Molecular identification and expression analysis of natural resistance associated macrophage protein (Nramp) cDNA from Japanese flounder (Paralichthys olivaceus)

Natural resistance associated macrophage protein (Nramp) controls partially innate resistance to intracellular parasites. Its function is to enhance the ability of macrophages to kill pathogens. However, little is known about the structure and function of Nramp in lower vertebrates such as teleosts. We have recently isolated a cDNA encoding Nramp from Japanese flounder (Paratichthys olivaceus). The full-length cDNA of the Nramp is 3066 bp in length, including 224 bp 5' terminal UTR, 1662 bp encoding region and 1180 bp 3' terminal UTR. The 1662-nt open reading frame was found to code for a protein with 554 amino acid residues. Comparison of amino acid sequence indicated that Japanese flounder Nramp consists of 12 transmembrane (TM) domains. A consensus transport motif (CTM) containing 20 residues was observed between transmembrane domains 8 and 9. The deduced amino acid sequence of Japanese flounder had 77.30%, 82.71%, 82.67%, 79.64%, 80.72%, 90.97%, 91.16%, 60.14%, 71.48%, 61.69%, 72.37% identity with that of rainbow trout Nramp alpha and beta, channel catfish Nramp, fathead minnow Nramp, common carp Nramp, striped sea bass Nramp, red sea bream Nramp, mouse Nramp 1 and 2, human Nramp 1 and 2, respectively. RT-PCR indicated that Nramp transcripts were highly abundant in spleen, head kidney, abundant in intestine, liver and gill, and less abundant in heart. The level of Nramp mRNA in embryos gradually increases during embryogenesis from 4 h (8 cell stage) to 80 h (hatched stage) after fertilization. (c) 2005 Elsevier Ltd. All rights reserved.

Haematopoietic lineage cell-specific protein 1 (HS1) promotes actin-related protein (Arp) 2/3 complex-mediated actin polymerization

HS1 (haematopoietic lineage cell-specific gene protein 1), a prominent substrate of intracellular protein tyrosine kinases in haematopoietic cells, is implicated in the immune response to extracellular stimuli and in cell differentiation induced by cytokines. Although HS1 contains a 37-amino acid tandem repeat motif and a C-terminal Src homology 3 domain and is closely related to the cortical-actin-associated protein cortactin, it lacks the fourth repeat that has been shown to be essential for cortactin binding to filamentous actin (F-actin). In this study, we examined the possible role of HS1 in the regulation of the actin cytoskeleton. Immunofluorescent staining demonstrated that HS1 co-localizes in the cytoplasm of cells with actin-related protein (Arp) 2/3 complex, the primary component of the cellular machinery responsible for de novo actin assembly. Furthermore, recombinant HS1 binds directly to Arp2/3 complex with an equilibrium dissociation constant (K-d) of 880 nM. Although HS1 is a modest F-actin-binding protein with a Kd of 400 nM, it increases the rate of the actin assembly mediated by Arp2/3 complex, and promotes the formation of branched actin filaments induced by Arp2/3 complex and a constitutively activated peptide of N-WASP (neural Wiskott-Aldrich syndrome protein). Our data suggest that HS1, like cortactin, plays an important role in the modulation of actin assembly.

Molecular evolution of the keratin associated protein gene family in mammals, role in the evolution of mammalian hair

Background: Hair is unique to mammals. Keratin associated proteins (KRTAPs), which contain two major groups: high/ultrahigh cysteine and high glycine-tyrosine, are one of the major components of hair and play essential roles in the formation of rigid and

Characterization and expression analysis of TNFR-associated factor 1 (TRAF1) in grass carp Ctenopharyngodon idella

TNF receptor associated factor 1 (TRAF1) plays an important role in regulating the TNF signaling and protecting cells from apoptosis. In the present study, a TRAF1 gene has been cloned from grass carp (Ctenopharyngodon idella) by reverse transcription (RT)-PCR and rapid amplification of cDNA ends (RACE). The full-length cDNA is 2235 bp, including a 250 bp 5' UTR (untranslated region), a 1659 bp open reading frame, and a 326 bp 3'UTR. The polyadenylation signal (AATAAA, AATAA) and one mRNA instability motif (AUUUA) were found followed by a poly (A) tail in the 3'UTR. No signal peptide or transmembrane region has been found in the putative amino acids of grass carp TRAF1 (gcTRAF1). The putative amino acids of gcTRAF1 share 72% identity with the homologue in zebrafish. It is characterized by a zinc finger at the N-terminus and a TRAF domain (contains one TRAF-C and one TRAF-N) at the C-terminus. The identity of the TRAF domain among all the TRAF1 homologues in vertebrates varies from 52% to 58%, while the identities of TRAF-C were almost the same as 70%. The recombinant gcTRAF1 has been constructed successfully and expressed in Escherichia coli by using pET-32a expression vector. The polyclonal antibody for rabbit has been successfully obtained. The expression of gcTRAF1 in different organs was examined by real-time quantitative PCR and Western blotting, respectively. It was widely distributed in heart, head kidney, thymus, brain, gill, liver, spleen, and trunk kidney. This is the first report of TRAF1 homologue molecule found in fish. (c) 2007 Elsevier B.V. All rights reserved.

Seasonal regulations of energetics, serum concentrations of leptin, and uncoupling protein 1 content of brown adipose tissue in root voles (Microtus oeconomus) from the Qinghai-Tibetan plateau

Survival of small mammals in winter requires proper adjustments in physiology, behavior and morphology. The present study was designed to examine the changes in serum leptin concentration and the molecular basis of thermogenesis in seasonally acclimatized root voles (Microtus oeconomus) from the Qinghai-Tibetan plateau. In January root voles had lower body mass and body fat mass coupled with higher nonshivering thermogenesis (NST) capacity. Consistently, cytochrome c oxidase activity and mitochondrial uncoupling protein-1 (UCP1) protein contents in brown adipose tissues were higher in January as compared to that in July. Circulating level of serum leptin was significantly lower in winter and higher in July. Correlation analysis showed that serum leptin levels were positively related with body mass and body fat mass while negatively correlated with UCP1 protein contents. Together, these data provided further evidence for our previous findings that root voles from the Qinghai-Tibetan plateau mainly depend on higher NST coupled with lower body mass to enhance winter survival. Further, fat deposition was significantly mobilized in cold winter and leptin was potentially involved in the regulation of body mass and thermogenesis in root voles. Serum leptin might act as a starvation signal in winter and satiety signal in summer.

小麦SKP1同源基因TSK1的克隆和功能分析

SKP1 (S-phase kinase-associated-protein 1) 家族蛋白是普遍存在于真核生物中的一类小分子量蛋白质，其主要的生物学功能在于参与SCF复合体的形成，从而调控生物体内泛素介导的蛋白质降解，并参与多方面的生物发育过程。SKP1蛋白能够同时和Cullin蛋白以及F-box蛋白结合，形成SCF复合体的核心部分。因此，SKP1正常功能的维持对于SCF复合体功能的实现至关重要。研究显示，植物中尤其是以拟南芥为代表模式植物中已经发现了21个SKP1基因成员，并发现其中的ASK1参与了多个SCF复合体的形成并调控着包括植物雄性减数分裂、生长素、赤霉素、茉莉酸和乙烯等生理和发育进程。但是来自高等植物尤其是小麦和水稻中的SKP1基因还鲜有报道，其功能还不为所知;此外，SKP1基因与ABA的关系还没有任何报道。 　　本文利用筛选小麦减数分裂期小花的cDNA文库结合RT-PCR的方法从小麦中分离到了一个SKP1同源基因，并命名为TSK1 (Triticum aestivum SKP1-Like 1)。序列比较结果显示TSK1与多个植物来源的SKP1基因有较高的同源性，对其推测的编码蛋白序列的分析发现TSK1与包括拟南芥来源的ASK1/ASK2等蛋白的羧基端存在非常高的保守性。 　　在对TSK1表达模式的研究中，本文发现TSK1主要是集中在小麦花序和幼根中表达。利用多种激素对小麦幼苗处理之后，发现TSK1的表达受ABA的抑制，但是当小麦中ABA合成受抑的情况下，TSK1的表达会有所增加，说明TSK1的表达受ABA的调控。RNA原位杂交显示TSK1基因在花顶端分生组织、花药以及幼根等分生较旺盛的组织中有较强的表达，暗示该基因可能参与了与细胞分裂相关的过程。 　　为了研究TSK1可能具有的功能，本文首先在ask1-1突变体背景上超表达TSK1，发现能够部分恢复ask1-1突变体雄性不育的表型，说明TSK1和ASK1在植物减数分裂过程中存在某种保守性。 　　在野生型拟南芥中超表达TSK1造成了拟南芥多个方面的变化，包括萌发和开花推迟，气孔开度减小等。进一步的观察发现，转基因植株的萌发和营养生长都呈现出对ABA的超敏感，后续证据证实这种ABA的超敏感性并不是由于转基因拟南芥中ABA合成途径的改变所造成的，而极有可能是影响了ABA的信号传导过程。RT-PCR的结果显示，转基因植株中多个ABA相关的已知基因表达量的发生了变化。 　　为了提供植物中SKP1家族成员参与调节植物ABA信号传导途径证据，本文对拟南芥ASK1/ask1 ASK12/ask2的杂合双突变体自交后代进行了研究。结果显示，ask1/ask1纯合突变体和ask1/ask1 ASK2/ask2植株表现出对ABA的弱敏感性。该结果从另一个侧面印证了TSK1超表达植株对ABA超敏感表型。 　　此外，TSK1超表达拟南芥也表现出生长素相关表型，也印证了该基因可能与ASK1类似，参与到生长素介导的根发育过程。 　　综上所述，本文认为TSK1参与了植物激素介导的植物发育过程，而且极有可能是形成了目前未知的某种SCF复合体。最重要的是，本文的结果为SCF复合体参与调节植物ABA信号传导途径提供了生理及遗传层面的证据。 　　 　　

A novel fusion gene, TRIMS-Cyclophilin A in the pig-tailed macaque determines its susceptibility to HIV-1 infection

Objective: In Old World monkeys, the tripartite motif Sec (TRIM5 alpha) protein confers resistance to HIV-1 infection following virus entry into host cells. However, the pig-tailed macaque (Macaca nemestrina) is an exception and is susceptible to HIV-1 in

Two-Dimensional Kinetics Of Beta(2)-Integrin And Icam-1 Bindings Between Neutrophils And Melanoma Cells In A Shear Flow

Cell adhesion, mediated by specific receptor-ligand interactions, plays an important role in biological processes such as tumor metastasis and inflammatory cascade. For example, interactions between beta(2)-integrin ( lymphocyte function-associated antigen-1 and/or Mac-1) on polymorphonuclear neutrophils (PMNs) and ICAM-1 on melanoma cells initiate the bindings of melanoma cells to PMNs within the tumor microenvironment in blood flow, which in turn activate PMN-melanoma cell aggregation in a near-wall region of the vascular endothelium, therefore enhancing subsequent extravasation of melanoma cells in the microcirculations. Kinetics of integrin-ligand bindings in a shear flow is the determinant of such a process, which has not been well understood. In the present study, interactions of PMNs with WM9 melanoma cells were investigated to quantify the kinetics of beta(2)-integrin and ICAM-1 bindings using a cone-plate viscometer that generates a linear shear flow combined with a two-color flow cytometry technique. Aggregation fractions exhibited a transition phase where it first increased before 60 s and then decreased with shear durations. Melanoma-PMN aggregation was also found to be inversely correlated with the shear rate. A previously developed probabilistic model was modified to predict the time dependence of aggregation fractions at different shear rates and medium viscosities. Kinetic parameters of beta(2)-integrin and ICAM-1 bindings were obtained by individual or global fittings, which were comparable to respectively published values. These findings provide new quantitative understanding of the biophysical basis of leukocyte-tumor cell interactions mediated by specific receptor-ligand interactions under shear flow conditions.

Identification of immune genes in grass carp Ctenopharyngodon idella in response to infection of the parasitic copepod Sinergasilus major

The parasitic copepod Sinergasilus major is an important pathogen of grass carp Ctenopharyngodon idella. To understand the immune response of grass carp to the copepod infection, suppression subtractive hybridization method was employed to characterize genes up-regulation during the copepod infection in liver and gills of the fish. One hundred and twenty-two dot blot positive clones from infected subtracted library were sequenced. Searching available databases by using these nucleotide sequences revealed that 23 genes are immune-related, including known acute-phase reactants, and four novel genes encoding proteins such as source of immunodominant MHC-associated peptides (SIMP), TNF receptor-associated factor 2 binding protein (T2BP), poliovirus receptor-related protein 1 precursor, glycoprotein A repetitions predominant (GARP). The differential expression of seven immune genes, i.e. GARP, alpha-2-macroglobulin, MHC class I, C3, SIMP, T2BP, transferrin, as a result of infection was further confirmed by RT-PCR, with the up-regulation of alpha-2-macroglobulin, MHC class I, C3, SIMP and T2BP in the liver of infected fish, and down-regulation of SIMP in the gills of infected fish. The present study provides foundation for understanding grass carp immune response and candidate genes for further analysis.

微生物对抗菌肽耐受的诱导及耐受机理初步研究

抗菌肽是一类具有强大杀菌能力的肽类分子，同时还具有离子调节、免疫调节、蛋白酶抑制剂和自由基清除等其他生物活性。现已鉴定的抗菌肽超过1,200 种，几乎存在于所有生物种类中。在抗生素耐受严重的今天，抗菌肽极有潜力成为新型的有效抗菌药物，许多抗菌肽已进入临床前研究或临床研究。在本论文中，我们选择了无指盘臭蛙（Odorrana grahami）来源的三种抗菌肽（Brevinin 2E-OG1、Nigrocin-OG4 和Palustrin-OG1），单独或组合使用，以藤黄微球菌（Micrococcus luteus）、枯草芽孢杆菌（Bacillus subtilis）和白假丝酵母菌（Candida albicans）为研究对象，进行微生物对抗菌肽耐受性的实验诱导；并通过检测胞外蛋白酶活性、蛋白质组学等方法对微生物耐受抗菌肽机制进行初步的研究。将微生物培养于含有低浓度抗菌肽（单独使用或组合使用）培养基中，每日转接一次，每十次酌情提高抗菌肽浓度。80 次转接后，除藤黄微球菌未对 Palustrin-OG1 产生耐受外，其余所有的实验菌株均表现出对所用三种抗菌肽的耐受。但是Palustrin-OG1 与Brevinin 2E-OG1 或Nigrocin-OG4 联合使用能在一定程度上降低耐受性。将诱导后细菌于不含抗菌肽条件下培养，转接5 次后，对耐受现象无影响，说明这种耐受是可以稳定遗传的。抗菌肽耐受机制之一是分泌蛋白酶水解胞外抗菌肽，我们通过两种方式检测胞外蛋白酶活性，一种是检测发酵液的酪蛋白水解活性，另一种是检测发酵液处理抗菌肽后对抗菌活性的影响。结果发现枯草芽孢杆菌和藤黄微球菌发酵液存在着蛋白酶活性，推测胞外蛋白酶可能与二者对抗菌肽的耐受有关；而白假丝酵母菌发酵液中未检测到蛋白酶活性。另外，我们还通过蛋白质组学的手段对枯草芽孢杆菌耐受机制进行了初步的研究，鉴定了5 个差异表达的蛋白，表达上调的蛋白有yraA（功能未知）、Tpx （巯基过氧化物酶，Thiol peroxidase）、pdhD（二氢硫辛酰胺脱氢酶，dihydrolipoamide dehydrogenase），表达下调的有cotN/TasA（芽孢膜相关蛋白，spore coat-associated protein）和gapA（三磷酸甘油醛脱氢酶，Glyceraldehyde 3-phosphate dehydrogenase 1 ，GAPDH）。yraA 和Tpx 都由Spx 调控，yraA 可以水解小肽增加自由氨基酸，而自由氨基酸增多时gapA、tasA 表达水平会下降，Spx 是由sigma-M 因子调控的，所以我们推测sigma-M 因子在B. subtiis 对抗菌肽耐受中起到了重要的作用。总之，本研究发现抗菌肽的联合作用会减缓微生物对其耐受的程度，为抗菌肽类药物研发提供了一种新思路；同时对抗菌肽耐受机制的初步研究也为今后的深入研究打下了基础。另外，我们还设计了一种新型的抗菌肽系统命名方法，并构建了昆明动物研究所抗菌肽数据库。

千金子和暖地大叶藓的化学成分研究

本论文由三部分共4 章组成。第一部分阐述了大戟科大戟属传统中药千金子（Euphorbia lathyris L.）化学成分、生物学活性以及千金子化学成分的HPLC、UPLC-MS、GC-MS 分析成果。第二部分介绍了民族药材暖地大叶藓（Rhodobryum giganteum (Schwaegr.) Par.）的化学成分研究和结构鉴定。第三部分概述了大戟属 植物中大环二萜酯的研究进展。 第一章包括1－3 节。在第1, 2 节中报道了千金子（Euphorbia lathyris L.）95% 乙醇提取物的化学成分分离鉴定。我们采用正、反相硅胶柱层析、重结晶等各种分离方法，凭借MS、IR、NMR、X-ray 等现代仪器手段,从中共分离鉴定22 个化合物。其中8 个是高活性化合物前体-续随子烷型大环二萜及3 个巨大戟烷型二萜，还有香豆素、生物碱、甾体等类型，其中完成对5 个大环二萜酯构型的确认，对2 个二萜酯构型进行了修正。第3 节中介绍对千金子化学成分的细胞毒性、α-葡萄糖苷酶抑制活性、P-gp 表达抑制活性的模型筛选结果。 第二章包括3 节，第1 节报道不同产地千金子高效液相色谱定量分析结果。第2 节介绍了各大环二萜酯的HPLC-MS/MS 的分析结果，并且对其质谱裂解规律、UPLC-MS 快速鉴定方法做了进一步讨论。第3 节介绍了千金子挥发油成分分析。采用传统水蒸气蒸馏方法提取千金子中的挥发油，并经气相色谱－质谱联用（GC-MS）技术共分离鉴定出 49 个化合物，占挥发油总量的90.48％。 第三章包括1, 2 两节，第1 节报道了暖地大叶藓化学成分。采用正、反相硅胶，凝胶柱层析等各种分离方法和MS、IR、NMR 等解析手段，共分离鉴定10个化合物，其中一个环肽化合物为新化合物。第2 节介绍了暖地大叶藓挥发油成分分析，共分离鉴定出 52 个化合物，占其挥发油总量的85.67％。 第四章概述了大戟科大戟属植物中大环二萜酯的研究进展。 This dissertation consists of three parts. In the first part, it is elaborated that the phytochemical investigation from the traditional Chinese medicine: seeds of Euphorbia lathyris L.. Biological activity and constituents analysis by HPLC、UPLC-MS、GC-MS were reported. In the second part, it is discussed that the chemical constituents were isolated and identificated from minority nationalitical herb－Rhodobryum giganteum (Schwaegr.) Par.. The third part is a review about the progress of studies on macrocyclic diterpenes from Euphorbia. The first part is composed of 1－3 sections. The section 1and 2 is focused on the isolation and identification of chemical constituents from seeds of E. lathyris. 22 compounds were isolated from the seeds of E. lathyris. by isolation methods of column chromatography (silica gel, including reversed phase) and recrystallisation on the basis of spectroscopic methods including IR, MS, NMR and X-ray. In 8 macrocyclic and 3 ingenane diterpenes, the relative configuration of 5 macrocyclic diterpenes were confirmed, in which 2 were amended. In the third section, cell cytotoxic activity, restraining activity of α-Glucosidase and multidrug resistance (MDR) reversing activity about P-gp were tested. 5 potential revsering reagents were found. The second part is composed of 1－3 sections. In first section it is described that the quality of the chemical constituents of E. lathyris from 5 sources , which were analyzed by high-performance liquid chromatography. In addition, the fractionation rules of some macrocyclic diterpenes were discussed and Ultra Performance Liquid Chromatography/ electrospray ionization mass spectrometry (UPLC-MS) was applied for quick determination of compounds in the second section. In the third section, chemical analysis of the essential oil from seeds of E. lathyris by GC-MS were reported. The essential oil from the seeds of E. lathyris L. in Sichuan was extracted by steam distillation and 49 compounds were isolated and identified from the essential oil by gas chromatography-mass spectrometer (GC-MS). These compounds are accounted for 90.46% of the total essential oil. The second part, including section 4 and 5, is about the phytochemical investigation of R. giganteum. In the former section, ten compounds were isolated and identified. Among them, a new peptide was characterized by spectroscopic analysis including IR, MS and NMR. In the other section, 52 compounds were isolated and identified from the essential oil by gas chromatography-mass spectrometer (GC-MS). These compounds are accounted for 85.67% of the total essential oil. The third part is a review about the progress of studies on macrocyclic diterpenes from Euphorbia.

Molecular determination of oxytetracycline-resistant bacteria and their resistance genes from mariculture environments of China

Aims: To assess the diversity of antibiotic-resistant bacteria and their resistance genes in typical maricultural environments. Methods nand Results: Multidrug-resistant bacteria and resistance genes from a mariculture farm of China were analysed via cultivation and polymerase chain reaction (PCR) methods. Oxytetracycline (OTC)-resistant bacteria were abundant in both abalone and turbot rearing waters, accounting for 3.7% and 9.9% of the culturable microbes. Multidrug resistance was common, with simultaneous resistance to OTC, chloramphenicol and ampicillin the most common resistance phenotype. 16S rDNA sequence analyses indicate that the typical resistant isolates belonged to marine Vibrio, Pseudoalteromonas or Alteromonas species, with resistance most common in Vibrio splendidus isolates. For OTC resistance, tet(A), tet(B) and tet(M) genes were detected in some multidrug-resistant isolates, with tet(D) being the most common molecular determinant. For chloramphenicol resistance, cat II was common, and floR was also detected, especially in marine Pseudoalteromonas strains. Conclusions: There is the risk of multidrug-resistant bacteria contamination in mariculture environments and marine Vibrio and Pseudoalteromonas species serve as reservoirs of specific antibiotic resistance determinants. Significance and Impact of the Study: This paper and similar findings from Korea and Japan indicate the potential for widespread distribution of antibiotic resistance genes in mariculture environments from the East Asian region of the world.

Molecular characterizations of chloramphenicol- and oxytetracycline-resistant bacteria and resistance genes in mariculture waters of China

In order to gain an understanding of the diversity and distribution of antimicrobial-resistant bacteria and their resistance genes in maricultural environments, multidrug-resistant bacteria were screened for the rearing waters from a mariculture farm of China. Both abalone Haliotis discus hannai and turbot Scophthalmus maximus rearing waters were populated with abundant chloramphenicol-resistant bacteria. These bacteria were also multidrug resistant, with Vibrio splendidus and Vibrio tasmaniensis being the most predominant species. The chloramphenicol-resistance gene cat II, cat IV or floR could be detected in most of the multidrug-resistant isolates, and the oxytetracycline-resistance gene tet(B), tet(D), tet(E) or tet(M) could also be detected for most of the isolates. Coexistence of chloramphenicol- and oxytetracycline-resistance genes partially explains the molecular mechanism of multidrug resistance in the studied maricultural environments. Comparative studies with different antimicrobial agents as the starting isolation reagents may help detect a wider diversity of the antimicrobial-resistant bacteria and their resistance genes. (C) 2009 Elsevier Ltd. All rights reserved.

Proteomic detection of changes in protein expression induced by cordycepin in human hepatocellular carcinoma BEL-7402 cells

The nucleoside analogue cordycepin (3'-deoxyodenosine, 3'-dA), one of the components of cordyceps militaris, has been shown to inhibit the growth of various tumor cells. However, the probable mechanism is still obscure. In this study, the inhibition of cell growth and changes in protein expression induced by cordycepin were investigated in BEL-7402 cells. Using the MTT assay and flow cytometry, we found that cordycepin inhibits cell viability and induces apoptosis in BEL 7402 cells. Additionally. the proteins were separated using two-dimensional polyacrylamide gel electrophoresis, and eight proteins were found to be significantly, affected by cordycepin compared to untreated control; among them, two were downregulated and six were upregulated. Of the eight proteins, six were identified with peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) after in-gel trypsin digestion. These proteins are involved in various aspects of cellular metabolism. It is suggested that the effect of cordycepin on the growth of tumor cells is significantly related to the metabolism-associated protein expression induced by cordycepin. Copyright 2008 Prous Science, S.A.U. or its licensors. All rights reserved.