Histological and cytological studies on the developing thymus of mandarin fish Siniperca chuatsi (Perciformes : Teleostei)

The thymus of the mandarin fish, Siniperca chuatsi, was examined by light and transmission electron microscopy to understand its formation and cellular composition. Larvae of the mandarin fish were collected and sectioned from 1 to 35 days post-hatching (dph). On dph 7 the thymus was packed with lymphocytes. From 12 dph onward, mucous cells were observed on the epithelial layer; from 23 dph, three zones could be differentiated in the thymic parenchyma. The thymus was connected with the extension of the third, fourth and fifth branchial pouches throughout early development, remaining in a superficial position in the adult S. chuatsi. In the thymus of the adult fish, thymic epithelial cells (TECs) characteristic of tonofilaments were observed, with limiting TECs (LECs) found in subcapsular, subseptal, perivascular and nurse-like TECs containing viable intact lymphocytes inside their vacuoles. In addition, three kinds of granulocytes were observed throughout the thymus, and an incomplete blood-thymus barrier was found in the inner zone. Other cell components such as cystic cells, macrophages and plasma cells, were also described in the thymus of the adult S. chuatsi. The thymus development in mandarin fish agrees, to some extent, with the ontogenetic patterns observed in other fish species.

Transgene for growth hormone in common carp (Cyprinus carpio L.) promotes thymus development

The transgenic carp were produced by micro-injection of CAgcGHc into the fertilized eggs. Observation of the thymus development between the transgenics and non-transgenic controls was carried out. The thymus of one-year-old transgenics F1 showed a great increase in both size and weight. The unilateral thymus of the transgenics weighed from 190 to 295 mg with average 218.6 mg, whereas the unilateral thymus of the controls weighed 20-81 mg with average 42.5 mg; i.e. the thymus weight in the transgenics was 5.14 fold over that in the controls. The index of thymus/body weight in the transgenics was 2.97 fold over the controls. Light microscopy observation indicated that the thymus of the transgenics; well developed with the thickened outer region and compactly arranged thymocytes, while the thymus in the controls were degenerating with the thinned outer region, scattered thymocytes and groups of fatty cells. Further analysis with the electron microscopy revealed that pro-liferous cells in the transgenics; were mainly small lymphocytes and no pathological changes were found. The results confirmed that the "All-fish" GH-transgene promotes thymus development and thymocyte proliferation, and retards thymus degeneration. The study has laid a foundation for further analysis of the immunobiological function in GH-transgenic carp.

Multiplex binding modes of toluidine blue with calf thymus DNA and conformational transition of DNA revealed by spectroscopic studies

It is noteworthy to understand the details of interactions between antitumor drugs and DNA because the binding modes and affinities affect their antitumor activities. Here, The interaction of toluidine blue (TB), a potential antitumor drug for photodynamic therapy of tumor, with calf thymus DNA (ctDNA) was explored by UV-vis, fluorescence, circular dichroism (CD) spectroscopy, UV-rnelting method and surface-enhance Raman spectroscopy (SERS). The experimental results suggest that TB could bind to ctDNA via both electrostatic interaction and partial intercalation.

A temperature-dependent interaction of neutral red with calf thymus DNA

Neutral red (NR) is used as a probe to study the temperature and concentration dependent interaction of a cationic dye with nucleic acid. A temperature-dependent interaction of NR with calf thymus DNA (CT DNA) has been studied by differential pulse voltammetry (DPV), UV-Visible absorption, circular dichroism (CD) and fluorescence spectroscopy. The experimental results of increasing peak current, changes in the UV-Visible absorption and fluorescence spectra of NR and decreasing the induced circular dichroism (ICD) intensity show that (i) the binding mode of NR molecules is changed from intercalating into DNA base pairs to aggregating along the DNA double helix and (ii) the orientation of NR chromophore in DNA double helix is also changed with the temperature.

Study on adsorption and oxidation of calf thymus DNA at glassy carbon electrode

The oxidation and adsorption of the temperature-denatured DNA at GC electrode are studied by differential pulse voltammetry and in situ FTIR spectroelectrochemistry. The temperature-denatured DNA is adsorbed and formed a DNA multilayer at electrode surface. The temperature-denatured DNA showing partly reversible process was first observed based on the reduction peaks appearing at negative scans and the reversible spectral change. The oxidation product of the temperature-denatured DNA can not diffuse away from the electrode surface easily due to the impediment of the DNA multilayer, so it can be partly reduced.

Voltammetric and spectroscopic studies on methyl green and cationic lipid bound to calf thymus DNA

DNA interaction with cationic lipids promises to be a versatile and effective synthetic transfection agent. This paper presents the study on binding of a simple artifical cationic lipid, cetyltrimethylammonium bromide (CTAB), to calf thymus DNA (CT DNA) prior to the condensation process, taking methyl green (MG) as a probe. The results show that the CTAB binds to DNA through electrostatic interaction forming a hydrophobic complex, thus changing the micro-environment of duplex of DNA, so the binding state of MG and DNA is changed, and a complex CTAB-CT DNA-MG is formed. This fact suggests a new way to mediate the conformation of molecular assemblies of DNA and lipids. (C) 2000 Elsevier Science B.V. All rights reserved.

In-situ FTIR study on adsorption and oxidation of native and thermally denatured calf thymus DNA at glassy carbon electrodes

In-situ Fourier transform infra-red (FTIR) spectra of native and thermally denatured calf thymus DNA (CT DNA) adsorbed and/or oxidized at a glassy carbon (GC) electrode surface are reported. The adsorption of native DNA occurs throughout the potential range (-0.2 similar to 1.3 V) studied, and the adsorbing state of DNA at electrode surface is changed from through the C=O band of bases and pyrimidine rings to through the C=O of cytosine and imidazole rings while the potential shifts negatively from 1.3 V to -0.2 V. An in-situ FTIR spectrum of native CT DNA adsorbed at GC electrode surface is similar to that of the dissolved DNA, indicating that the structure of CT DNA is not distorted while it is adsorbed at the GC electrode surface. In the potential range of -0.2 similar to 1.30 V, the temperature-denatured CT DNA is adsorbed at the electrode surface first, then undergoes electrochemical oxidation reaction and following that, diffuses away from the electrode surface. (C) 2001 Elsevier Science B.V. All rights reserved.

鄂尔多斯高原沙地草地的生态异质性

空间异质性的重要性在于其在自然生态系统中的普遍性和它所引起的生态学效应。空间异质性不仅在生态学理论中占有重要位置，而且也是生态系统管理和景观规划等实践中需要解决的基本问题。本文对鄂尔多斯高原(37038，～40052'N，106027'～111028'E)沙地草地的主要景观单元一沙地和风蚀沙化梁地植被与土壤的空间异质性格局与动态进行了定量的描述和实验研究。 在鄂尔多斯高原沙地景观中，通过对两条相互垂直的Skm样线上植被盖度、植物物种数的半方差分析和分形分析，揭示了植被的依赖于空间尺度的自相关特征和等级结构;沙地景观植被结构以小尺度空间变异占优势，植被斑块多集中在5-10 m的尺度上。对植被盖度低于20%的流沙斑点或斑块沿样线分布的空隙度分析表明流沙斑点或斑块呈适度聚集的格局。植被盖度和植物物种数与土壤有机碳和全氮含量的空间异质性之间的关系随空间尺度和生境类型的不同而有所不同。对植物群落内风蚀沙化斑块中的植物再生动态和克隆植物沙鞭的克隆生长观测实验表明，克隆植物的存在有利于风蚀沙化斑块的固定，跨越不同斑块边界的克隆植物的生长导致了斑块间的物质能量流动，从而影响了景观空间异质性的动态。 在风蚀沙化梁地中，梁坡处的土壤最为瘠薄，土壤颗粒组成以中细沙为主，这反映了风沙活动在梁坡处形成的沙粒堆积。相应地，梁坡处是以油蒿为优势种的沙生植物群落，与梁顶的典型草原植物（本氏针茅（Stipa bungeana)+百里香（Thymus serpyllum）+冷蒿（Artemisiafrigida））群落截然不同。从梁顶到梁底，植物群落受控于较大尺度上的与地形有关的环境梯度，因而其在较大尺度上的空间异质性占优势。

五助百里香植物愈伤组织培养及挥发油成份的生物合成研究

高等植物具有合成各种化合物的特殊功能。早在本世纪二十年代初期，许多有机化学家对这些复杂的天然次生代谢物提出了各种生源假说，阐述这些化合物的结构关系及其可能的生源途径。 目前，对于次生代谢产物生物合成研究主要有二个途径，整体方法和离体方法，其中离体方法包括非细胞系以及植物组织和细胞培养。 经过近一个世纪的努力，植物组织和细胞培养成了今天研究次生代谢产物生物合成的一个重要工具。目前这方面研究主要包括三个方面;﹤一﹥通过植物组织和细胞培养获得有价值的天然次生代谢产物;﹤二﹥研究次生代谢产物的生物合成途径;﹤三﹥生物合成新的有特殊价值的天然产物。 近二十年来，这一领域发展迅速，尤其是在寻找用植物细胞，像微生物那样，工业生产有价值的天然产物方面，为许多科学家所瞩目。 为了从植物组织和细胞培养中获得原植物特有的次生代谢产物，目前，主要包括四个方面的研究。﹤一﹥营养调节;﹤二﹥激素调节;﹤三﹥一级代谢对二级代谢的调节;﹤四﹥一些物理因子的调节。这些工作均在不同程度上取得一定的进展。 目前，在本领域中存在的主要问题是：﹤1﹥在植物组织培养中，许多植物特有的次生产物只能在在具有一定程度分化的细胞中才能产生;﹤2﹥在植物组织或细胞培养中，合成的次生产物含量较低;﹤3﹥许多植物组织或细胞培养不能合成原植物特有的次生代谢产物。 在我国，应用植物组织或细胞培养研究次生代谢产物合成的工作开展不多，尤其是有关精油的生物合成研究，目前才刚刚开始。 本实验应用两种化学型（芳樟醇型和香芹酚型）五肋百里香（Thymus guinguecostatus celak)植物的叶、茎、子房、花粉等诱导愈伤组织，以期探讨萜类及有关成份在愈伤组织中合成的规律;比较不同愈伤组织合成萜类及有关成份的差异;探讨不同化学型的遗传信息在愈伤组织中合成的作用。 在本实验中，我们的结果如下： 1.愈伤组织诱导 不同培养基对愈伤组织诱导不同，其中以添加6ppm 2,4-D和0.5ppm激动素的staba培养基的诱导率最高，接种17天后，诱导率达70%。老茎和老叶均不易产生愈伤组织。光照下培养也不易产生愈伤组织，诱导愈伤组织是在暗处进行的。 2.愈伤组织培养 愈伤组织在含有0.2ppm NAA和1ppm激动素的 staba 和 Ms 培养基中生长较好，五周后可长成约1克重。在光照下培养，茎的愈伤组织是绿色的，叶的愈伤组织为白色，无叶绿素，子房和花药愈伤组织含有少量叶绿素。光照下培养比暗处生长较快。在这两种培养基中添加200毫克/升亮氨酸时，茎的愈伤组织出现分化的不定芽，叶的愈伤组织不发生分化，但生长加快。 3.原植物精油成份 移栽于本所北京植物园的五肋百里香精油成份，经气-质联用鉴定,与原产地的精油成份相似。从山东烟台地区移栽的芳樟醇型，经气-质联用鉴定有13个化合物，除前已坚定的五个原产地精油成份，如芳樟醇，龙脑，莰烯，蒎烯和β-红波药烯外，本实验鉴定的成份。 从山东泰安地区移栽的为香芹酚型经气-质联用鉴定的精油成份有12个，除前已鉴定的香芹酚，百里香酚等9个成份外，本实验鉴定的成份有桧烯，α-侧柏烯和新异侧柏醇等。 4.愈伤组织中萜类和有关成份及其生源 芳樟醇型叶愈伤组织，经提取分离，气-质联合检出4个化合物：异戌酰胺，苯甲酸乙酯，橙花叔醇和香兰素。主要成份是异戌酰胺和苯甲酸乙酯。 在香芹酚型愈伤组织中，鉴定了一个化合物，异戌酰胺，它是该愈伤组织的主要成份。 在本实验中，没有检出单萜成份，但检出一个倍半萜，橙花叔醇。因此，我们认为单萜和倍半萜的生物合成部位可能是相互分离的。倍半萜可能是在细胞质中合成的。 在本实验中，我们认为主要成份异戌酰胺可能是由亮氨酸转化的;香兰素和苯甲酸乙酯可能是从肉桂酸途径衍生的。

Ontogeny of IgM-producing cells in the mandarin fish Siniperca chuatsi identified by in situ hybridisation

The ontogeny of IgM-producing cells was studied in juvenile mandarin fish Simperca chuatsi, an important fish in China's aquaculture sector. The IgM-producing cells were localised through in situ hybridisation with a probe complementary to the Ig mu-chain in lymphoid-related tissues, including head kidney, spleen, thymus, intestine and gills. In head kidney, transcripts of Ig mu were first detected at 20 days post-hatching (dph) with a few positive signals. and the number of IgM-producing cells increased obviously from 39 dph onwards. At 136 dph, a large amount of positive cells were observed in the entire organ with clusters of these cells located around the blood vessels. In spleen, IgM-producing cells were found from 26 dph onwards, followed by an increase until 67 dph: clusters of positive cells were also detected around blood vessels at 102 dph. In thymus, IgM-producing cells were first observed at 39 dph; thereafter, no obvious increase was detected until 78 dph. The positive cells in thymus were distributed mainly in the outer zone of thymus. A few IgM-producing cells were still observed in thymus of 1-year-old mandarin fish. IgM-producing cells were not detected in the intestine until 87 dph, with several discrete positively stained cells distributed in the lamina propria. IgM-producing cells, scattered mainly in primary gill filaments around blood vessels, were detected in gills from 90 dph. As in other teleosts, these results indicated that the head kidney appears to be the primary organ for IgM production in mandarin fish, and IgM-producing cells exist in all organs examined in the present study, implying their lymphoid role in fish. In addition, it is suggested that vaccination after 20 dph may be much more effective in mandarin fish. (C) 2009 Elsevier B.V. All rights reserved.

Expression regulation and functional characterization of a novel interferon inducible gene Gig2 and its promoter

Grass carp hemorrhagic virus (GCHV)-induced gene 2 (Gig2) is a novel gene previously identified from UV-inactivated GCHV-treated Carassius auratus blastulae embryonic (CAB) cells, suggesting that it should play a pivotal role in the interferon (IFN) antiviral response. In this study, a polyclonal anti-Gig2 antiserum was generated and used to study the inductive expression pattern by Western blot analysis, showing no basal expression in normal CAB cells but a significant up-regulation upon UV-inactivated GCHV, polyinosinic:polycytidylic acid (Poly I:Q and recombinant IFN (rIFN). However, constitutive expression of Gig2 is observed in all tested tissues from grass carp (Ctenopharyngodon idellus), and Poly I:C injection increases the relative amount of Gig2 protein in skin, spleen, trunk kidney, gill, hindgut and thymus. Moreover, the genomic sequence covering the whole Gig2 ORF and the upstream promoter region were amplified by genomic walking. Significantly, the Gig2 promoter contains three IFN-stimulated response elements (ISREs), nine GAAA/TfTC motifs and five gamma-IFN activating sites (GAS), which are the characteristics of genes responsive to both type I IFN and type 11 IFN. Subsequently, the complete Gig2 promoter sequence was cloned into pGL3-Basic vector, and its activity was measured by luciferase assays in the transfected CAB cells. The Gig2 promoter-driven construct is highly induced in CAB cells after treatment with Poly I:C or rIFN, and the functional capability is dependent on IFN regulatory factor 7 (IRF7), because its activity can be stimulated by IRF7. Collectively, the data provide strong evidence that Gig2 is indeed a novel IFN inducible gene and its expression is likely dependent on IRF7 upon Poly I:C or IFN. (C) 2009 Elsevier Ltd. All rights reserved.

Structure, organization and expression of common carp (Cyprinus carpio L.) NKEF-B gene

Natural killer (NK) cell enhancing factor (NKEF) belongs to the newly defined peroxiredoxin (Prx) family. Its functions are to enhance NK cell cytotoxicity and to protect DNA and proteins from oxidative damage. In this study, a partial cDNA sequence of carp NKEF-B was isolated from thymus cDNA library. Subsequently, the full-length cDNA of carp NKEF-B was obtained by means of 3' and 5' RACE, respectively. The full-length cDNA of carp NKEF-B was 1022 bp, consisting of a 73 bp 5'-terminal untranslated region (UTR), a 355 bp T-terminal UTR, and a 594 bp open reading frame coding for a protein of 197 amino acids. Carp NKEF-B contained two consensus Val-Cys-Pro (VCP) motifs and three consensus cysteine (Cys-51, Cys-70 and Cys-172) residues. Sequence comparison showed that the deduced amino acid sequence of carp NKEF-B had an overall similarity of 74-96% to that of other species homologues. Phylogenetic analysis revealed that carp NKEF-B forms a cluster with other known teleost NKEF-Bs. Then, by PCR we obtained a 5.1 -k long genomic DNA of carp NKEF-B containing six exons and five introns. Realtime RT-PCR results showed that carp NKEF-B gene was predominantly detected in kidney and head kidney under un-infected conditions. Whereas under SVCV-infection condition, the expression of NKEF-B gene was significantly increased in blood cells, gill, intestine and spleen, but maintained in liver, and decreased significantly in kidney and head kidney. Finally, the rNKEF-B was constructed and expressed in Escherichia coli. By using an antibody against carp rNKEF-B, immunohistochemical study further indicated that NKEF-B positive cells are mainly some RBCs and a few epithelial cells in gill and intestine, and that under SVCV-infection condition, these positive cells or positive products in their cytoplasm were mainly increased in gill and spleen sections of carp. The results obtained in the present study will help to understand the function of NKEF-B in teleost innate immunity. (C) 2008 Elsevier Ltd. All rights reserved.

Cloning, characterization and expression analysis of SIMP (source of immunodominant MHC-associated peptides) in grass carp Ctenopharyngodon idella

SIMP (source of immunodominant MHC-associated peptides) plays a key rote in N-linked glycosylation with the active site of oligosaccharyltransferase, being the source of MHC-peptides in the MHC I presentation pathway. In the present study, the SIMP gene has been cloned from grass carp Ctenopharyngodon idella by rapid amplification of cDNA ends (RACE). The full length of the cDNA sequence is 4384 bp, including a 1117 bp 5' UTR (untranslated region), a 2418 bp open reading frame, and a 849 bp 3' UTR. The deduced amino acids of the grass carp SIMP (gcSIMP) are a highly conserved protein with a STT3 domain and 11 transmembrane regions. The gcSIMP spans over more than 24,212 bp in length, containing 16 exons and 15 introns. Most encoding exons, except the first and the 15th, have the same length as those in human and mouse. The gcSIMP promoter contains many putative transcription factor binding sites, such as Oct-1, GCN4, YY1, Sp1, Palpha, TBP, GATA-1, C/EBP beta, and five C/EBP alpha binding sites. The mRNA expression of gcSIMP in different organs was examined by real-time PCR. The gcSIMP was distributed in all the organs examined, with the highest level in brain, followed by the level in the heart, liver, gill, trunk kidney, muscle, head kidney, thymus, and the lowest level in spleen. Furthermore, the recombinant gcSIMP has been constructed successfully and expressed in Escherichia coli by using pQE-40 vector, and the polyclonal antibody for rabbit has been successfully obtained, which was verified to be specific. Identification of gcSIMP will help to explore the function in fish innate immunity. (c) 2007 Elsevier Ltd. All rights reserved.

Structure, organization and expression of common carp (Cyprinus carpio L.) SLP-76 gene

SLP-76 is an important member of the SLP-76 family of adapters, and it plays a key role in TCR signaling and T cell function. Partial cDNA sequence of SLP-76 of common carp (Cyprinus carpio L.) was isolated from thymus cDNA Library by the method of suppression subtractive hybridization (SSH). Subsequently, the full length cDNA of carp SLP-76 was obtained by means of 3' RACE and 5' RACE, respectively. The full Length cDNA of carp SLP-76 was 2007 bp, consisting of a T-terminal untranslated region (UTR) of 285 bp, a T-terminal. UTR of 240 bp, and an open reading frame of 1482 bp. Sequence comparison showed that the deduced amino acid sequence of carp SLP-76 had an overall similarity of 34-73% to that of other species homotogues, and it was composed of an NH2-terminal domain, a central proline-rich domain, and a C-terminal SH2 domain. Amino acid sequence analysis indicated the existence of a Gads binding site R-X-X-K, a 10-aa-long sequence which binds to the SH3 domain of LCK in vitro, and three conserved tyrosine-containing sequence in the NH2-terminal domain. Then we used PCR to obtain a genomic DNA which covers the entire coding region of carp SLP-76. In the 9.2 k-long genomic sequence, twenty one exons and twenty introns were identified. RT-PCR results showed that carp SLP-76 was expressed predominantly in hematopoietic tissues, and was upregulated in thymus tissue of four-month carp compared to one-year old carp. RT-PCR and virtual northern hybridization results showed that carp SLP-76 was also upregulated in thymus tissue of GH transgenic carp at the age of four-months. These results suggest that the expression level of SLP-76 gene may be related to thymocyte development in teleosts. (c) 2007 Published by Elsevier Ltd.

Molecular cloning and characterization of common carp (Cyprinus carpio L.) TCR gamma and CD3 gamma/delta chains

Partial cDNA sequences of TCR gamma and CD3 gamma/delta were isolated from the thymus of common carp (Cyprinus carpio L.) by the method of suppression subtractive hybridization (SSH). Subsequently the full length cDNAs of carp TCR gamma and CD3 gamma/delta were obtained by means of 3' RACE and 5' RACE, respectively. The full length of carp TCR gamma chain is 1368 bp and encodes 326 amino acids including a signal peptide region of 19 amino acids and a transmembrane region of 23 amino acids at the C-terminal region from aa 291 to 313. The V region of carp TCR gamma contains 109 amino acids, the core motif FGXG in J segment was also found in carp TCR gamma. The C region of carp TCR gamma contains the characteristic CX6PX6WX45C motif. The CP region of carp TCR C gamma contains 37 amino acids. The full length of carp CD3 gamma/delta is 790 bp and encodes 175 amino acids including a signal peptide region of 17 amino acids and a transmembrane region of 23 amino acids from aa 93 to 115. Similar to other known CD3 gamma/delta s, four cysteine residues in the extracellular domain and an immunoreceptor tyrosine-based activation motif ITAM (YxxL/Ix6-8YxxL/I) in the intracellular domain are also included in carp CD3 gamma/delta. Differing from other known CD3 gamma/delta s, carp CD3 gamma/delta tacks the CXXCXE motif in the extracellular domain. RTPCR analysis demonstrated that the expression of TCR gamma gene was mainly in the thymus and gill of 6-month carp, but in 18-month carp, TCR gamma gene was detected in all the examined tissues. The expression of CD3 gamma/delta gene was detected in all examined tissues of 6 and 18-month carp; among them, the highest expression level was in the thymus of 6-month carp. In situ hybridization showed that CD3 gamma/delta-expressing cells were widely distributed in the head kidney, spleen and kidney of carp, whereas in the thymus, they were densely distributed in the lymphoid outer zone and scattered in the epithelioid inner zone. (c) 2007 Published by Etsevier Ltd.