A serpin from Chinese shrimp Fenneropenaeus chinensis is responsive to bacteria and WSSV challenge

Arthropod defence responses (e.g. prophenoloxidase (proPO) activation and Toll pathway initiation) are mediated by serine proteinase cascades and regulated by serpins in haemolymph. A serpin (Fc-serpin) cDNA was cloned from the haemocytes of Fenneropenaeus chinensis by rapid amplification of cDNA ends (RACE) PCR and haemocyte cDNA library screening. The full-length cDNA consists of 1734 bp, encoding 411 amino acids with a calculated molecular mass of 46.55 kDa and a theoretical isoelectric point of 7.70. Fc-serpin contains a typical serpin-like homologue (serine proteinase inhibitors domain). The deduced protein contains a putative signal peptide of 19 amino acids and the serpin's signature sequence ((FHCNRPFLFLI389)-F-379). Fc-serpin showed some identity with Pacifastacus leniusculus serpin (42%) and Manduca sexta serpin-6 (34%). The reactive centre loop (RCL) sequences of Fc-serpin, P leniusculus serpin, M. sexta serpin-6 and Bombyx mori serpin-2 are highly similar. An Arg at the PI position of the reactive site indicates that Fc-serpin may have inhibitory activity against prophenoloxidase activating proteinase (PAP) and clotting enzyme. Transcripts of Fc-serpin mRNA were mainly detected in haemocytes and the lymphoid organ by RT-PCR. The variation of the mRNA transcription level in haemocytes followed by artificial infection with bacteria OF white spot syndrome virus (WSSV) was quantified by SYBR Green real-time PCR analysis. Expression profiles of Fc-serpin greatly fluctuated after challenge. This work represents the first report Of a serpin in penaeid shrimp. The data provide clues that Fc-serpin might play potential roles in the innate immunity of shrimp. (C) 2008 Elsevier Ltd. All rights reserved.

Characterization of an ETS transcription factor in the sea scallop Chlamys farreri

We have cloned and characterized a cDNA encoding a putative ETS transcription factor, designated Cf-ets. The Cf-ets encodes a 406 amino acid protein containing a conserved ETS domain and a Pointed domain. Phylogenetic analysis revealed that Cf-ets belongs to the ESE group of ETS transcription factor family. Real-time PCR analysis of Cf-ets expression in adult sea scallop tissues revealed that Cf-ets was expressed mainly in gill and hemocytes, in a constitutive manner. Cf-ets mRNA level in hemocytes increased drastically after microbial challenge indicated its indispensable role in the anti-infection process. Simultaneously, the circulating hemocyte number decreased. In mammals, most ETS transcription factors play indispensable roles in blood cell differentiation and linage commitment during hematopoisis. Cf-ets is therefore likely to be a potential biomarker for hematopoiesis studies in scallops. (C) 2009 Elsevier Ltd. All rights reserved.

Cortactin mediated morphogenic cell movements during zebrafish (Danio rerio) gastrulation

Cell migration is essential to direct embryonic cells to specific sites at which their developmental fates are ultimately determined. However, the mechanism by which cell motility is regulated in embryonic development is largely unknown. Cortactin, a filamentous actin binding protein, is an activator of Arp2/3 complex in the nucleation of actin cytoskeleton at the cell leading edge and acts directly on the machinery of cell motility. To determine whether cortactin and Arp2/3 mediated actin assembly plays a role in the morphogenic cell movements during the early development of zebrafish, we initiated a study of cortactin expression in zebrafish embryos at gastrulating stages when massive cell migrations occur. Western blot analysis using a cortactin specific monoclonal antibody demonstrated that cortactin protein is abundantly present in embryos at the most early developmental stages. Immunostaining of whole-mounted embryo showed that cortactin immunoreactivity was associated with the embryonic shield, predominantly at the dorsal side of the embryos during gastrulation. In addition, cortactin was detected in the convergent cells of the epiblast and hypoblast, and later in the central nervous system. Immunofluorescent staining with cortactin and Arp3 antibodies also revealed that cortactin and Arp2/3 complex colocalized at the periphery and many patches associated with the cell-to-cell junction in motile embryonic cells. Therefore, our data suggest that cortactin and Arp2/3 mediated actin polymerization is implicated in the cell movement during gastrulation and perhaps the development of the central neural system as well.

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.

Biological effect of R-phycoerythrin-mediated photosensitization on DNA.

R-phycoerythrin (R-PE) is one of important proteins involved in capturing light during photosynthesis in red algae, and it is highly fluorescent, and water-soluble chromophores. In vivo, it can transfer the light energy into photosynthetic center, however, it can deliver the captured light energy captured to the surrounding oxygen in vitro and produce reactive oxygen species such as singlet oxygen, which is toxic to tumor cells. R-PE was added to the culture medium of tumor cells, subsequently with irradiation of 488 nm, Argon laser of 25.6 J/cm(2). The result by MTT assay showed that the survival rate decreased with the increase of R-PE concentration from 1 to 100 mg/L. The result from H-3-TdR incorporation demonstrated that the synthesis of DNA reduced when the concentration of R-PE increased from 0.01 to 0.32 mg/L. Besides, pUC18 DNA showed a conversion from supercoiled into linear conformation. The conclusion comes that R-PE mediated PDT can influence the conformation of DNA, and it may be one of the mechanisms of R-PE mediated photodynamic therapy.

Scophthalmus maximus cystatin B enhances head kidney macrophage-mediated bacterial killing

Cystatins form a large family of cysteine protease inhibitors found in a wide arrange of organisms. Studies have indicated that mammalian cystatins play important roles under both physiological and pathological conditions. However, much less is known about fish cystatins. In this report, we described the identification and analysis of a cystatin B homologue, SmCytB, from turbot Scophthalmus maximus. The open reading frame of SmCytB is 300 bp, which encodes a 99-residue protein that shares high levels of sequence identities with the cystatin B of a number of fish species and contains the conserved cysteine protease inhibitor motif of cystatin B. Constitutive expression of SmCytB is high in muscle, brain, heart and liver, and low in spleen. blood, gill and kidney. Bacterial infection upregulates SmCytB expression in kidney, spleen, liver and brain but not in muscle or heart. Functional analysis showed that recombinant SmCytB purified from Escherichia colt exhibits apparent cysteine protease inhibitor activity. Transient overexpression of SmCytB in head kidney macrophages enhances macrophage bactericidal activity probably through a nitric oxide-independent mechanism. These results indicate that SmCytB is involved in the immune defense of turbot against bacterial infection. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and analysis of a Scophthalmus maximus ferritin that is regulated at transcription level by oxidative stress and bacterial infection

Ferritins are conserved Iron storage proteins that exist in most living organisms and play an essential role in Iron homeostasis. In this study, we reported the identification and analysis a ferritin M subunit, SmFerM, from turbot Scophthalmus maximus. The full length cDNA of SmFerM contains a 5'-untranslated region (UTR) of 232 bp, an open reading frame (ORF) of 531 bp, and a 3'-UTR of 196 bp The ORF encodes a putative protein of 176 amino acids, which shares extensive sequence identities with the M terrains of several fish species. In silico analysis identified in SmFerM both the ferroxidase center of mammalian H ferritins and the iron nucleation site of mammalian L ferritins. Quantitative real time reverse transcriptase-PCR analysis indicated that SmFerM expression was highest in muscle and lowest in heart and responded positively to experimental challenges with bacterial pathogens and poly(I center dot C) Exposure of cultured turbot hepatocytes to treatment of stress inducers (iron, copper, and H2O2) significantly upregulated the expression of SmFerM in a dose dependent manner. Iron chelating analysis showed that recombinant SmFerM purified from Escherichia coli exhibited apparent iron binding activity. These results suggest that SmFerM is a functional M ferritin and is likely to play a role in iron sequestration and protection against oxidative stress and microbial infection (C) 2010 Elsevier Inc All rights reserved

Sensitive and Rapid Detection of Genetic Modified Soybean (Roundup Ready) by Loop-Mediated Isothermal Amplification

Using the LAMP method, a highly specific and sensitive detection system for genetically modified soybean (Roundup Ready) was designed. In this detection system, a set of four primers was designed by targeting the exogenous 35S epsps gene. Target DNA was amplified and visualized on agarose gel within 45 min under isothermal conditions at 65 degrees C. Without gel electrophoresis, the LAMP amplicon was visualized directly in the reaction tube by the addition of SYBR Green I for naked-eye inspection. The detection sensitivity of LAMP was 10-fold higher than the nested PCR established in our laboratory. Moreover, the LAMP method was much quicker, taking only 70 min, as compared with 300 min for nested PCR to complete the analysis of the GM soybean. Compared with traditional PCR approaches, the LAMP procedure is faster and more sensitive, and there is no need for a special PCR machine or electrophoresis equipment. Hence, this method can be a very useful tool for GMO detection and is particularly convenient for fast screening.

Decline in medicinal and forage species with warming is mediated by plant traits on the Tibetan Plateau

Experimental studies of how global changes and human activities affect plant diversity often focus on broad measures of diversity and discuss the implications of these changes for ecosystem function. We examined how experimental warming and grazing affected species within plant groups of direct importance to Tibetan pastoralists: medicinal plants used by humans and palatable plants consumed by livestock. Warming resulted in species losses from both the medicinal and palatable plant groups; however, differential relative vulnerability to warming occurred. With respect to the percent of warming-induced species losses, the overall plant community lost 27%, medicinal plants lost 21%, and non-medicinal plants lost 40% of species. Losses of palatable and non-palatable species were similar to losses in the overall plant community. The deep-rootedness of medicinal plants resulted in lowered sensitivity to warming, whereas the shallow-rootedness of non-medicinal plants resulted in greater sensitivity to warming; the variable rooting depth of palatable and non-palatable plants resulted in an intermediate response to warming. Predicting the vulnerability of plant groups to human activities can be enhanced by knowledge of plant traits, their response to specific drivers, and their distribution within plant groups. Knowledge of the mechanisms through which a driver operates, and the evolutionary interaction of plants with that driver, will aid predictions. Future steps to protect ecosystem services furnished by medicinal and palatable plants will be required under the novel stress of a warmer climate. Grazing may be an important tool in maintaining some of these services under future warming.