Overlay Assay Research Articles

Identification of a 37.6 kDa membrane protein from flounder gill cells involved in binding and infection to lymphocystis disease virus

Flounder gill (FG) cell line derived from the gill tissue of Japanese flounder (Paralichthys olivaceus) was employed as a target cell for investigating the molecules involved in lymphocystis disease virus (LCDV) binding and infection. Indirect immunofluorescence assay showed that LCDV could attach to the FG cells membrane susceptibly. By virus overlay protein binding assay (VOPBA), a 37.6kDa LCDV-binding protein was detected in FG cells' membrane. Two dimensional gel electrophoresis analysis revealed that the 37.6kDa protein was a single polypeptide with PI of 6.0. Mass spectrometric analysis showed that the 37.6kDa protein had a strong association with a coiled-coil domain-containing protein. While treated with trypsin or sodium periodate, the binding ability of the 37.6kDa protein to LCDV was inhibited, which suggested that sugar chains of the 37.6kDa glycosylated protein were involved in virus binding. Polyclonal antibodies were obtained by immunizing the rabbit with electroeluted 37.6kDa protein, which showed a dose-dependent blocking effect to the binding between LCDV and 37.6kDa protein in modified VOPBA, and also inhibited LCDV infection to FG cells in virus infection assay. These results demonstrated that the 37.6kDa trypsin-sensitive glycoprotein might be a potential receptor for LCDV infection to FG cells.

Protein-anchoring Strategy for Delivering Acetylcholinesterase to the Neuromuscular Junction

Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is anchored to the synaptic basal lamina via a triple helical collagen Q (ColQ). Congenital defects of ColQ cause endplate AChE deficiency and myasthenic syndrome. A single intravenous administration of adeno-associated virus serotype 8 (AAV8)-COLQ to Colq(-/-) mice recovered motor functions, synaptic transmission, as well as the morphology of the NMJ. ColQ-tailed AChE was specifically anchored to NMJ and its amount was restored to 89% of the wild type. We next characterized the molecular basis of this efficient recovery. We first confirmed that ColQ-tailed AChE can be specifically targeted to NMJ by an in vitro overlay assay in Colq(-/-) mice muscle sections. We then injected AAV1-COLQ-IRES-EGFP into the left tibialis anterior and detected AChE in noninjected limbs. Furthermore, the in vivo injection of recombinant ColQ-tailed AChE protein complex into the gluteus maximus muscle of Colq(-/-) mice led to accumulation of AChE in noninjected forelimbs. We demonstrated for the first time in vivo that the ColQ protein contains a tissue-targeting signal that is sufficient for anchoring itself to the NMJ. We propose that the protein-anchoring strategy is potentially applicable to a broad spectrum of diseases affecting extracellular matrix molecules.

A Tenebrio molitor GPI-anchored alkaline phosphatase is involved in binding of Bacillus thuringiensis Cry3Aa to brush border membrane vesicles

Bacillus thuringiensis Cry toxins recognizes their target cells in part by the binding to glycosyl–phosphatidyl–inositol (GPI) anchored proteins such as aminopeptidase-N (APN) or alkaline phosphatases (ALP). Treatment of Tenebrio molitor brush border membrane vesicles (BBMV) with phospholipase C that cleaves out GPI-anchored proteins from the membranes, showed that GPI-anchored proteins are involved in binding of Cry3Aa toxin to BBMV. A 68kDa GPI-anchored ALP was shown to bind Cry3Aa by toxin overlay assays. The 68kDa GPI-anchored ALP was preferentially expressed in early instar larvae in comparison to late instar larvae. Our work shows for the first time that GPI-anchored ALP is important for Cry3Aa binding to T. molitor BBMV suggesting that the mode of action of Cry toxins is conserved in different insect orders.

Differential effects of a soluble or immobilized VEGFR-binding peptide

Regulating endothelial cell behavior is a key step in understanding and controlling neovascularization for both pro-angiogenic and anti-angiogenic therapeutic strategies. Here, we characterized the effects of a covalently immobilized peptide mimic of vascular endothelial growth factor, herein referred to as VEGF receptor-binding peptide (VR-BP), on human umbilical vein endothelial cell (HUVEC) behavior. Self-assembled monolayer arrays presenting varied densities of covalently immobilized VR-BP and varied densities of the fibronectin-derived cell adhesion peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) were used to probe for changes in HUVEC attachment, proliferation and tubulogenesis. In a soluble form, VR-BP exhibited pro-angiogenic effects in agreement with previous studies, indicated by increases in HUVEC proliferation. However, when presented to cells in an insoluble context, covalently immobilized VR-BP inhibited several pro-angiogenic HUVEC behaviors, including attachment and proliferation, and also inhibited HUVEC response to soluble recombinant VEGF protein. Furthermore, substrates with covalently immobilized VR-BP also modulated HUVEC tubulogenesis when a matrigel overlay assay was used to provide cells with a pseudo-three dimensional environment. Taken together, these results demonstrate that the context in which ligands are presented to cell surface receptors strongly influences their effects, and that the same ligand can be an agonist or an antagonist depending on the manner of presentation to the cell.

Involvements of the ABC protein ABCF2 and α‐actinin‐4 in regulation of cell volume and anion channels in human epithelial cells

After osmotic swelling, cell volume is regulated by a process called regulatory volume decrease (RVD). Although actin cytoskeletons are known to play a regulatory role in RVD, it is not clear how actin-binding proteins are involved in the RVD process. In the present study, an involvement of an actin-binding protein, α-actinin-4 (ACTN4), in RVD was examined in human epithelial HEK293T cells. Overexpression of ACTN4 significantly facilitated RVD, whereas siRNA-mediated downregulation of endogenous ACTN4 suppressed RVD. When the cells were subjected to hypotonic stress, the content of ACTN4 increased in a 100,000 × g pellet, which was sensitive to cytochalasin D pretreatment. Protein overlay assays revealed that ABCF2, a cytosolic member of the ABC transporter superfamily, is a binding partner of ACTN4. The ACTN4-ABCF2 interaction was markedly enhanced by hypotonic stimulation and required the NH(2) -terminal region of ABCF2. Overexpression of ABCF2 suppressed RVD, whereas downregulation of ABCF2 facilitated RVD. We then tested whether ABCF2 has a suppressive effect on the activity of volume-sensitive outwardly rectifying anion channel (VSOR), which is known to mediate Cl(-) efflux involved in RVD, because another ABC transporter member, CFTR, was shown to suppress VSOR activity. Whole-cell VSOR currents were largely reduced by overexpression of ABCF2 and markedly enhanced by siRNA-mediated depletion of ABCF2. Thus, the present study indicates that ACTN4 acts as an enhancer of RVD, whereas ABCF2 acts as a suppressor of VSOR and RVD, and suggests that a swelling-induced interaction between ACTN4 and ABCF2 prevents ABCF2 from suppressing VSOR activity in the human epithelial cells.

Sequence-Specific Binding of Recombinant Zbed4 to DNA: Insights into Zbed4 Participation in Gene Transcription and Its Association with Other Proteins

Zbed4, a member of the BED subclass of Zinc-finger proteins, is expressed in cone photoreceptors and glial Müller cells of human retina whereas it is only present in Müller cells of mouse retina. To characterize structural and functional properties of Zbed4, enough amounts of purified protein were needed. Thus, recombinant Zbed4 was expressed in E. coli and its refolding conditions optimized for the production of homogenous and functionally active protein. Zbed4’s secondary structure, determined by circular dichroism spectroscopy, showed that this protein contains 32% α-helices, 18% β-sheets, 20% turns and 30% unordered structures. CASTing was used to identify the target sites of Zbed4 in DNA. The majority of the DNA fragments obtained contained poly-Gs and some of them had, in addition, the core signature of GC boxes; a few clones had only GC-boxes. With electrophoretic mobility shift assays we demonstrated that Zbed4 binds both not only to DNA and but also to RNA oligonucleotides with very high affinity, interacting with poly-G tracts that have a minimum of 5 Gs; its binding to and GC-box consensus sequences. However, the latter binding depends on the GC-box flanking nucleotides. We also found that Zbed4 interacts in Y79 retinoblastoma cells with nuclear and cytoplasmic proteins Scaffold Attachment Factor B1 (SAFB1), estrogen receptor alpha (ERα), and cellular myosin 9 (MYH9), as shown with immunoprecipitation and mass spectrometry studies as well as gel overlay assays. In addition, immunostaining corroborated the co-localization of Zbed4 with these proteins. Most importantly, in vitro experiments using constructs containing promoters of genes directing expression of the luciferase gene, showed that Zbed4 transactivates the transcription of those promoters with poly-G tracts.

Abstract 362: Akt-Dependent Interaction with EBP50 Regulates Skp2 Localization and Stability

The PDZ domain-containing scaffolding protein, Ezrin-Radixin-Moesin-Binding Phosphoprotein 50 (EBP50) regulates vascular stenosis following endoluminal vessel injury. Its expression in vascular smooth muscle cells (VSMC) increases after wire injury, and neointima formation is significantly reduced in EBP50 knockout mice. We previously reported that the mitogenic effect of EBP50 is Akt-dependent and mediated by the S-phase kinase-associated protein 2 (Skp2), a component of the E3 ligase complex that degrades the cell cycle inhibitor p21cip1. The mechanisms by which Akt directly increases Skp2 stability are controversial. We hypothesize that EBP50 regulates Akt-dependent stabilization of Skp2. The C-terminal four amino acids of Skp2 (PSCL) are a canonical PDZ-binding sequence. Co-immunoprecipitation and in-gel overlay assays demonstrated the direct interaction between EBP50 and Skp2. Mutation of the C-terminal Leu to Ala (L424A-Skp2) abrogated the interaction with EBP50. Akt potentiated the association between EBP50 and Skp2. Sequence analysis indicated the presence of an Akt phosphorylation motif (RPRLCT) in EBP50 between residues 151 and 156. Indeed, in vitro assays using purified EBP50 and constitutively active Akt showed that Akt phosphorylates EBP50. This effect was abrogated by a naturally occurring mutation R153Q in EBP50 that disrupts the Akt consensus sequence. EBP50 was expressed exclusively in the cytoplasm of VSMC, where it associated with the cytoskeleton. Cell fractionation and fluorescence microscopy studies showed that expression of EBP50, but not of R153Q, induced relocalization of Skp2 from the nucleus to the cytoplasm. EBP50 expression increased Skp2 stability and, consequently, VSMC proliferation. Collectively, these experiments show that Akt-mediated EBP50 phosphorylation increases the interaction with Skp2, regulates Skp2 cellular localization and increases Skp2 stability. These studies identify a novel function for EBP50 in the direct regulation of the cell cycle and provide the mechanistic basis for the effect of this scaffolding protein on VSMC proliferation and neointima formation.

The human H3N2 influenza viruses A/Victoria/3/75 and A/Hiroshima/52/2005 preferentially bind to α2-3-sialylated monosialogangliosides with fucosylated poly-N-acetyllactosaminyl chains

Among influenza A viruses, subtype H3N2 is the major cause of human influenza morbidity and is associated with seasonal epidemics causing annually half million deaths worldwide. Influenza A virus infection is initiated via hemagglutinin that binds to terminally sialylated glycoconjugates exposed on the surface of target cells. Gangliosides from human granulocytes were probed using thin-layer chromatography overlay assays for their binding potential to H3N2 virus strains A/Victoria/3/75 and A/Hiroshima/52/2005. Highly polar gangliosides with poly-N-acetyllactosaminyl chains showing low chromatographic mobility exhibited strong virus adhesion which was entirely abolished by sialidase treatment. Auxiliary overlay assays using anti-sialyl Lewis(x) (sLe(x)) monoclonal antibodies showed identical binding patterns compared with those performed with the viruses. A comprehensive structural analysis of fractionated gangliosides by electrospray ionization quadrupole time-of-flight mass spectrometry revealed sLe(x) gangliosides with terminal Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAc epitope and extended neolacto (nLc)-series core structures as the preferential virus binding gangliosides. More precisely, sLe(x) gangliosides with nLc8, nLc10 and nLc12Cer cores, carrying sphingosine (d18:1) and a fatty acid with variable chain length (mostly C24:0, C24:1 or C16:0) in the ceramide moiety and one or two additional internal fucose residues in the oligosaccharide portion, were identified as the preferred receptors recognized by H3N2 virus strains A/Victoria/3/75 and A/Hiroshima/52/2005. This study describes glycan-binding requirements of hemagglutinin beyond binding to glycans with a specific sialic acid linkage of as yet undefined neutrophil receptors acting as ligands for H3N2 viruses. In addition, our results pose new questions on the biological and clinical relevance of this unexpected specificity of a subtype of influenza A viruses.

Characterization of Ffh of Mycobacterium tuberculosis and its interaction with 4.5S RNA

Signal recognition particle (SRP) mediates targeting of proteins to appropriate cellular compartments, which is an important process in all living organisms. In prokaryotes, SRP consists of Ffh, a protein, and 4.5S RNA that recognizes signal peptide emerging from ribosomes. The SRP (Ffh) of one the most successful intracellular pathogen, Mycobacterium tuberculosis, has been investigated with respect to biochemical properties. In the present study, Ffh of M. tuberculosis was overexpressed and was confirmed to be a GTPase using thin layer chromatography and malachite green assay. The GTP binding ability was confirmed by GTP overlay assay. The 4.5S RNA sequence of M. tuberculosis was synthesized by in vitro transcription assay. The interaction between Ffh and 4.5S RNA was confirmed by overlay assay and RNA gel shift assay. The results show that the biochemical properties of M. tuberculosis Ffh have been conserved, and this is the first report that shows the interaction of components of SRP in M. tuberculosis, namely Ffh protein and 4.5S RNA.

Abstract 4227: Sensitizing resistant ovarian cancer to chemotherapy through inhibition of small GTPases

Abstract Platinum- or paclitaxel-resistance is a major problem in women with advanced ovarian cancer (OVCA) and that confers a poor prognosis for patients. Consequently there is a great need to identify new drugs and drug targets for the management of residual and recurrent metastatic OVCA. About 90% of OVCA starts on the ovarian surface epithelium, but individual cells or multicellular aggregates (MCAs) shed into the peritoneal cavity and contribute to OVCA's metastasis into adjacent organs. These MCAs are resistant to apoptosis and to radio- and chemo-therapy. In OVCA, resistance to chemotherapy and apoptosis has been linked to the small GTPases, Rac1 and Cdc42, and their downstream effectors. Identifying compounds that disperse MCAs through disrupting adhesions would sensitize this resistant subpopulation to chemotherapy. We hypothesize that inhibition of one or both of these GTPases disrupts MCAs and sensitizes drug-resistant OVCA cells to respond to chemotherapeutic agents. We assessed if chemical inhibition of Rac1 or Cdc42 would sensitize drug-resistant OVCA monolayers and in vitro MCAs to cisplatin's cytotoxicity. The OVCA cell lines evaluated were OVCA 429 and SKOV-3ip. The inhibitors we used included: (1) NSC 23766, a Rac1-specific inhibitor, (2) ML141, a Cdc42-specific inhibitor, (3) Toxin B, an inhibitor of Rac, Cdc42 and Rho, and (4) R-naproxen, an inhibitor of Rac1 and Cdc42 (based on our data). We found that Rac1 and Cdc42 inhibitors disperse MCAs in a liquid overlay assay. Furthermore, these inhibitors sensitized OVCA cells to cisplatin's cytotoxic effect. Finding drugs that increase efficacy of chemotherapeutic agents against resistant OVCA cell populations is of particular importance. Thus far, GTPases have not been extensively examined as therapeutic targets, but they offer new opportunities for OVCA therapy. Our findings indicate that Rac1 and Cdc42 inhibitors disperse MCAs and improve their response to chemotherapeutic agents. This could increase the effectiveness of intraperitoneal chemotherapy and improve patient outcomes and survival. Since one of these inhibitors is R-Naproxen (an FDA approved off-patent drug), it has potential for rapid clinical application. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4227. doi:1538-7445.AM2012-4227

Abstract 3805: Identification of a distinctive pattern of lipid composition in Irvalec® resistant tumor cells

Abstract Irvalec® is a marine derived synthetic depsipeptide showing cytotoxic activity against a wide diversity of tumor cells both in vitro and in vivo. The compound is currently undergoing Phase II clinical studies in patients with solid tumors. The existing data on the mechanism of action of Irvalec® indicate that the compound inserts and self-organize in the plasma membrane of tumor cells, rapidly inducing loss of membrane integrity and cell permeabilization, giving rise to a fast and dramatic necrotic death. By continuous exposure of human colon carcinoma HCT-116 cells to stepwise increasing concentratrions of Irvalec®, a resistant subclone of the cell line was established, named HCT-116-Irv. In dose-response cytotoxicity assays, HCT-116-Irv cells showed a GI50 value >50 μM, representing a specific resistance index to the compound greater than 10.8 as compared to their wt counterpart. The resistant cells did not show any of the morphological and physiological responses to Irvalec® observed in parental cells and no overexpression of Pgp efflux pump or significant cross-resistance to other antitumoral drugs was detected. Due to the presumably direct effect of Irvalec® on the cell membrane, we searched for possible changes in the lipid composition of the resistant cells, acquired during the process of resistance development. After fractionation of the lipid extracts using thin layer cromatography (TLC), two differential fractions, that were completely absent in the resistant cells as compared to wt cells, were identified and purified. Interestingly, one of these fractions, named F4, was found to be able to interact with a biotinilated derivative of Irvalec®, using a standard overlay assay. These results suggested that the lipid composition could be directly related to the sensitivity of tumor cells to Irvalec®. Efforts are now focused on the chemical characterization of the selected lipid fraction and its putative biological role mediating the cytotoxic response to Irvalec®. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3805. doi:1538-7445.AM2012-3805

β-Microseminoprotein Endows Post Coital Seminal Plasma with Potent Candidacidal Activity by a Calcium- and pH-Dependent Mechanism

The innate immune factors controlling Candida albicans are mostly unknown. Vulvovaginal candidiasis is common in women and affects approximately 70–75% of all women at least once. Despite the propensity of Candida to colonize the vagina, transmission of Candida albicans following sexual intercourse is very rare. This prompted us to investigate whether the post coital vaginal milieu contained factors active against C. albicans. By CFU assays, we found prominent candidacidal activity of post coital seminal plasma at both neutral and the acid vaginal pH. In contrast, normal seminal plasma did not display candidacidal activity prior to acidification. By antifungal gel overlay assay, one clearing zone corresponding to a protein band was found in both post coital and normal seminal plasma, which was subsequently identified as β-microseminoprotein. At neutral pH, the fungicidal activity of β-microseminoprotein and seminal plasma was inhibited by calcium. By NMR spectroscopy, amino acid residue E71 was shown to be critical for the calcium coordination. The acidic vaginal milieu unleashed the fungicidal activity by decreasing the inhibitory effect of calcium. The candidacidal activity of β-microseminoprotein was mapped to a fragment of the C-terminal domain with no structural similarity to other known proteins. A homologous fragment from porcine β-microseminoprotein demonstrated calcium-dependent fungicidal activity in a CFU assay, suggesting this may be a common feature for members of the β-microseminoprotein family. By electron microscopy, β-microseminoprotein was found to cause lysis of Candida. Liposome experiments demonstrated that β-microseminoprotein was active towards ergosterol-containing liposomes that mimic fungal membranes, offering an explanation for the selectivity against fungi. These data identify β-microseminoprotein as an important innate immune factor active against C. albicans and may help explain the low sexual transmission rate of Candida.

Phagosomes Induced by Cytokines Function as anti-Listeria Vaccines

Phagosomes are critical compartments for innate immunity. However, their role in the protection against murine listeriosis has not been examined. We describe here that listericidal phago-receptosomes are induced by the function of IFN-γ or IL-6 as centralized compartments for innate and adaptive immunity because they are able to confer protection against murine listeriosis. These phago-receptosomes elicited LLO(91-99)/CD8(+)- and LLO(189-201)/CD4(+)-specific immune responses and recruited mature dendritic cells to the vaccination sites controlled by T cells. Moreover, they present exceptional features as efficient vaccine vectors. First, they compartmentalize a novel listericidal STAT-1-mediated signaling pathway that confines multiple innate immune components to the same environment. Second, they show features of MHC class II antigen-loading competent compartments for cathepsin-D-mediated LLO processing. Third, murine cathepsin-D deficiencies fail to develop protective immunity after vaccination with listericidal phago-receptosomes induced by IFN-γ or IL-6. Therefore, it appears that the connection of STAT-1 and cathepsin-D in a single compartment is relevant for protection against listeriosis.

Efficacy of Lactobacillus plantarum C5 Cell and Their Supernatant Against Colletotrichum gloeosporioides on Germination Rate of Chilli Seeds

Lactic Acid Bacteria (LAB) were isolated from different fermented fruits and vegetables and screened for antifllllgal activity against chilli phytopathogen Colletotrichum gloeosporioides. Seven LAB showed antifrmgal activity as observed in the dual overlay assay method. These LAB grew between 10 and 45°C, pH 4.4 and 9.6 (except 1 isolate), 6.5 and 18% NaCl. Fungi infected seeds from four species of chilli were evaluated for germination rate with and without LAB treatments. LAB isolated C5 showed good fungal inhibition and allowed good seed germination and was identified as Lactobacillus plantarum using API CH50L kit. The results indicate that C5 has the potential to be used as a biological control for treatment of chilli seeds against C. gloeosporioides to replace the use of chemical fllllgicides.

Hydrophobic residues in small ankyrin 1 participate in binding to obscurin

Abstract Small ankyrin-1 is a splice variant of the ANK1 gene that binds to obscurin A. Previous studies have identified electrostatic interactions that contribute to this interaction. In addition, molecular dynamics (MD) simulations predict four hydrophobic residues in a 'hot spot' on the surface of the ankyrin-like repeats of sAnk1, near the charged residues involved in binding. We used site-directed mutagenesis, blot overlays and surface plasmon resonance assays to study the contribution of the hydrophobic residues, V70, F71, I102 and I103, to two different 30-mers of obscurin that bind sAnk1, Obsc₆₃₁₆₋₆₃₄₅ and Obsc₆₂₃₁₋₆₂₆₀. Alanine mutations of each of the hydrophobic residues disrupted binding to the high affinity binding site, Obsc₆₃₁₆₋₆₃₄₅. In contrast, V70A and I102A mutations had no effect on binding to the lower affinity site, Obsc₆₂₃₁₋₆₂₆₀. Alanine mutagenesis of the five hydrophobic residues present in Obsc₆₃₁₆₋₆₃₄₅ showed that V6328, I6332, and V6334 were critical to sAnk1 binding. Individual alanine mutants of the six hydrophobic residues of Obsc₆₂₃₁₋₆₂₆₀ had no effect on binding to sAnk1, although a triple alanine mutant of residues V6233/I6234/I6235 decreased binding. We also examined a model of the Obsc₆₃₁₆₋₆₃₄₅-sAnk1 complex in MD simulations and found I102 of sAnk1 to be within 2.2Å of V6334 of Obsc₆₃₁₆₋₆₃₄₅. In contrast to the I102A mutation, mutating I102 of sAnk1 to other hydrophobic amino acids such as phenylalanine or leucine did not disrupt binding to obscurin. Our results suggest that hydrophobic interactions contribute to the higher affinity of Obsc₆₃₁₆₋₆₃₄₅ for sAnk1 and to the dominant role exhibited by this sequence in binding.

Structural Requirements of Membrane Phospholipids for M-type Potassium Channel Activation and Binding

M-channels are voltage-gated potassium channels that regulate cell excitability. They are heterotetrameric assemblies of Kv7.2 and Kv7.3 subunits. Their opening requires the presence of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)). However, the specificity of PI(4,5)P(2) as a binding and activating ligand is unknown. Here, we tested the ability of different phosphoinositides and lipid phosphates to activate or bind to M-channel proteins. Activation of functional channels was measured in membrane patches isolated from cells coexpressing Kv7.2 and Kv7.3 subunits. Channels were activated to similar extents (maximum open probability of ∼0.8 at 0 mV) by 0.1-300 μM dioctanoyl homologs of the three endogenous phosphoinositides, PI(4)P, PI(4,5)P(2), and PI(3,4,5)P(3), with sensitivity increasing with increasing numbers of phosphates. Non-acylated inositol phosphates had no effect up to 100 μM. Channels were also activated with increasing efficacy by 1-300 μM concentrations of the monoacyl monophosphates fingolimod phosphate, sphingosine 1-phosphate, and lysophosphatidic acid but not by phosphate-free fingolimod or sphingosine or by phosphate-masked phosphatidylcholine or phosphatidylglycerol. An overlay assay confirmed that a fusion protein containing the full-length C terminus of Kv7.2 could bind to a broad range of phosphoinositides and phospholipids. A mutated Kv7.2 C-terminal construct with reduced sensitivity to PI(4,5)P showed significantly less binding to most polyphosphoinositides. We concluded that M-channels bind to, and are activated by, a wide range of lipid phosphates, with a minimum requirement for an acyl chain and a phosphate headgroup. In this, they more closely resemble inwardly rectifying Kir6.2 potassium channels than the more PI(4,5)P(2)-specific Kir2 channels. Notwithstanding, the data also support the view that the main endogenous activator of M-channels is PI(4,5)P(2).

Characterization of putative Japanese encephalitis virus receptor molecules on microglial cells

Japanese encephalitis virus (JEV) a mosquito-borne flavivirus is a major cause of viral encephalitis in Asia. While the principle target cells for JEV in the central nervous system are believed to be neurons, microglia are activated in response to JEV and have been proposed to act as a long lasting virus reservoir. Viral attachment to a host cell is the first step of the viral entry process and is a critical mediator of tissue tropism. This study sought to identify molecules associated with JEV entry to microglial cells. Virus overlay protein-binding assay (VOPBA) and liquid chromatography-mass spectrometry (LC/MS/MS) identified the 37/67 kDa high-affinity laminin receptor protein and nucleolin as a potential JEV-binding proteins. These proteins were subsequently investigated for a contribution to JEV entry to mouse microglial BV-2 cells together with other possible candidate receptor molecules including Hsp70, Hsp90, GRP78, CD14, and CD4. In antibody mediated inhibition of infection experiments, both anti-laminin receptor and anti-CD4 antibodies significantly reduced virus entry while anti-Hsp70 and 90 antibodies produced a slight reduction. Significant inhibition of virus entry (up to 80%) was observed in the presence of lipopolysaccharide (LPS) which resulted in a complete down-regulation of CD4 and moderate down-regulation of CD14. These results suggest that multiple receptor proteins may mediate the entry of JEV to microglial cells, with CD4 playing a major role.

Receptor for Activated C Kinase 1 (RACK1) Inhibits Function of Transient Receptor Potential (TRP)-type Channel Pkd2L1 through Physical Interaction

Pkd2L1 (also called TRPP3) is a non-selective cation channel permeable to Ca(2+), Na(+), and K(+) and is activated by Ca(2+). It is also part of an acid-triggered off-response cation channel complex. We previously reported roles of the Pkd2L1 C-terminal fragments in its channel function, but the role of the N terminus remains unclear. Using a yeast two-hybrid screening, we found that the Pkd2L1 N terminus interacts with the receptor for activated C kinase 1 (RACK1), a scaffolding/anchoring protein implicated in various cellular functions. This interaction requires the last two Trp-Asp (WD) motifs of RACK1 and fragment Ala(19)-Pro(45) of Pkd2L1. The interaction was confirmed by GST pulldown, blot overlay, and co-immunoprecipitation assays. By (45)Ca tracer uptake and two-microelectrode voltage clamp electrophysiology, we found that in Xenopus oocytes with RACK1 overexpression Pkd2L1 channel activity is abolished or substantially reduced. Combining with oocyte surface biotinylation experiments, we demonstrated that RACK1 inhibits the function of Pkd2L1 channel on the plasma membrane in addition to reducing its total and plasma membrane expression. Overexpressing Pkd2L1 N- or C-terminal fragments as potential blocking peptides for the Pkd2L1-RACK1 interaction, we found that Pkd2L1 N-terminal fragment Met(1)-Pro(45), but not Ile(40)-Ile(97) or C-terminal fragments, abolishes the inhibition of Pkd2L1 channel by overexpressed and oocyte-native RACK1 likely through disrupting the Pkd2L1-RACK1 association. Taken together, our study demonstrated that RACK1 inhibits Pkd2L1 channel function through binding to domain Met(1)-Pro(45) of Pkd2L1. Thus, Pkd2L1 is a novel target channel whose function is regulated by the versatile scaffolding protein RACK1.

Production and Characterization of Antifungal Compounds Produced by Lactobacillus plantarum IMAU10014

Lactobacillus plantarum IMAU10014 was isolated from koumiss that produces a broad spectrum of antifungal compounds, all of which were active against plant pathogenic fungi in an agar plate assay. Two major antifungal compounds were extracted from the cell-free supernatant broth of L. plantarum IMAU10014. 3-phenyllactic acid and Benzeneacetic acid, 2-propenyl ester were carried out by HPLC, LC-MS, GC-MS, NMR analysis. It is the first report that lactic acid bacteria produce antifungal Benzeneacetic acid, 2-propenyl ester. Of these, the antifungal products also have a broad spectrum of antifungal activity, namely against Botrytis cinerea, Glomerella cingulate, Phytophthora drechsleri Tucker, Penicillium citrinum, Penicillium digitatum and Fusarium oxysporum, which was identified by the overlay and well-diffusion assay. F. oxysporum, P. citrinum and P. drechsleri Tucker were the most sensitive among molds.

Regulation of Bacteriocin Production and Cell Death by the VicRK Signaling System in Streptococcus mutans

The VicRK two-component signaling system modulates biofilm formation, genetic competence, and stress tolerance in Streptococcus mutans. We show here that the VicRK modulates bacteriocin production and cell viability, in part by direct modulation of competence-stimulating peptide (CSP) production in S. mutans. Global transcriptome and real-time transcriptional analysis of the VicK-deficient mutant (SmuvicK) revealed significant modulation of several bacteriocin-related loci, including nlmAB, nlmC, and nlmD (P < 0.001), suggesting a role for the VicRK in producing mutacins IV, V, and VI. Bacteriocin overlay assays revealed an altered ability of the vic mutants to kill related species. Since a well-conserved VicR binding site (TGTWAH-N(5)-TGTWAH) was identified within the comC coding region, we confirmed VicR binding to this sequence using DNA footprinting. Overexpression of the vic operon caused growth-phase-dependent repression of comC, comDE, and comX. In the vic mutants, transcription of nlmC/cipB encoding mutacin V, previously linked to CSP-dependent cell lysis, as well as expression of its putative immunity factor encoded by immB, were significantly affected relative to the wild type (P < 0.05). In contrast to previous reports that proposed a hyper-resistant phenotype for the VicK mutant in cell viability, the release of extracellular genomic DNA was significantly enhanced in SmuvicK (P < 0.05), likely as a result of increased autolysis compared with the parent. The drastic influence of VicRK on cell viability was also demonstrated using vic mutant biofilms. Taken together, we have identified a novel regulatory link between the VicRK and ComDE systems to modulate bacteriocin production and cell viability of S. mutans.