Characterization of the first insect prostaglandin (PGE2) receptor: MansePGE2R is expressed in oenocytoids and lipoteichoic acid (LTA) increases transcript expression.

In arthropods, eicosanoids derived from the oxygenated metabolism of arachidonic acid are significant in mediating immune responses. However, the lack of information about insect eicosanoid receptors is an obstacle to completely decipher immune mechanisms underlying both eicosanoid downstream signal cascades and their relationship to immune pathogen-associated molecular patterns (PAMPs). Here, we cloned and sequenced a G protein-coupled receptor (MW 46.16 kDa) from the model lepidopteran, Manduca sexta (Sphingidae). The receptor shares similarity of amino acid motifs to human prostaglandin E2 (PGE2) receptors, and phylogenetic analysis supports its classification as a prostaglandin receptor. In agreement, the recombinant receptor was activated by PGE2 resulting in intracellular cAMP increase, and therefore designated MansePGE2R. Expression of MansePGE2R in Sf9 cells in which the endogenous orthologous receptor had been silenced showed similar cAMP increase upon PGE2 challenge. Receptor transcript expression was identified in various tissues in larvae and female adults, including Malpighian tubules, fat body, gut and hemocytes, and in female ovaries. In addition to the cDNA cloned that encodes the functional receptor, an mRNA was found featuring the poly-A tail but lacking the predicted transmembrane (TM) regions 2 and 3, suggesting the possibility that internally deleted receptor proteins exist in insects. Immunocytochemistry and in situ hybridization revealed that among hemocytes, the receptor was exclusively localized in the oenocytoids. Larval immune challenges injecting bacterial components showed that lipoteichoic acid (LTA) increased MansePGE2R expression in hemocytes. In contrast, injection of LPS or peptidoglycan did not increase MansePGE2R transcript levels in hemocytes, suggesting the LTA-associated increase in receptor transcript is regulated through a distinct pathway. This study provides the first characterization of an eicosanoid receptor in insects, and paves the way for establishing the hierarchy in signaling steps required for establishing insect immune responses to infections.

The red imported fire ant (Solenopsis invicta Buren) kept Y not F: predicted sNPY endogenous ligands deorphanize the short NPF (sNPF) receptor.

Neuropeptides and their receptors play vital roles in controlling the physiology and behavior of animals. Short neuropeptide F (sNPF) signaling regulates several physiological processes in insects such as feeding, locomotion, circadian rhythm and reproduction, among others. Previously, the red imported fire ant (Solenopsis invicta) sNPF receptor (S. invicta sNPFR), a G protein-coupled receptor, was immunolocalized in queen and worker brain and queen ovaries. Differential distribution patterns of S. invicta sNPFR protein in fire ant worker brain were associated both with worker subcastes and with presence or absence of brood in the colony. However, the cognate ligand for this sNPFR has not been characterized and attempts to deorphanize the receptor with sNPF peptides from other insect species which ended in the canonical sequence LRLRFamide, failed. Receptor deorphanization is an important step to understand the neuropeptide receptor downstream signaling cascade. We cloned the full length cDNA of the putative S. invicta sNPF prepropeptide and identified the putative "sNPF" ligand within its sequence. The peptide ends with an amidated Tyr residue whereas in other insect species sNPFs have an amidated Phe or Trp residue at the C-terminus. We stably expressed the HA-tagged S. invicta sNPFR in CHO-K1 cells. Two S. invicta sNPFs differing at their N-terminus were synthesized that equally activated the sNPFR, SLRSALAAGHLRYa (EC50 = 3.2 nM) and SALAAGHLRYa (EC50 = 8.6 nM). Both peptides decreased the intracellular cAMP concentration, indicating signaling through the Gαi-subunit. The receptor was not activated by sNPF peptides from other insect species, honey bee long NPF (NPY) or mammalian PYY. Further, a synthesized peptide otherwise identical to the fire ant sequence but in which the C-terminal amidated amino acid residue 'Y' was switched to 'F', failed to activate the sNPFR. This discovery will now allow us to investigate the function of sNPY and its cognate receptor in fire ant biology.

Molecular and functional characterization of the first tick CAP2b (periviscerokinin) receptor from Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

The cDNA of the receptor for CAP(2b)/periviscerokinin (PVK) neuropeptides, designated Rhimi-CAP(2b)-R, was cloned from synganglia of tick Rhipicephalus (Boophilus) microplus. This receptor is the ortholog of the insect CAP(2b)/PVK receptor, as concluded from analyses of the predicted protein sequence, phylogenetics and functional expression. Expression analyses of synganglion, salivary gland, Malpighian tubule, and ovary revealed Rhimi-CAP(2b)-R transcripts. The expression in mammalian cells of the open reading frame of Rhimi-CAP(2b)-R cDNA fused with a hemagglutinin tag at the receptor N-terminus was confirmed by immunocytochemistry. In a calcium bioluminescence assay the recombinant receptor was activated by the tick Ixodes scapularis CAP(2b)/PVK and a PVK analog with EC50s of 64 nM and 249 nM, respectively. Tick pyrokinins were not active. This is the first report on the functional characterization of the CAP(2b)/PVK receptor from any tick species which will now permit the discovery of the physiological roles of these neuropeptides in ticks, as neurohormones, neuromodulators and/or neurotransmitters.

Effect of microencapsulation of Lactobacillus salivarus 29 into alginate/chitosan/alginate microcapsules on viability and cytokine induction.

Harsh gastric condition causes low bioavailability of probiotics when supplied orally. Polymeric encapsulation has successfully protected bacteria from harsh gastric condition and ultimately increased persistency and multiplication at the targeted region. In this study, we encapsulated LS29 into ACA microcapsules and characterized them. The survivability and release of LS29 from LS29-loaded ACA microcapsules in SGF and SIF were studied. Encapsulation efficiency of LS29 in ACA microcapsules was 99.9%. Approximately 70% of bacteria survived at pH 2 by 120 min after encapsulation. Although not much difference of the survivability of LS29 encapsulated into ACA and FDACA was observed, freeze-drying improved the controlled-release of LS29 in SIF and also showed better storage survivability at 4°C for 8 weeks. Furthermore, investigation of in vitro production of cytokines in RAW264.7 showed high level of induction of TNF-α and IL-10. These in vitro results support that the LS29 might have a balanced immunomodulatory effect.

Mucoadhesive alginate/poly (L-lysine)/thiolated alginate microcapsules for oral delivery of Lactobacillus salivarius 29.

In this study, thiolated alginate was synthesized by introduction of cysteine to alginate to prepare mucoadhesive alginate/poly (L-lysine)/thiolated alginate (APTA) microcapsules for efficient oral delivery of Lactobacillus salivarius 29 (LS29), a novel therapeutic Lactobacillus strain, in vitro and in vivo. About 759 +/- 32.4 microM of cysteine per gram of alginate was introduced by estimation of Ellman's reagent reaction. LS29-loaded APTA microcapsules provided suitable morphology, size, and a high loading content and efficiency. LS29 in LS29-loaded APTA microcapsules were effectively protected from simulated gastric condition (pH 2.0) than that of unprotected LS29. LS29 were released from APTA microcapsules in simulated intestinal condition (pH 7.2) with a time-dependent manner. The in vitro and in vivo mucoadhesion study suggested that APTA microcapsules had remarkably stronger mucoadhesive property and provided a promising delivery system for oral administration of LS29.

Effects of gender-specific adult bovine serum on myogenic satellite cell proliferation, differentiation and lipid accumulation.

The study was performed to explore the effects of adult bovine male serum (MS), female serum (FS), and castrated male serum (C-MS) on myogenic satellite cells (MSCs) proliferation and differentiation into myotubes or into adipocyte-like cells (ALCs). MSC proliferation and differentiation was highest in the medium supplemented with MS, implying the important role of male steroid hormones. Myogenin and desmin were highly upregulated in cells cultured in MS-supplemented medium. In contrast, lipid accumulation in ALCs was highest in the medium supplemented with FS. Fatty acid transporter (FAT/CD36) was upregulated in FS-supplemented cultures. Detection of higher FAT/CD36 inducing fatty acids (arachidic acid and eicosapentaenoic acid) in FS compared with MS and C-MS suggests that these fatty acids may have influenced the enhanced formation of lipid droplets in ALCs. Effect of sex steroids on cell proliferation and cell growth of bovine MSCs and C2C12 cell in C-MS was greater than charcoal-dextran-treated fetal bovine serum (CDFBS). Concluding the above facts, the results indicate that each gender-specific bovine serum constitutes of different component, which leads to unique effects on cell behavior.

Effect of different ingredients in traditional Korean medicine for human uterine leiomyoma on normal myometrial and leiomyomal smooth muscle cell proliferation.

Crude water extracts of 13 traditional Korean medicinal ingredients used for leiomyomal treatment were prepared and used to treat human uterine normal myometrial and leiomyomal cell cultures. All the ingredients inhibited proliferation and altered the morphology of both myometrial and leiomyomal cells. Among the 13 ingredients, n-hexane-, chloroform-, and ethylacetate-soluble fractions were extracted from seven ingredients that potently inhibited cell proliferation in their water extract form. Among these, the ethylacetate-fraction of Phlomis umbrosa and Spatholobus suberectus, and the chloroform-fraction of Curcuma zedoaria and S. suberectus inhibited leiomyomal cell proliferation significantly compared to myometrial cell proliferation. Similarly, immunohistochemical analysis showed the inhibition of transforming growth factor-beta receptor 2 in leiomyomal tissue after treatment with the fractions of the ingredients. Moreover, the chloroform-fraction of C. zedoaria was subfractionated by open column chromatography. Two of the eight subfractions (fractions 6 and 7) potently inhibited cell proliferation in leiomyoma compared to myometrium. Further study will be performed with the goal of isolating specific compounds from two effective subfractions of C. zedoaria, ethylacetate-fraction of P. umbrosa, and the ethylacetate and chloroform-fractions of S. suberectus. The present study may be helpful in developing an alternative remedy to leiomyoma with minimal side-effects compared to the current treatments.