Molecular cloning and expression patterns of FK506‐binding protein 12, an immunophilin from the cabbage butterfly,Pieris rapae

FK506‐binding protein (FK506BP) class belonging to immunophilin protein family has been known to play key roles in modulating T‐cell activation, regulation of cell cycle and protein folding. However, little is known about the involvement of FK506BP during viral pathogenesis in insect host. In this study, an attempt has been made to focus on the involvement of FK506BP in antiviral innate immunity, by cloning the full‐length cDNA of FK506BP12 (PrFK506BP12) from the cabbage butterfly, Pieris rapae. It comprised of 532 bp (excluding poly‐A tail) with a longest open reading frame (ORF) of 327 bp encoding 108 amino acids. In silico analysis of PrFK506BP12 ORF revealed a highly conserved FK506‐binding domain (FKBD). As expected, it showed high homology to other FK506BPs identified from Bombyx mori (92%), Manduca sexta (91%), Suberites domuncula (82%), Tribolium castaneum (81%) and Aedes aegypti (74%) . Expression of PrFK506BP12 was observed during developmental stages of P. rapae, but was pronounced in late pupal and adult stage. In addition, spatial expression pattern analysis indicated its high expression in the head and fat body. Furthermore, PrFK506BP12 mRNA was induced 12 h after LTA, Poly I:C treatment and 3h after Pieris rapae granulovirus (PrGV) treatment in carcass. It suggests that PrFK506BP12 appears to be involved in immune responses and also play an important role in the fat body, although it remains to be clarified about their precise role in response to granulovirus.     

A new culture system for in situ observation of the growth and development of Eucyclops serrulatus (Copepoda: Cyclopoida)

A practical and convenient method of rearing Eucyclops serrulatus in a microculture environment is described. A complete life cycle of E. serrulatus was maintained in a narrow space on a microscope slide glass on which a cover glass of 22 x 40 mm in size was mounted at a height of 0.8 mm. The culture medium was constituted by bottled mineral water boiled with grains of Glycine max (soybean). Chilomonas paramecium, a free-living protozoan organism, was provided as live food. Growth of nauplii hatched from eggs to the first stage of copepodite took an average of 7.7 days, and the growth of copepodite 1 to the egg-bearing adult female took an average of 20.1 days in the microculture cell with an average life time of 44.7 days. Continuous passage of copepods was successfully maintained as long as sufficient medium and food were provided. The microculture method enables an in situ microscopic observation on the growth and developmental process of helminth larvae experimentally infected to copepods as well as of copepod itself. Furthermore, it does not require anesthetization and, therefore, minimize the amount of stress exposed to copepods during the handling process.     

Directed evolution of Escherichia coli farnesyl diphosphate synthase (IspA) reveals novel structural determinants of chain length specificity

Directed evolution of farnesyl diphosphate (FPP, C15) synthase (IspA) of Escherichia coli was carried out by error-prone PCR with a color complementation screen utilizing C40 carotenoid pathway enzymes. This allowed IspA mutants with enhanced production of the C40 carotenoid precursor geranylgeranyl diphosphate (GGPP, C20) to be readily identified. Analysis of these mutants was carried out in order to better understand the mechanisms of product chain length specificity in this enzyme. The 12 evolved clones having enhanced C20 GGPP production have characteristic mutations in the conserved regions of prenyl diphosphate synthases (designated regions I through VII). Some of these mutations (I76T, Y79S, Y79H, C75Y, H83Y, and H83Q) are found near or before the conserved first aspartate rich motif (FARM), which is involved in the mechanism for chain elongation reaction of all prenyl synthases. Molecular modeling suggested a mechanism for chain length determination for these mutations including substitutions at the 1st and 9th amino acids upstream of the FARM that have not been reported previously. In addition, a mutation on a helix adjacent to the FARM within the substrate-binding pocket (D115G) suggests a novel mechanism for chain length determination. One mutant IspA clone carries a mutation of C155G at the 2nd amino acid upstream of conserved region IV (GQxxDL), which was recently found to be an important region controlling the chain elongation of a Type III GGPP synthase. One IspA clone carries mutations (T234A and T249I) near the conserved second aspartate rich motif (SARM). As a verification of the in vivo activity of the mutant clones (represented as C40 carotenoid formation), we confirmed the product distribution of wild-type and mutant IspA using an in vitro assay.     

Secretory phospholipases A2 induce neurite outgrowth in PC12 cells through lysophosphatidylcholine generation and activation of G2A receptor

We previously demonstrated that secretory phospholipase A2 (sPLA2) and lysophosphatidylcholine (LPC) exhibit neurotrophin-like neuritogenic activity in the rat pheochromocytoma cell line PC12. In this study, we further analyzed the mechanism whereby sPLA2 displays neurite-inducing activity. Exogenously added mammalian group X sPLA2 (sPLA2-X), but not group IB and IIA sPLA2s, induced neuritogenesis, which correlated with the ability of sPLA2-X to liberate LPC into the culture media. In accordance, blocking the effect of LPC by supplementation of bovine serum albumin or phospholipase B attenuated neuritogenesis by sPLA2 or LPC. Overproduction or suppression of G2A, a G-protein-coupled receptor involved in LPC signaling, resulted in the enhancement or reduction of neuritogenesis induced by sPLA2 treatment. These results indicate that the neuritogenic effect of sPLA2 is mediated by generation of LPC and subsequent activation of G2A.     

Prostaglandin E2 augments IL-10 signaling and function

In inflamed joints of rheumatoid arthritis, PGE(2) is highly expressed, and IL-10 and IL-6 are also abundant. PGE(2) is a well-known activator of the cAMP signaling pathway, and there is functional cross-talk between cAMP signaling and the Jak-STAT signaling pathway. In this study, we evaluated the modulating effect of PGE(2) on STAT signaling and its biological function induced by IL-10 and IL-6, and elucidated its mechanism in THP-1 cells. STAT phosphorylation was determined by Western blot, and gene expression was analyzed using real-time PCR. Pretreatment with PGE(2) significantly augmented IL-10-induced STAT3 and STAT1 phosphorylation, as well as suppressors of cytokine signaling 3 (SOCS3) and IL-1R antagonist gene expression. In contrast, PGE(2) suppressed IL-6-induced phosphorylation of STAT3 and STAT1. These PGE(2)-induced modulating effects were largely reversed by actinomycin D. Pretreatment with dibutyryl cAMP augmented IL-10-induced, but did not change IL-6-induced STAT3 phosphorylation. Misoprostol, an EP2/3/4 agonist, and butaprost, an EP2 agonist, augmented IL-10-induced STAT3 phosphorylation and SOCS3 gene expression, but sulprostone, an EP1/3 agonist, had no effect. H89, a protein kinase A inhibitor, and LY294002, a PI3K inhibitor, diminished PGE(2)-mediated augmentation of IL-10-induced STAT3 phosphorylation. In this study, we found that PGE(2) selectively regulates cytokine signaling via increased intracellular cAMP levels and de novo gene expression, and these modulating effects may be mediated through EP2 or EP4 receptors. PGE(2) may modulate immune responses by alteration of cytokine signaling in THP-1 cells.     

Expression, purification and transduction of PEP-1-botulinum neurotoxin type A (PEP-1-BoNT/A) into skin

Botulinum neurotoxin A (BoNT/A) has been used therapeutically to treat muscular hypercontractions and sudomotor hyperactivity and it has been reported that BoNT/A might have analgesic properties in headache. PEP-1 peptide is a known carrier peptide that delivers full-length native proteins in vitro and in vivo. In this study, a BoNT/A gene were fused with PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame PEP-1-BoNT/A fusion protein. The expressed and purified PEP-1-BoNT/A fusion proteins were efficiently transduced into cells in a time- and dose-dependent manner when added exogenously in a culture medium. In addition, immunohistochemical analysis revealed that PEP-1-BoNT/A fusion protein efficiently penetrated into the epidermis as well as the dermis of the subcutaneous layer, when sprayed on mice skin. These results suggest that PEP-1-BoNT/A fusion protein provide an efficient strategy for therapeutic delivery in various human diseases related to this protein.     

Detection of an intermediate during the unfolding process of the dimeric ketosteroid isomerase

Failure to detect the intermediate in spite of its existence often leads to the conclusion that two-state transition in the unfolding process of the protein can be justified. In contrast to the previous equilibrium unfolding experiment fitted to a two-state model by circular dichroism and fluorescence spectroscopies, an equilibrium unfolding intermediate of a dimeric ketosteroid isomerase (KSI) could be detected by small angle X-ray scattering (SAXS) and analytical ultracentrifugation. The sizes of KSI were determined to be 18.7A in 0M urea, 17.3A in 5.2M urea, and 25.1A in 7M urea by SAXS. The size of KSI in 5.2M urea was significantly decreased compared with those in 0M and 7M urea, suggesting the existence of a compact intermediate. Sedimentation velocity as obtained by ultracentrifugation confirmed that KSI in 5.2M urea is distinctly different from native and fully-unfolded forms. The sizes measured by pulse field gradient nuclear magnetic resonance (NMR) spectroscopy were consistent with those obtained by SAXS. Discrepancy of equilibrium unfolding studies between size measurement methods and optical spectroscopies might be due to the failure in detecting the intermediate by optical spectroscopic methods. Further characterization of the intermediate using (1)H NMR spectroscopy and Kratky plot supported the existence of a partially-folded form of KSI which is distinct from those of native and fully-unfolded KSIs. Taken together, our results suggest that the formation of a compact intermediate should precede the association of monomers prior to the dimerization process during the folding of KSI.