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.     

Prawn lipocalin: characterization of a color shift induced by gene knockdown and ligand binding assay

The lipocalin family of proteins functions in the transport of steroids, carotenoids, retinoids, and other small hydrophobic molecules. Recently, a lipocalin (MrLC) was isolated from the prawn Macrobrachium rosenbergii and its expression varied with the molting cycle. In this study, knockdown of the MrLC gene by RNA interference (RNAi) was performed and resulted in a shift in body color from blue to orangish red over the entire carapace. By immune-gold electron microscopy, MrLC was found to co-localize with the lipid droplets in subepidermal adiose tissue that were found to be decreased dramatically in MrLC knockdown prawns, in which a reduction in relative fat content was also quantified. Furthermore, MrLC was found to specifically bind astaxanthin and molt hormone (20-hydroxyecdysone) in both in vitro ligand binding assay and in vivo native ligand detection. These results suggested that MrLC plays roles in the regulation of coloration through its association with astaxanthin and may also be involved in the regulation of molting in crustacean.     

Molecular mechanisms regulating molting in a crustacean

Crustacean growth and development is characterized by periodic shedding (ecdysis) and replacement of the rigid exoskeleton. Secretions of the X-organ sinus gland complex control the cellular events that lead to growth and molting. Western blot and ELISA results showed a progressive increase in growth arrest-specific protein (Gas7) from early postmolt stage to a maximum at late postmolt stage. Phosphorylation of ERK2, a downstream signaling protein, was also identified in the subsequent stages. ERK2 phosphorylation resulted in the expression of molt-inhibiting hormone (MIH). Specific ERK inhibitors (PD98059 and UO126) exhibited the ability to reduce the molting duration of Fenneropenaeus indicus from 12-14 days to 7-8 days, suggesting that the ERK1/2 signaling pathway is responsible for the expression of MIH, which controls the molt cycle. We have identified the stage-specific expression of Gas7 (approximately 48 kDa) in the X-organ sinus gland complex of eyestalk which is involved in the downstream signaling of the ERK1/2 pathway regulating the expression of MIH during the molt cycle of the white shrimp, F. indicus. These are the first data showing an association between the Gas7 signal-transduction process and regulation of the molt cycle and provides an alternative molecular intervention mechanism to the traditional eyestalk ablation in crustaceans.     

Inhibition of Human UDP-Glucose Dehydrogenase Expression Using siRNA Expression Vector in Breast Cancer Cells

UDP-glucose dehydrogenase (UGDH) catalyzes two oxidations of UDP-glucose to yield UDP-glucuronic acid. Pathological over-production of extracellular matrix components may be linked to the availability of UDP-glucuronic acid, therefore UGDH is a potential therapeutic target. RNA interference (RNAi) has been adapted to knock down the expression of human UGDH. A UGDH siRNA plasmid was constructed using a pRNA-U6.1/Neo vector and transfected into breast cancer cells, ZR-75-1, with an efficiency of up to 50%. Western blot analysis showed that the UGDH expression was efficiently knocked down at protein levels by RNAi in ZR-75-1 cells.     

Naegleria fowleri: nfa1 gene knock-down by double-stranded RNAs

Nfa1 protein expressed by the nfa1 gene that was cloned recently from pathogenic Naegleria fowleri was found in pseudopodia, especially food-cups, and concerned with a mechanism of pathogenicity of N. fowleri. In the present study, N. fowleri nfa1 gene was knocked down using double-stranded RNAs, and the expression of Nfa1 protein was observed. Using synthetic double-stranded RNA of the nfa1 gene in vitro, the nfa1 gene and Nfa1 protein were knocked down about 50.4+/-3.1% and 52+/-2%, respectively. These results suggest that RNA interference (RNAi) may be an effective technique for gene knock-down in N. fowleri trophozoites.     

Choice of the adequate detection time for the accurate evaluation of the efficiency of siRNA-induced gene silencing

RNA interference (RNAi) mediated by small interfering RNA (siRNA) has become a popular tool of examining the function of various genes. However, many studies have failed to identify any inhibitory effect of the siRNAs on the expression of the target gene, even though the siRNA being tested had been designed sequence-specifically. In order to determine if this failure is due to the incorrect choice of observation time rather than that of the target site of the gene of interest, this study examined the RNAi efficiency of a vector-driven siRNA targeting two different reporter proteins, EGFP and d2EGFP, whose targeted sequences were identical but the half-lives within the cells differed remarkably from each other (>24h versus 2h), during the time course after transfection. The EGFP expression levels in both cells were reduced in time-dependent manner but the reduction patterns were quite different from each other. The RNAi efficiency varied among the different observation time points and the time required for the maximum RNAi efficiency was proportional to the half-life of the target protein. Stable knocked down cell lines for EGFP expression were then established and the reduced EGFP expression levels in these cell lines were retained for a long period. These results suggest that the choice of an adequate observation time or the establishment of stable knocked down cells by antibiotic selection might be required for making an accurate evaluation of the RNAi effect on the target protein possessing a long half-life.     

Comparative immunohistochemistry and cellular distribution of farnesoic acid O-methyltransferase in the shrimp and the crayfish

Farnesoic acid O-methyltransferase (FAMeT) catalyzes the conversion of farnesoic acid (FA) to methylfarnesoate (MF) by the mandibular organ (MO) of crustaceans. Here we report the cellular localization of FAMeT and radiochemical assay of endogenous FAMeT activity in shrimp (Metapenaeus ensis) and crayfish (Procambarus clarkii) tissues. As in the eyestalk (ES), FAMeT is concentrated in specific neurosecretory cells of the ventral nerve cord (VNC) whereas only weak FAMeT immunoreactivity was observed in the MO. FAMeT was also detected in the ventral nerve cord, heart (HET), eyestalk, and muscle of the juvenile shrimp. Although the VNC shows the greatest FAMeT immunoreactivity, the heart extract exhibited the highest FAMeT enzymatic activity. These results suggest that FAMeT in the VNC may be inactive or inactivated at the stages of development tested. Contrary to the previous reports in other crustaceans, MO extract in shrimp shows only low FAMeT activity. The eyestalk, epidermis, ovary and testis show appreciable FAMeT activity. The presence of FAMeT in neurosecretory cells of VNC and eyestalk of shrimp and crayfish implies a possible interaction of FAMeT with the eyestalk CHH-family of neuropeptides. The widespread activity of FAMeT suggests that it has a wide spectrum of action in many tissues that contribute to the function and regulation of MF synthesis in shrimp and crayfish.     

白背飞虱气味结合蛋白SfOBP11与寄主选择行为的相关性研究

【目的】研究白背飞虱Sogatella furcifera气味结合蛋白的生物学功能,筛选与寄主植物气味感知过程相关的关键基因,为研发基于RNAi的白背飞虱防控策略提供理论依据。【方法】利用显微注射法对触角中特异性高表达的SfOBP11进行RNA干扰,并使用实时荧光定量PCR检测干扰效率,最后采用H型嗅觉选择仪观察白背飞虱对寄主水稻识别能力的变化。【结果】SfOBP11 mRNA相对表达水平在注射48 h后显著下降,但试虫的存活率未受影响。注射SfOBP11 dsRNA的若虫对寄主水稻的识别能力降低。【结论】SfOBP11可能在白背飞虱识别寄主水稻挥发物的过程中发挥重要作用。     

The shrimp FAMeT cDNA is encoded for a putative enzyme involved in the methylfarnesoate (MF) biosynthetic pathway and is temporally expressed in the eyestalk of different sexes

Methylfarnesoate (MF), an analogue of the insect juvenile hormone III, has been implicated to play a vital role in the regulation of the growth and reproductive development in crustaceans. Farnesoic acid O-methyltransferase (FAMeT) is the key enzyme involved in catalyzing the final step in the MF biosynthetic pathway. In this study, we report the cloning and characterization of the cDNA encoding the putative FAMeT of the shrimp Metapenaeus ensis. FAMeT comprises 280 amino acid residues with a predicted molecular weight of 32kDa. The predicted putative FAMeT protein reveals a high degree of structural conservation of FAMeT with the lobsters. It shares 79 and 70% sequence identities with the putative FAMeTs of Homarus americanus and Panulirus interruptus, respectively. As revealed by the Southern blot analysis and genomic PCR, only one gene exists in the shrimp genome and the gene is uninterrupted in the coding region. The shrimp FAMeT mRNA is widely distributed in many tissues with the highest expression level observed in the central nervous system. A constant level of FAMeT expression is recorded in the ventral nerve cord of the juveniles and the mature females during the reproductive cycle. Unlike the ventral nerve cord, the eyestalk of the juvenile male, but not the female, expresses FAMeT. Further study shows that the eyestalk of the mature female expresses FAMeT during all stages of ovarian maturation. We speculate that FAMeT may be important for the regulation of eyestalk neuropeptides. This is the first extensive study on the molecular characterization, structural analysis and expression of the crustacean FAMeT.     

Double-stranded RNA induces sequence-specific antiviral silencing in addition to nonspecific immunity in a marine shrimp: convergence of RNA interference and innate immunity in the invertebrate antiviral response?

Double-stranded RNA (dsRNA) is a common by-product of viral infections and a potent inducer of innate antiviral immune responses in vertebrates. In the marine shrimp Litopenaeus vannamei, innate antiviral immunity is also induced by dsRNA in a sequence-independent manner. In this study, the hypothesis that dsRNA can evoke not only innate antiviral immunity but also a sequence-specific antiviral response in shrimp was tested. It was found that viral sequence-specific dsRNA affords potent antiviral immunity in vivo, implying the involvement of RNA interference (RNAi)-like mechanisms in the antiviral response of the shrimp. Consistent with the activation of RNAi by virus-specific dsRNA, endogenous shrimp genes could be silenced in a systemic fashion by the administration of cognate long dsRNA. While innate antiviral immunity, sequence-dependent antiviral protection, and gene silencing could all be induced by injection of long dsRNA molecules, injection of short interfering RNAs failed to induce similar responses, suggesting a size requirement for extracellular dsRNA to engage antiviral mechanisms and gene silencing. We propose a model of antiviral immunity in shrimp by which viral dsRNA engages not only innate immune pathways but also an RNAi-like mechanism to induce potent antiviral responses in vivo.     

Regulation of methyl farnesoate production by mandibular organs in the crayfish, Procambarus clarkii: a possible role for allatostatins

Decapod crustaceans do not appear to produce juvenile hormone, but rather its immediate precursor, methyl farnesoate (MF). Both MF and its immediate precursor, farnesoic acid (FA) are produced by the mandibular organs (MO) in crustaceans. The MO are homologous to the insect corpora allata (CA), the site of juvenile hormone biosynthesis. However, the FGLamide allatostatin (ASTs) peptides, of which there are about 60 distinct forms reported from crustaceans, have previously been found to have no effect on MO activity in crustaceans. We have identified by immunocytochemistry the presence of FGLamide-like AST immunoreactivity in neurosecretory cells throughout the CNS as well as in neurohaemal structures such as the sinus gland and pericardial organs. The ASTs are likely delivered to the MO hormonally and/or by local neurohaemal release. Using MO from adult males, we have found wide variability between animals in the in vitro rates of MF and FA biosynthesis. Treatment with Dippu-ASTs has a statistically significant stimulatory effect on MF synthesis, but only in MO that are initially producing MF at lower rates. No effect on FA production was observed, suggesting that the FGLamide ASTs exert their effect on the o-methyl transferase, the enzyme responsible for the conversion of FA to MF.     

针对leader序列的siRNA能够有效抑制SARS冠状病毒多个mRNA的表达

小干扰RNAs(siRNAs)能够有效降解具有互补序列的RNA.在SARS-CoV的基因组RNA和所有亚基因组RNA的5′端均有一段共同的leader序列,而且该leader序列在不同的病毒分离物中高度保守,因此leader序列可作为一个用于抑制SARS-CoV复制的有效靶点.研究表明,针对leader序列化学合成的siRNA和DNA载体表达的shRNA都可以有效抑制SARS-CoV mRNA的表达.Leader序列特异的siRNA或shRNA不仅可以有效抑制leader与报告基因EGFP融合基因的表达,而且还可以有效抑制leader与刺突蛋白(spikeprotein)、膜蛋白(membrane protein)和核衣壳蛋白(nucleocapsid protein)基因的融合转录产物的表达.结果表明,针对leader序列的RNA干扰可以发展成为一种抗SARS-CoV治疗的有效策略.     

RNAi studies reveal a conserved role for RXR in molting in the cockroach Blattella germanica

Ecdysteroids play a major role during developmental growth in insects. The more active form of these hormones, 20-hydroxyecdysone (20E), acts upon binding to its heterodimeric receptor, formed by the two nuclear receptors, EcR and RXR/USP. Functional characterization of USP has been exclusively conducted on the holometabolous insect Drosophila melanogaster. However, it has been impossible to extend such analysis to primitive-hemimetabolous insects since species of this group are not amenable to genetic analysis. The development of methodologies based on gene silencing using RNA interference (RNAi) after treatment with double-stranded RNA (dsRNA) in vivo has resolved such limitations. In this paper, we show that injection of dsRNA into the haemocoel of nymphs and adults of the cockroach Blattella germanica can be used to silence gene function in vivo. In our initial attempt to test RNAi techniques, we halted the expression of the adult-specific vitellogenin gene. We then used the same technique to silence the expression of the B. germanica RXR/USP (BgRXR) gene in vivo during the last nymphal instar. BgRXR knockdown nymphs progressed through the instar correctly but they arrested development at the end of the stage and were unable to molt into adults. The results described herein suggest that RXR/USP function, in relation to molting, is conserved across the insect Class.     

Setogenesis and molting in planktonic crustaceans

The formation of new setae, termed setogenesis, is describedfor two taxa of planktonic crustaceans: euphausiids, Euphausiapacifica and Thysanoessa spinifera, and a calanoid copepod,Calanus marshallae. Characteristics of setal formation and eversionat ecdysis are described from two time-series of animals preservedat known intervals in their molt cycle. Results from these laboratoryreference series indicate that previous interpretations of setogenesisin the literature can by synthesized to describe a dynamic processof setal formation which is common to all crustacean taxa. The morphological characteristics of developing setae are usedto designate three specific phases in the molt cycle of planktoniccrustaceans (premolt, postmolt, and intermolt). This stagingtechnique may be used to study field-oriented problems relatedto molting in small planktonic crustaceans.