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

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

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?

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

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

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

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

<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

Comparative functional analysis of aquaporins/glyceroporins in mammals and anurans

Maintenance of fluid homeostasis is critical to establishing and maintaining normal physiology. The landmark discovery of membrane water channels (aquaporins; AQPs) ushered in a new area in osmoregulatory biology that has drawn from and contributed to diverse branches of biology, from molecular biology and genomics to systems biology and evolution, and from microbial and plant biology to animal and translational physiology. As a result, the study of AQPs provides a unique and integrated backdrop for exploring the relationships between genes and genome systems, the regulation of gene expression, and the physiologic consequences of genetic variation. The wide species distribution of AQP family members and the evolutionary conservation of the family indicate that the control of membrane water flux is a critical biological process. AQP function and regulation is proving to be central to many of the pathways involved in individual physiologic systems in both mammals and anurans. In mammals, AQPs are essential to normal secretory and absorptive functions of the eye, lung, salivary gland, sweat glands, gastrointestinal tract, and kidney. In urinary, respiratory, and gastrointestinal systems, AQPs are required for proper urine concentration, fluid reabsorption, and glandular secretions. In anurans, AQPs are important in mediating physiologic responses to changes in the external environment, including those that occur during metamorphosis and adaptation from an aquatic to terrestrial environment and thermal acclimation in anticipation of freezing. Therefore, an understanding of AQP function and regulation is an important aspect of an integrated approach to basic biological research

Anastrozole versus tamoxifen for the prevention of locoregional and contralateral breast cancer in postmenopausal women with locally excised ductal carcinoma in situ (IBIS-II DCIS): a double-blind, randomised controlled trial

Background Third-generation aromatase inhibitors are more effective than tamoxifen for preventing recurrence in postmenopausal women with hormone-receptor-positive invasive breast cancer. However, it is not known whether anastrozole is more effective than tamoxifen for women with hormone-receptor-positive ductal carcinoma in situ (DCIS). Here, we compare the efficacy of anastrozole with that of tamoxifen in postmenopausal women with hormone-receptor-positive DCIS. Methods In a double-blind, multicentre, randomised placebo-controlled trial, we recruited women who had been diagnosed with locally excised, hormone-receptor-positive DCIS. Eligible women were randomly assigned in a 1:1 ratio by central computer allocation to receive 1 mg oral anastrozole or 20 mg oral tamoxifen every day for 5 years. Randomisation was stratified by major centre or hub and was done in blocks (six, eight, or ten). All trial personnel, participants, and clinicians were masked to treatment allocation and only the trial statistician had access to treatment allocation. The primary endpoint was all recurrence, including recurrent DCIS and new contralateral tumours. All analyses were done on a modified intention-to-treat basis (in all women who were randomised and did not revoke consent for their data to be included) and proportional hazard models were used to compute hazard ratios and corresponding confidence intervals. This trial is registered at the ISRCTN registry, number ISRCTN37546358. Results Between March 3, 2003, and Feb 8, 2012, we enrolled 2980 postmenopausal women from 236 centres in 14 countries and randomly assigned them to receive anastrozole (1449 analysed) or tamoxifen (1489 analysed). Median follow-up was 7·2 years (IQR 5·6–8·9), and 144 breast cancer recurrences were recorded. We noted no statistically significant difference in overall recurrence (67 recurrences for anastrozole vs 77 for tamoxifen; HR 0·89 [95% CI 0·64–1·23]). The non-inferiority of anastrozole was established (upper 95% CI <1·25), but its superiority to tamoxifen was not (p=0·49). A total of 69 deaths were recorded (33 for anastrozole vs 36 for tamoxifen; HR 0·93 [95% CI 0·58–1·50], p=0·78), and no specific cause was more common in one group than the other. The number of women reporting any adverse event was similar between anastrozole (1323 women, 91%) and tamoxifen (1379 women, 93%); the side-effect profiles of the two drugs differed, with more fractures, musculoskeletal events, hypercholesterolaemia, and strokes with anastrozole and more muscle spasm, gynaecological cancers and symptoms, vasomotor symptoms, and deep vein thromboses with tamoxifen. Conclusions No clear efficacy differences were seen between the two treatments. Anastrozole offers another treatment option for postmenopausal women with hormone-receptor-positive DCIS, which may be be more appropriate for some women with contraindications for tamoxifen. Longer follow-up will be necessary to fully evaluate treatment differences