Evidence that the C‐terminal domain (CtD) autoinhibits neural repression by Drosophila E(spl)M8

Analysis of the retinal defects of a CK2 phosphomimetic variant of E(spl)M8 (M8S¹⁵⁹D) and the truncated protein M8* encoded by the E(spl)D allele, suggest that the nonphosphorylated CtD “autoinhibits” repression. We have investigated this model by testing for inhibition (in “trans”) by the CtD fragment in its nonphosphorylated (M8‐CtD) and phosphomimetic (M8SD‐CtD) states. In N⁺ flies, ectopic M8‐CtD compromises lateral inhibition, i.e., elicits supernumerary bristles as with loss of N signaling. This antimorphic activity of M8‐CtD strongly rescues the reduced eye and/or bristle loss phenotypes that are elicited by ectopic M8SD or wild type M8. Additionally, the severely reduced eye of N^spl/Y; E(spl)D/+ flies is also rescued by M8‐CtD. Rescue is specific to the time and place, the morphogenetic furrow, where “founding” R8 photoreceptors are specified. In contrast, the phosphomimetic M8SD‐CtD that is predicted to be deficient for autoinhibition, exhibits significantly attenuated or negligible activity. These studies provide evidence that autoinhibition by the CtD regulates M8 activity in a phosphorylation‐dependent manner. genesis 48:44–55, 2010. © 2009 Wiley‐Liss, Inc.     

Identification of aquaporin-5 and lipid rafts in human resting saliva and their release into cevimeline-stimulated saliva

BACKGROUND: It is unknown whether AQP5 and lipid rafts are released into human unstimulated (resting) saliva and saliva in response to secretagogues. METHODS: In order to quantitate the salivary concentration of AQP5, we produced a polyclonal antibody for human AQP5 and developed an enzyme-like immunosorbent assay (ELISA). RESULTS: AQP5 and lipid rafts were identified in human resting saliva. The amount of AQP5 in resting saliva showed a diurnal variation with high levels during waking hours, and an age-related decrease in AQP5 was coincident with the volume of resting saliva. Cevimeline, a muscarinic acetylcholine receptor (mAChR) agonist, induced the release of AQP5 with lipid rafts, amylase, mucin, and lysozyme. Changes in saliva AQP5 levels after cevimeline administration occurred simultaneously with changes in saliva flow rates. Confocal microscopy revealed that AQP5 was located in the apical plasma membrane and showed a diffuse pattern in parotid glands under resting conditions. Following cevimeline administration, AQP5 was predominantly associated with the APM and was localized in the lumen. GENERAL SIGNIFICANCE: AQP5 and lipid rafts were released with salivary proteins from human salivary glands by the stimulation of M3 mAChRs, and that changes in saliva AQP5 levels can be used as an indicator of salivary flow rate and also as a useful index of M3 mAChR agonist's action on human salivary glands.     

Reduced Neural Synchrony in Patients with Restless Legs Syndrome during a Visual Oddball Task

Background

Restless legs syndrome (RLS) is a sensorimotor neurological disorder characterized by an irresistible urge to move the legs. It has been reported that RLS patients show cognitive deficits, presumably due to hyperactivity causing loss of attention, or malfunctions in the frontal region resulting from sleep deprivation. However, the mechanism underlying cognitive deficits in RLS patients is mostly unknown. As an effort to clarifying this, we investigated the differences in neural activity and phase synchrony between healthy controls and RLS patients during cognitive task performances.

Methodology/Principal Findings

Seventeen female drug-naive RLS patients were enrolled in the study, and an age-matched group of thirteen healthy female volunteers served as controls. Multichannel event-related potentials (ERPs) were recorded from RLS patients and normal controls while performing a visual oddball task. In addition to conventional analyses of ERP waveforms and spectra, interregional gamma-band phase synchrony (GBPS) was investigated to observe the differences in interregional neural synchronies between normal and RLS patient groups. Strong GBPS was observed primarily between anterior and posterior regions along the midline for both groups. Along with significant reduction and delay of P300 ERP and induced gamma-band activity (GBA), the GBPS was considerably decreased in RLS patients compared to normal subjects, especially at frontal region.

Conclusions

Overall, our results support that cognitive dysfunction in RLS patients is associated with reduced interregional neural synchrony as well as alterations in local neural activity.     

Analysis of population structure among Korean and Japanese Legionella pneumophila isolates using hsp60 sequences

The population structure of Korean (150 strains) and Japanese (92 strains) Legionella pneumophila isolates along with 18 reference strains were investigated using hsp60 sequence (1647 bp) analysis. Twelve clonal subgroups (hsP-I to hsP-X and hsF-I and hsF-II) were designated on the hsp60 tree, inferred from representative sequences using the neighbor-joining method. Some of the isolates showed unique subgroups depending on the source of isolates, including hsP-I, hsF-I, and hsF-II from cooling tower water, and subgroups hsP-VIII and hsP-X from circulating hot water bath. These subgroups may be useful for epidemiological studies to chase or specify sources of infection in Korea and Japan.     

Stress-induced expression and structure of the putative gene hyp-1 for hypericin biosynthesis

The phenolic oxidative coupling protein (Hyp-1) with proposed activity in the biosynthesis of hypericin in Hypericum perforatum shares about 50 % sequence similarity with Bet.v.1-like/PR-10 proteins. In our previous study, we showed that this protein is not a limiting factor in hypericin biosynthesis. To ascertain the role of Hyp-1 in defense mechanisms, we have analyzed some structural features of the hyp-1 gene in 14 Hypericum species with different abilities to synthesise hypericin. We show that the hyp-1 gene possesses characteristics typical for genes encoding plant PR-10 proteins. The coding sequence of the hyp-1 gene is interrupted by a single 86- to 125-bp intron localised strictly in codon 62, which is a typical feature of the dicot PR-10 subfamily. The localisation of the intron is conserved in all 14 tested Hypericum species indicating a common evolutionary history with genes encoding PR-10 proteins. In addition, we report that the hyp-1 gene exhibits a similar response to stress conditions as the PR-10 proteins encoding genes. Following either wounding or infection by Agrobacterium tumefaciens, all analysed Hypericum species exhibited rapid and significant upregulation of hyp-1 gene expression; this was particularly observed in hypericin-producing species. On the other hand, in the presence of high levels of abscisic acid, different levels of gene expression were observed.     

PAB-1, a Caenorhabditis elegans Poly(A)-Binding Protein,Regulates mRNA Metabolism in germline by Interacting with CGH-1 and CAR-1

Poly(A)-binding proteins are highly conserved among eukaryotes and regulate stability of mRNA and translation. Among C. elegans homologues, pab-1 mutants showed defects in germline mitotic proliferation. Unlike pab-1 mutants, pab-1 RNAi at every larval stage caused arrest of germline development at the following stage, indicating that pab-1 is required for the entire postembryonic germline development. This idea is supported by the observations that the mRNA level of pab-1 increased throughout postembryonic development and its protein expression was germline-enriched. PAB-1 localized to P granules and the cytoplasm in the germline. PAB-1 colocalized with CGH-1 and CAR-1 and affected their localization, suggesting that PAB-1 is a component of processing (P)-bodies that interacts with them. The mRNA and protein levels of representative germline genes, rec-8, GLP-1, rme-2, and msp-152, were decreased after pab-1 RNAi. Although the mRNA level of msp-152 was increased in cgh-1 mutant, it was also significantly reduced by pab-1 RNAi. Our results suggest that PAB-1 positively regulates the mRNA levels of germline genes, which is likely facilitated by the interaction of PAB-1 with other P-body components, CGH-1 and CAR-1.     

Cutting edge: a potent adjuvant effect of ligand to receptor activator of NF-kappa B gene for inducing antigen-specific CD8+ T cell response by DNA and viral vector vaccination

The ligand to receptor activator of NF-kappaB (RANK-L)/RANK interaction has been implicated in CD40 ligand/CD40-independent T cell priming by dendritic cells. In this report, we show that the coadministration of the RANK-L gene with a Trypanosoma cruzi gene markedly enhances the induction of Trypanosoma Ag-specific CD8(+) T cells and improves the DNA vaccine efficacy. A similarly potent adjuvant effect of the RANK-L gene on the induction of Ag-specific CD8(+) T cells was also observed when recombinant influenza virus expressing murine malaria Ag was used as an immunogen. In contrast, the coadministration of the CD40L gene was not effective in these systems. Our results demonstrated, for the first time, the potent immunostimulatory effect of the RANK-L gene to improve the CD8(+) T cell-mediated immunity against infectious agents.     

Engineering tolerance and accumulation of lead and cadmium in transgenic plants

We have studied the utility of the yeast protein YCF1, which detoxifies cadmium by transporting it into vacuoles, for the remediation of lead and cadmium contamination. We found that the yeast YCF1-deletion mutant DTY167 was hypersensitive to Pb(II) as compared with wild-type yeast. DTY167 cells overexpressing YCF1 were more resistant to Pb(II) and Cd(II) than were wild-type cells, and accumulated more lead and cadmium. Analysis of transgenic Arabidopsis thaliana plants overexpressing YCF1 showed that YCF1 is functionally active and that the plants have enhanced tolerance of Pb(II) and Cd(II) and accumulated greater amounts of these metals. These results suggest that transgenic plants expressing YCF1 may be useful for phytoremediation of lead and cadmium.     

Efficacy of poly (ADP-ribose) polymerase inhibitor olaparib against head and neck cancer cells: Predictions of drug sensitivity based on PAR–p53–NF-κB interactions

Poly (ADP-ribose) polymerase (PARP) is a key molecule in the DNA damage response (DDR), which is a major target of both chemotherapies and radiotherapies. PARP inhibitors therefore comprise a promising class of anticancer therapeutics. In this study, we evaluated the efficacy of the PARP inhibitor olaparib, and also sought to identify the mechanism and predictive marker associated with olaparib sensitivity in head and neck cancer (HNC) cells. A total of 15 HNC cell lines, including AMC HNC cells, were tested. AMC-HN3 and HN4 exhibited stronger responses to olaparib. Among cisplatin-resistant cell lines, only AMC HN9-cisR cells were significantly suppressed by olaparib. We found that basal poly (ADP-ribose) (PAR) levels, but not PARP-1 levels, correlated with olaparib sensitivity. AMC-HN3 and HN4 cells exhibited higher basal levels of NF-κB that decreased significantly after olaparib treatment. In contrast, apoptotic proteins were intrinsically expressed in AMC-HN9-cisR cells. As interference with p53 expression led to NF-κB reactivation, we concluded that elevated basal PAR and NF-κB levels are predictive of olaparib responsiveness in HNC cells; in addition, olaparib inhibits HNC cells via PAR–p53–NF-κB interactions.