Regulated RalBP1 Binding to RalA and PSD-95 Controls AMPA Receptor Endocytosis and LTD

Long-term depression (LTD) is a long-lasting activity-dependent decrease in synaptic strength. NMDA receptor (NMDAR)–dependent LTD, an extensively studied form of LTD, involves the endocytosis of AMPA receptors (AMPARs) via protein dephosphorylation, but the underlying mechanism has remained unclear. We show here that a regulated interaction of the endocytic adaptor RalBP1 with two synaptic proteins, the small GTPase RalA and the postsynaptic scaffolding protein PSD-95, controls NMDAR-dependent AMPAR endocytosis during LTD. NMDAR activation stimulates RalA, which binds and translocates widespread RalBP1 to synapses. In addition, NMDAR activation dephosphorylates RalBP1, promoting the interaction of RalBP1 with PSD-95. These two regulated interactions are required for NMDAR-dependent AMPAR endocytosis and LTD and are sufficient to induce AMPAR endocytosis in the absence of NMDAR activation. RalA in the basal state, however, maintains surface AMPARs. We propose that NMDAR activation brings RalBP1 close to PSD-95 to promote the interaction of RalBP1-associated endocytic proteins with PSD-95-associated AMPARs. This suggests that scaffolding proteins at specialized cellular junctions can switch their function from maintenance to endocytosis of interacting membrane proteins in a regulated manner.     

Repair activities of human 8-oxoguanine DNA glycosylase are stimulated by the interaction with human checkpoint sensor Rad9–Rad1–Hus1 complex

Rad9–Rad1–Hus1 (9–1–1) is a checkpoint protein complex playing roles in DNA damage sensing, cell cycle arrest, DNA repair or apoptosis. Human 8-oxoguanine DNA glycosylase (hOGG1) is the major DNA glycosylase responsible for repairing a specific aberrantly oxidized nucleotide, 7,8-dihydro-8-oxoguanine (8-oxoG). In this study, we identified a novel interaction between hOGG1 and human 9–1–1, and investigated the functional consequences of this interaction. Co-immunoprecipitation assays using transiently transfected HEK293 cells demonstrated an interaction between hOGG1 and the 9–1–1 proteins. Subsequently, GST pull-down assays using bacterially expressed and purified hOGG1-His and GST-fused 9–1–1 subunits (GST-hRad9, GST-hRad1, and GST-hHus1) demonstrated that hOGG1 interacted directly with the individual subunits of the human 9–1–1 complex. In vitro excision assay, which employed a DNA duplex containing an 8-oxoG/C mismatch, showed that hRad9, hRad1, and hHus1 enhanced the 8-oxoG excision and β-elimination activities of hOGG1. In addition, the presence of hRad9, hRad1, and hHus1 enhanced the formation of covalently cross-linked hOGG1–8-oxoG/C duplex complexes, as determined by a trapping assay using NaBH₄. A trimeric human 9–1–1 complex was purified from Escherichia coli cell transformed with hRad9, His-fused hRad1, or His-fused hHus1 expressing vectors. It also showed the similar activity to enhance in vitro hOGG1 glycosylase activity, compared with individual human 9–1–1 subunits. Detection of 8-oxoG in HEK293 cells using flow cytometric and spectrofluorometric analysis revealed that over-expression of hOGG1 or human 9–1–1 reduced the formation of 8-oxoG residues following the H₂O₂ treatment. The highest 8-oxoG reduction was observed in HEK293 cells over-expressing hOGG1 and all the three subunits of human 9–1–1. These indicate that individual human 9–1–1 subunits and human 9–1–1 complex showed almost the same abilities to enhance the in vitro 8-oxoG excision activity of hOGG1, but that the greatest effect to remove 8-oxoG residues in H₂O₂-treated cells was derived from the 9–1–1 complex as a whole.     

Piezoelectric biosensor using olfactory receptor protein expressed in Escherichia coli

An olfactory receptor protein of C. elegans, ODR-10, was expressed in Escherichia coli as a fusion protein, with GST and 6x His-tag. The expression of the target protein was analyzed by SDS-PAGE and Western blot, and was confirmed to be expressed at the membrane fraction of the host E. coli. The surface of a quartz crystal microbalance (QCM) was coated with crude membrane extracts, containing the expressed receptor protein, and the interaction between the olfactory receptor and various odorant molecules examined. Compared with other odorants, diacetyl (2,3-butanedione), known as a natural ligand for the ODR-10 receptor, interacted most strongly with the expressed protein. Various concentrations of diacetyl were applied to the expressed ODR-10 receptor, and the response of the QCM showed a linear relationship to the logarithmic value of the odorant concentration. This piezoelectric biosensor system, using olfactory receptor proteins expressed in E. coli, can be used in diagnostics, toxic chemical detection and the quality control of food.     

Exocytosis unbound

The accepted theory of vesicular release of neurotransmitter posits that only a single vesicle per synapse can fuse with the membrane following action potential invasion, and this exocytotic event is limited to the ultrastructurally defined presynaptic active zone. Neither of these dictums is universally true. At certain synapses, more than a single vesicle can be released per action potential, and there is growing evidence that neuronal exocytosis can occur from sites that are unremarkable in electron micrographs. The first discrepancy extends the dynamic range of synapses, whereas the second enables faster and more robust chemical transmission at sites distant from morphologically defined synapses. Taken together, these attributes expand the capabilities of cellular communication in the nervous system.     

Specificity of the myotubularin family of phosphatidylinositol-3-phosphatase is determined by the PH/GRAM domain

Myotubularins (MTM) are a large subfamily of lipid phosphatases that specifically dephosphorylate at the D3 position of phosphatidylinositol 3-phosphate (PI(3)P) in PI(3)P and PI(3,5)P2. We recently found that MTMR6 specifically inhibits the Ca2+-activated K+ channel, KCa3.1, by dephosphorylating PI(3)P. We now show that inhibition is specific for MTMR6 and other MTMs do not inhibit KCa3.1. By replacing either or both of the coiled-coil (CC) and pleckstrin homology/GRAM (PH/G) domains of MTMs that failed to inhibit KCa3.1 with the CC and PH/G domains of MTMR6, we found that chimeric MTMs containing both the MTMR6 CC and PH/G domains functioned like MTMR6 to inhibit KCa3.1 channel activity, whereas chimeric MTMs containing either domain alone did not. Immunofluorescent microscopy demonstrated that both the MTMR6 CC and PH/G domains are required to co-localize MTMR6 to the plasma membrane with KCa3.1. These findings support a model in which two specific low affinity interactions are required to co-localize MTMR6 with KCa3.1: 1) between the CC domains on MTMR6 and KCa3.1 and (2) between the PH/G domain and a component of the plasma membrane. Our inability to detect significant interaction of the MTMR6 G/PH domain with phosphoinositides suggests that this domain may bind a protein. Identifying the specific binding partners of the CC and PH/G domains on other MTMs will provide important clues to the specific functions regulated by other MTMs as well as the mechanism(s) whereby loss of some MTMs lead to disease.     

Plasma leptin response to oral glucose tolerance and fasting/re-feeding tests in rats with fructose-induced metabolic derangements

The aim of this study was to compare the postprandial leptin response in rats with and without metabolic syndrome induced by a fructose-enriched diet. The effect of aging and the association between variations in metabolic substrates was also evaluated. Oral glucose tolerance test (OGTT) and fasting/re-feeding test were used to evaluate the responses of leptin and to explore the dynamic relationship between endogenous leptin and metabolic substrates, including glucose, insulin and triglycerides (TG). At the 7th week, plasma leptin was unchanged in control rats after oral glucose loading. However, plasma leptin levels increased in fructose-fed rats with insulin resistant OGTT curves. At the 11th month, plasma leptin level was reduced during starvation and returned to the level prior to starvation during re-feeding in control rats. In contrast, the starvation-induced reduction in leptin showed a potentially larger rebound effect during re-feeding in fructose-fed rats. Analysis of covariance demonstrated that there alone was no interactive effect of dietary manipulation between leptin and TG, suggesting that fructose diet-induced insulin resistance-related metabolic syndrome may concomitantly elevate leptin and TG. Furthermore, multiple regression analysis suggests TG was the primary correlative determinant of endogenous leptin concentration. Our data showed that there are different patterns of leptin response to OGTT and fasting/re-feeding tests in rats with and without metabolic syndrome. The results suggest that these effects may be related to a TG-mediated impairment of leptin function and a protective mechanism to reduce lipid-induced tissue damage in patients with metabolic syndrome.     

Manipulation of light and CO2 environments of the primary leaves of bean (Phaseolus vulgaris L.) affects photosynthesis in both the primary and the first trifoliate leaves: involvement of systemic regulation.

Possible involvement of systemic regulation of the photosynthetic properties of young leaves by the local environments and/or photosynthate production of the mature leaves were examined using Phaseolus vulgaris plants. When primary leaves (PLs) were treated with air containing 150 microL CO2 L(-1) with the other plant parts in ambient air at a photosynthetic photon flux density (PPFD) of 300 micromol photon m(-2) s(-1), decreases in the photosynthetic rate measured at 360 microL CO2 L(-1) and a PPFD of 300 micromol photon m(-2) s(-1) (A360) were markedly retarded in both PLs and the first trifoliate leaves (TLs) as compared to plants treated with 400 microL CO2 L(-1). Conversely, when PLs were treated with 1000 microL CO2 L(-1), decreases in A360 were accelerated in both PLs and TLs. Shading of PLs accelerated the decrease in PL A360, and delayed the decrease in TLs. In the CO2 treatments, changes in A360 in TLs were mainly attributed to the changes in ribulose bisphosphate (RuBP) carboxylation rate, while the shading of PLs caused increases in both the RuBP carboxylation and regeneration rates in TLs. The ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco) activity on chlorophyll basis, an indicator of sun/shade acclimation, differed both among PLs and among TLs in accordance with the redox state of photosystem II (PSII) in PLs. Although carbohydrate contents of TLs were not affected by any manipulation of PLs, changes in the photosynthetic capacities of TLs acted to compensate for changes in PL photosynthesis. These results clearly indicate that the CO2 and shade treatments of PLs not only affect photosynthetic properties of the PLs themselves, but also systemically affected the photosynthetic properties of TLs. Possible roles of the redox state and photosynthate concentration in PLs in regulation of photosynthesis in PLs and TLs are discussed.     

Lichens of Barla Mountain in Isparta,Turkey: Diversity study and ecological assessment of the area

This study reports on 230 infrageneric lichenized and lichenicolous taxa from Barla Mountain, Isparta, Turkey and assesses the ecological features of the area using the distribution of the lichens in the region and their poleophoby and solar irradiation ecological indicator values. One lichenized fungus, Protoblastenia terricola, and one lichenicolous fungus, Zwackhiomyces dispersus, are recorded as new in Turkey and 194 taxa are reported for the first time from Barla Mountain. After this research, number of the lichenized and lichenicolous fungi taxa of Barla Mountain rose to 241. Based on assessments using the ecological indicator values, the area is dominated by natural or semi-natural and well-preserved habitats.     

The role of the saliva antioxidant barrier to reactive oxygen species with regard to caries development

Objectives: The aim of this study was to evaluate the role of the antioxidant barrier in the saliva of children with caries, and its impact on the colonization of cariogenic bacteria.

Methods: This is a cross-sectional study of 81 children aged 1–5 years. Antioxidant levels and salivary bacterial profiles were measured. Patients were divided into two groups as follows: initial stage decay, termed non-cavitated (1–2 in International Caries Detection and Assessment System (ICDAS)), and extensive decay, termed cavitated lesions (5–6 in ICDAS). The control group includes children without caries.

Results: The linear regression model demonstrated that the GSH, GSSG, GSH/GSSG, and total antioxidant capacity levels are influenced (P?P?Discussion: Our results indicate that the high levels of antioxidants in saliva increase significantly in children in line with the salivary cariogenic bacterial profiles and caries progression.