152 research outputs found

    Mapping conformational changes of a type IIb Na+/Pi cotransporter by voltage clamp fluorometry.

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    The fluorescence of a fluorophore depends on its environment, and if attached to a protein it may report on conformational changes. We have combined two-electrode voltage clamp with simultaneous fluorescence measurements to detect conformational changes in a type IIb Na(+)/P(i) cotransporter expressed in Xenopus oocytes. Four novel Cys labelled with a fluorescent probe yielded voltage-and substrate-dependent changes in fluorescence (F). Neither Cys-substitution nor labelling significantly altered the mutants' electrogenic properties. Different F responses to voltage and substrate were recorded at the four sites. S155C, located in an intracellular re-entrant loop in the first half of the protein, and E451C, located in an extracellular re-entrant loop in the second half of the protein both showed Na(+), Li(+)- and P(i)-dependent F signals. S226C and Q319C, located at opposite ends of a large extracellular loop in the middle of the protein, mainly responded to changes in Na(+) and Li(+). Hyperpolarization increased F for S155C and S226C, but decreased F for Q319C and E451C. The labelling and F response of S155C, confirmed that the intracellular loop containing Ser-155 is re-entrant as it is accessible from the extracellular milieu. The behavior of S155C and E451C indicates a strong involvement of the two re-entrant loops in conformational changes during the transport cycle. Moreover, the data for S226C and Q319C suggest that also the large extracellular loop is associated with transport function. Finally, the reciprocal voltage-dependencies of the S155C-E451C and S226C-Q319C pairs suggest reciprocal conformational changes during the transport cycle for their respective local environments

    Temperature dependence of steady-state and presteady-state kinetics of a type IIb Na+/Pi cotransporter

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    The temperature dependence of the transport kinetics of flounder Na(+)-coupled inorganic phosphate (P(i)) cotransporters (NaPi-IIb) expressed in Xenopus oocytes was investigated using radiotracer and electrophysiological assays. (32)P(i) uptake was strongly temperature-dependent and decreased by approximately 80% at a temperature change from 25 degrees C to 5 degrees C. The corresponding activation energy (E (a)) was approximately 14 kcal mol(-1) for the cotransport mode. The temperature dependence of the cotransport and leak modes was determined from electrogenic responses to 1 mM P(i) and phosphonoformic acid (PFA), respectively, under voltage clamp. The magnitude of the P(i)- and PFA-induced changes in holding current decreased with temperature. E (a) at -100 mV for the cotransport and leak modes was approximately 16 kcal mol(-1) and approximately 11 kcal mol(-1), respectively, which suggested that the leak is mediated by a carrier, rather than a channel, mechanism. Moreover, E (a) for cotransport was voltage-independent, suggesting that a major conformational change in the transport cycle is electroneutral. To identify partial reactions that confer temperature dependence, we acquired presteady-state currents at different temperatures with 0 mM P(i) over a range of external Na(+). The relaxation time constants increased, and the peak time constant shifted toward more positive potentials with decreasing temperature. Likewise, there was a depolarizing shift of the charge distribution, whereas the total available charge and apparent valency predicted from single Boltzmann fits were temperature-independent. These effects were explained by an increased temperature sensitivity of the Na(+)-debinding rate compared with the other voltage-dependent rate constants

    Diminished salivary epidermal growth factor secretion : a link between Sjogren's syndrome and autoimmune gastritis?

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    Objectives: Healthy human labial salivary glands produce epidermal growth factor (EGF). In Sjogren's syndrome (SS), EGF staining is diminished. SS is also associated with chronic autoimmune corpus gastritis. We therefore hypothesized that EGF secretion would be diminished in SS and that this could affect gastric target cells.Methods: Salivary EGF secretion in SS was compared to that in healthy controls using an enzyme-linked immunosorbent assay (ELISA). EGF receptor (EGFR) immunoreactive cells in the gastric corpus of healthy human subjects were analysed using immunostaining.Results: Salivary secretion of EGF was diminished in SS patients (232.4, range 52.6-618.4, vs. 756.6, range 105.3-1631.6 pg/min, p=0.002). Proton-pump positive parietal cells were mostly EGFR immunoreactive whereas very few pepsinogen I (PGI)-positive cells were EGFR positive.Conclusions: As EGF is relatively acid resistant, salivary gland-derived EGF might participate in an exo/endocrine mode of parietal cell maintenance in the gastric corpus. Deficiency of salivary gland-derived EGF in SS patients may cause impairment of gastric parietal cells resulting in exposure of immunogenic cryptic antigens and loss of immunological self-tolerance.Peer reviewe

    Diminished salivary epidermal growth factor secretion : a link between Sjogren's syndrome and autoimmune gastritis?

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    Objectives: Healthy human labial salivary glands produce epidermal growth factor (EGF). In Sjogren's syndrome (SS), EGF staining is diminished. SS is also associated with chronic autoimmune corpus gastritis. We therefore hypothesized that EGF secretion would be diminished in SS and that this could affect gastric target cells.Methods: Salivary EGF secretion in SS was compared to that in healthy controls using an enzyme-linked immunosorbent assay (ELISA). EGF receptor (EGFR) immunoreactive cells in the gastric corpus of healthy human subjects were analysed using immunostaining.Results: Salivary secretion of EGF was diminished in SS patients (232.4, range 52.6-618.4, vs. 756.6, range 105.3-1631.6 pg/min, p=0.002). Proton-pump positive parietal cells were mostly EGFR immunoreactive whereas very few pepsinogen I (PGI)-positive cells were EGFR positive.Conclusions: As EGF is relatively acid resistant, salivary gland-derived EGF might participate in an exo/endocrine mode of parietal cell maintenance in the gastric corpus. Deficiency of salivary gland-derived EGF in SS patients may cause impairment of gastric parietal cells resulting in exposure of immunogenic cryptic antigens and loss of immunological self-tolerance.Peer reviewe

    Shape model and spin-state analysis of PHA contact binary (85990) 1999 JV6 from combined radar and optical observations

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    Context. The potentially hazardous asteroid (85990)1999 JV6 has been a target of previously published thermal-infrared observations and optical photometry. It has been identified as a promising candidate for possible Yarkovsky-O’Keefe-Radzievskii-Paddack (YORP) effect detection. Aims. The YORP effect is a small thermal-radiation torque considered to be a key factor in spin-state evolution of small Solar System bodies. In order to detect YORP on 1999 JV6 we developed a detailed shape model and analysed the spin-state using both optical and radar observations. Methods. For 1999 JV6, we collected optical photometry between 2007 and 2016. Additionally, we obtained radar echo-power spectra and imaging observations with Arecibo and Goldstone planetary radar facilities in 2015, 2016, and 2017. We combined our data with published optical photometry to develop a robust physical model. Results. We determine that the rotation pole resides at negative latitudes in an area with a 5◦ radius close to the south ecliptic pole. The refined sidereal rotation period is 6.536787±0.000007 h. The radar images are best reproduced with a bilobed shape model. Both lobes of 1999 JV6 can be represented as oblate ellipsoids with a smaller, more spherical component resting at the end of a larger, more elongated component. While contact binaries appear to be abundant in the near-Earth population, there are only a few published shape models for asteroids in this particular configuration. By combining the radar-derived shape model with optical light curves we determine a constant-period solution that fits all available data well. Using light-curve data alone we determine an upper limit for YORP of 8.5×10−8 rad day−2. Conclusions. The bifurcated shape of 1999 JV6 might be a result of two ellipsoidal components gently merging with each other, or a deformation of a rubble pile with a weak-tensile-strength core due to spin-up. The physical model of 1999 JV6 presented here will enable future studies of contact binary asteroid formation and evolution

    The receptors for gibbon ape leukemia virus and amphotropic murine leukemia virus are not downregulated in productively infected cells

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    <p>Abstract</p> <p>Background</p> <p>Over the last several decades it has been noted, using a variety of different methods, that cells infected by a specific gammaretrovirus are resistant to infection by other retroviruses that employ the same receptor; a phenomenon termed receptor interference. Receptor masking is thought to provide an earlier means of blocking superinfection, whereas receptor down regulation is generally considered to occur in chronically infected cells.</p> <p>Results</p> <p>We used replication-competent GFP-expressing viruses containing either an amphotropic murine leukemia virus (A-MLV) or the gibbon ape leukemia virus (GALV) envelope. We also constructed similar viruses containing fluorescence-labeled Gag proteins for the detection of viral particles. Using this repertoire of reagents together with a wide range of antibodies, we were able to determine the presence and availability of viral receptors, and detect viral envelope proteins and particles presence on the cell surface of chronically infected cells.</p> <p>Conclusions</p> <p>A-MLV or GALV receptors remain on the surface of chronically infected cells and are detectable by respective antibodies, indicating that these receptors are not downregulated in these infected cells as previously proposed. We were also able to detect viral envelope proteins on the infected cell surface and infected cells are unable to bind soluble A-MLV or GALV envelopes indicating that receptor binding sites are masked by endogenously expressed A-MLV or GALV viral envelope. However, receptor masking does not completely prevent A-MLV or GALV superinfection.</p

    Poor correlation between large-scale environmental flow violations and freshwater biodiversity: implications for water resource management and the freshwater planetary boundary

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    The freshwater ecosystems around the world are degrading, such that maintaining environmental flow1 (EF) in river networks is critical to their preservation. The relationship between streamflow alterations (subsequent EF violations2) and the freshwater biodiversity response is well established at the scale of stream reaches or small basins (∼&lt;100 km2). However, it is unclear if this relationship is robust at larger scales, even though there are large-scale initiatives to legalize the EF requirement. Moreover, EFs have been used in assessing a planetary boundary3 for freshwater. Therefore, this study intends to conduct an exploratory evaluation of the relationship between EF violation and freshwater biodiversity at globally aggregated scales and for freshwater ecoregions. Four EF violation indices (severity, frequency, probability of shifting to a violated state, and probability of staying violated) and seven independent freshwater biodiversity indicators (calculated from observed biota data) were used for correlation analysis. No statistically significant negative relationship between EF violation and freshwater biodiversity was found at global or ecoregion scales. These findings imply the need for a holistic bio-geo-hydro-physical approach in determining the environmental flows. While our results thus suggest that streamflow and EF may not be the only determinant of freshwater biodiversity at large scales, they do not preclude the existence of relationships at smaller scales or with more holistic EF methods (e.g., including water temperature, water quality, intermittency, connectivity, etc.) or with other biodiversity data or metrics.</p
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