Characterization of heat shock protein 90 in the shrimp Metapenaeus ensis: Evidence for its role in the regulation of vitellogenin synthesis

Estrogen hormones play a vital role in the regulation of female reproductive maturation. In oviparous vertebrates, the synthesis of vitellogenin (VTG) is tightly controlled by estrogen hormone signal transduction pathway, which is mediated by estrogen receptor and heat shock protein 90 (Hsp90). In order to investigate whether a similar mechanism exists in crustaceans, the Hsp90 gene was cloned and isolated from the shrimp Metapenaeus ensis by homology cloning strategy. The Hsp90 is 2,524 bp in length, containing an open reading frame of 2,163 bp that encodes a 720 amino acid polypeptide (83 kD). The Hsp90-coding region is interrupted by four introns. MeHsp90 is differentially expressed in eyestalk, ovary, and hepatopancreas at different ovarian maturation stages, and consistently expressed in other tissues including heart, gill, gut, muscle, and central nervous system. In vitro ovary explant assay reveals that MeHsp90 expression in immature ovary can be induced by the addition of exogenous estradiol-17beta, but expression in fully mature ovary exhibits no response to estradiol-17beta treatment. In situ hybridization shows that MeHsp90 is highly expressed in previtellogenic oocytes and its expression decreases with the progress of maturation, and finally stops in late-vitellogenic oocytes. Our results indicate a strong correlation between estrogen hormones and Hsp90 expression in shrimp, suggesting that the expression of VTG may be under the regulation of estrogen hormones through a mechanism similar to that in vertebrates. The result provides insights on the control of vitellogenesis in invertebrates.     

Function and cellular localization of farnesoic acid O-methyltransferase (FAMeT) in the shrimp, Metapenaeus ensis.

The isoprenoid methyl farnesoate (MF) has been implicated in the regulation of crustacean development and reproduction in conjunction with eyestalk molt inhibiting hormones and ecdysteroids. Farnesoic acid O-methyltransferase (FAMeT) catalyzes the methylation of farnesoic acid (FA) to produce MF in the terminal step of MF synthesis. We have previously cloned and characterized the shrimp FAMeT. In the present study, recombinant FAMeT (rFAMeT) was produced for bioassay and antiserum generation. FAMeT is widely distributed in shrimp tissues with the highest concentration observed in the ventral nerve cord. Interestingly, an additional larger protein in the eyestalk also showed immunoreactivity to anti-FAMeT serum. FAMeT was localized in the neurosecretory cells of the X-organ-sinus gland complex of the eyestalk. As shown by RT-PCR, FAMeT mRNA is constitutively expressed throughout the molt cycle in the eyestalk and the ventral nerve cord. To show that our cloned gene product had FAMeT activity, we demonstrated that expressed rFAMeT gene product catalyzed the conversion of FA to MF in a radiochemical assay. The ubiquitous distribution of FAMeT suggests that this enzyme is involved in physiological processes in addition to gametogenesis, oocyte maturation and development and metamorphosis of the shrimp. We hypothesize that FAMeT directly or indirectly (through MF) modulates the reproduction and growth of crustaceans by interacting with the eyestalk neuropeptides as a consequence of its presence in the neurosecretory cells of the X-organ-sinus gland.     

Larval ecology and reproductive activity of Metapenaeus ensis and M.endeavouri in the Gulf of Carpentaria, Australia, assessed from the distribution and abundance of the protozoeal stages

Plankton samples were collected during six cruises in the Gulfof Carpentaria, Australia, between May 1976 and March 1977.Prawn larvae were identified to genus level, and then Metapenaeusprotozoeae were identified using discriminant analysis of morphologicalcharacters. The majority of larvae identified were either Metapenaeusensis or Metapenaeus endeuvouri. Metapenaeus endeavouri larvaewere only found in coastal regions. Although numbers of thisspecies were low during most months sampled, abundance peakedduring January and March 1977. Metapenaeus ensis larvae werewidely distributed, in both coastal and offshore areas, andwere present throughout the year. Highest abundances were observedin January 1977. The Gulf-wide distribution of adult prawnswas also assessed by exploratory trawling during one cruisein May 1976; during this cruise, adult M.ensis were also foundoffshore, in areas not frequented by commercial fishing vessels.Therefore, this species may be more widely distributed thanwas previously thought.     

Generation of recombinant monoclonal antibodies to study structure-function of envelope protein VP28 of white spot syndrome virus from shrimp

White spot syndrome virus (WSSV) is a major pathogen in shrimp aquaculture. VP28 is one of the most important envelope proteins of WSSV. In this study, a recombinant antibody library, as single-chain fragment variable (scFv) format, displayed on phage was constructed using mRNA from spleen cells of mice immunized with full-length VP28 expressed in Escherichia coli. After several rounds of panning, six scFv antibodies specifically binding to the epitopes in the N-terminal, middle, and C-terminal regions of VP28, respectively, were isolated from the library. Using these scFv antibodies as tools, the epitopes in VP28 were located on the envelope of the virion by immuno-electron microscopy. Neutralization assay with these antibodies in vitro suggested that these epitopes may not be the attachment site of WSSV to host cell receptor. This study provides a new way to investigate the structure and function of the envelope proteins of WSSV.     

Therapy for prion diseases: Insights from the use of RNA interference

Insights into the molecular basis and the temporal evolution of neurotoxicity in prion disease are increasing, and recent work in mice leads to new avenues for targeting treatment of these disorders. Using lentivirally mediated RNA interference (RNAi) against native prion protein (PrP), White et al report the first therapeutic intervention that results in neuronal rescue, prevents symptoms and increases survival in mice with established prion disease.1 Both the target, and the timing, of treatment here are crucial to the effectiveness of this strategy: the formation of the neurotoxic prion agent is prevented at a point when diseased neurons can still be saved from death. But the data also give new insights into the timing of treatment in the context of the pattern of spread of prion infection throughout the brain, with implications for developing the most effective treatments.

This perspective considers developments in the field that led to the rationale for targeting endogenous prion protein (PrP) in prion therapeutics and to the discovery of a window of reversibility of early neuronal damage in prion disease. It introduces RNA interference (RNAi) and its therapeutic use in this context and discusses insights into prion pathogenesis and future treatment strategies and goals. A key concept is targeting the critical brain regions for the spread of prion replication. This may have relevance in other neurodegenerative diseases due to protein misfolding, which recent literature suggests may also propagate throughout the brain in disease-specific patterns.     

Therapy for prion diseases: Insights from the use of RNA interference

Insights into the molecular basis and the temporal evolution of neurotoxicity in prion disease are increasing, and recent work in mice leads to new avenues for targeting treatment of these disorders. Using lentivirally mediated RNA interference (RNAi) against native prion protein (PrP), White et al. report the first therapeutic intervention that results in neuronal rescue, prevents symptoms and increases survival in mice with established prion disease.¹ Both the target and the timing of treatment here are crucial to the effectiveness of this strategy: the formation of the neurotoxic prion agent is prevented at a point when diseased neurons can still be saved from death. But the data also give new insights into the timing of treatment in the context of the pattern of spread of prion infection throughout the brain, with implications for developing the most effective treatments.Key words: prion, RNA interference, gene therapy, neurodegeneration, synapticThis perspective considers developments in the field that led to the rationale for targeting endogenous prion protein (PrP) in prion therapeutics and to the discovery of a window of reversibility of early neuronal damage in prion disease. It introduces RNA interference (RNAi) and its therapeutic use in this context and discusses insights into prion pathogenesis and future treatment strategies and goals. A key concept is targeting the critical brain regions for the spread of prion replication. This may have relevance in other neurodegenerative diseases due to protein misfolding, which recent literature suggests may also propagate throughout the brain in disease-specific patterns.     

The importance of reproductive interference in ecology and evolution: from organisms to communities

Knowledge of species interactions is vital to understand ecological and evolutionary patterns in nature. Traditional species interactions (e.g., competition, predation, symbiosis) have received a great deal of deserved attention and their general importance in shaping the evolution of populations and structure of communities is unquestioned. Recently, reproductive interference has been receiving attention as an important species interaction. Reproductive interference is defined as interspecific reproductive activities that decrease the fitness of at least one of the species involved in the interaction. Reproductive interference has the potential to affect the evolutionary trajectories of populations and structure of communities. Here, I comment on seven papers that make up this special feature on reproductive interference. Along the way I highlight key discoveries of these studies and areas of research that may contribute to our understanding of the causes and consequences of reproductive interference.     

RNA interference unveils functions of the hypertrehalosemic hormone on cyclic fluctuation of hemolymph trehalose and oviposition in the virgin female Blattella germanica

Hypertrehalosemic hormone (HTH) is a neuropeptide within the adipokinetic hormone (AKH) family that induces a release of trehalose from fat body into hemolymph in a number of insects. In this study, we first showed that female adult German cockroach, Blattella germanica, displayed a cyclic fluctuation of hemolymph trehalose levels correlated to the maturation of oocytes in the reproductive cycle. After cloning the HTH cDNA from the German cockroach (Blage-HTH), expression studies indicated that Blage-HTH mRNA showed the cyclic changes during the first reproductive cycle, where peak values occurred in 8-day-old virgin female cockroaches, which were going to produce oothecae. The functions of Blage-HTH were studied using RNA interference (RNAi) to knockdown its expression. Adult virgin females of B. germanica injected with Blage-HTH dsRNA increased hemolymph trehalose levels in the late period of vitellogenesis more slowly than control. Furthermore, RNAi of Blage-HTH delayed oviposition time and some (10%) individuals did not produce the first ootheca until 15 days after eclosion, whereas the control group produced ootheca before 9 days in all cases.     

The use of diachronic spatial approaches and predictive modelling to study the vegetation dynamics of a managed heathland

According to the EU Habitats Directive, heathlands are a semi-natural habitat type of community interest. This status aims at conserving these habitats, especially where and when they are threatened by various changes, including natural vegetation succession. We present results of a study of the dynamics of a typical dry heathland plot located in the Fontainebleau massif (France). An exhaustive observation of vegetation changes were made on this area of four hectares between 2000 and 2008, employing a spatial approach. We recorded the expansion of Molinia caerulea (L.) Moench at the expense of Ericaceae. The potential future vegetation of the site was modelled using Markov chains coupled to a GIS programme. This model predicted a gradual change in the floristic composition of heathland in favour of M. caerulea at the expense of Calluna vulgaris (L.) Hull and Erica tetralix L., and the expansion of Pinus sylvestris L. The study demonstrates how spatial methods can contribute to the design of reliable management methods of habitats such as the heathlands.     

山羊PTHrP基因干扰重组腺病毒的制备与鉴定

甲状旁腺激素相关蛋白 (Parathyroid hormone related protein,PTHrP) 具有广泛生物学功能,对调控钙代谢具有重要作用。采用RNA干扰和重组腺病毒技术,对山羊乳腺上皮细胞中PTHrP基因表达进行沉默,为进一步研究该基因在乳腺上皮细胞中的功能奠定基础。采用BLOCK-iT shRNA腺病毒干扰系统,将设计好的寡聚shRNA-322/357经退火复性后插入穿梭质粒pENTR/CMV-GFP/U6中。经Western blotting检测干扰效率后,选择pENTR/CMV-GFP/U6-322与病毒骨架质粒pAd/PL-DEST进行同源重组,成功获得重组干扰腺病毒载体pAD/PL-DEST/CMV-GFP/U6-322,并转染HEK-293细胞,通过包装、扩增后产生干扰重组腺病毒AD-PTHrP-322,TCID50法测定其滴度为2.0×109 PFU/mL。用AD-PTHrP-322感染山羊原代乳腺上皮细胞(MOI=200),经实时定量PCR及Western blotting检测表明在病毒感染24 h、48 h和72 h后,山羊乳腺上皮细胞中PTHrP mRNA表达量分别下调了29.2％、68.1％和82.6％ (P＜0.05),其蛋白表达水平也明显下调,干扰效果明显。     

The use of RNA interference for the metabolic engineering of plants (Review)

The metabolic engineering of plants is aimed at the realization of new biochemical reactions by transgenic cells. These reactions are determined by enzymes encoded by foreign or self-modified genes. Plants are considered to be the most interesting objects for metabolic engineering. Although they are characterized by the same pathways for the synthesis of basic biological compounds, plants differ by the astonishing diversity of their products: sugars, aromatic compounds, fatty acids, steroid compounds, and other biologically active substances. RNA interference aimed at modifying metabolic pathways is a powerful tool that allows for the obtainment of plants with new valuable properties. The present review discusses the main tendencies for research development directed toward the obtainment of transgenic plants with altered metabolism.