对作物安全性评价的重要指标。 5倍田间用量乙草胺处理虽然对玉米产生了一定的影响，但玉米经一个月左右能恢复正常生长，说明乙草胺对玉米的安全性较高。但由于乙草胺在土壤中的最终残留量与初始浓度密切相关，且对土壤（根际和非根际）微生物群落会产生一定影响，所以应严格控制乙草胺的用量，以降低对土壤环境、水体环境和人类生活健康的风险性。

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.