P2X receptors: epithelial ion channels and regulators of salt and water transport.

When the results from electrophysiological studies of renal epithelial cells are combined with data from in vivo tubule microperfusion experiments and immunohistochemical surveys of the nephron, the accumulated evidence suggests that ATP-gated ion channels, P2X receptors, play a specialized role in the regulation of ion and water movement across the renal tubule and are integral to electrolyte and fluid homeostasis. In this short review, we discuss the concept of P2X receptors as regulators of salt and water salvage pathways, as well as acknowledging their accepted role as ATP-gated ion channels

Altered urothelial ATP signaling in a major subset of human overactive bladder patients with pyuria

Overactive Bladder (OAB) is an idiopathic condition, characterized by urgency, urinary frequency, and urgency incontinence, in the absence of routinely traceable urinary infection. We have described microscopic pyuria (≥10 wbc/μl) in patients suffering from the worst symptoms. It is established that inflammation is associated with increased ATP release from epithelial cells, and extracellular ATP originating from the urothelium following increased hydrostatic pressure is a mediator of bladder sensation. Here, using bladder biopsy samples, we have investigated urothelial ATP signaling in OAB patients with microscopic pyuria. Basal, but not stretch-evoked, release of ATP was significantly greater from the urothelium of OAB patients with pyuria than from non-OAB patients or OAB patients without pyuria (<10 wbc/μl). Basal ATP release from the urothelium of OAB patients with pyuria was inhibited by the P2 receptor antagonist suramin and abolished by the hemichannel blocker carbenoxolone, which differed from stretch-activated ATP release. Altered P2 receptor expression was evident in the urothelium from pyuric OAB patients. Furthermore, intracellular bacteria were visualized in shed urothelial cells from ∼80% of OAB patients with pyuria. These data suggest that increased ATP release from the urothelium, involving bacterial colonization, may play a role in the heightened symptoms associated with pyuric OAB patients

Pharmacological Properties and Physiological Function of a P2X-Like Current in Single Proximal Tubule Cells Isolated from Frog Kidney

Although previous studies have provided evidence for the expression of P2X receptors in renal proximal tubule, only one cell line study has provided functional evidence. The current study investigated the pharmacological properties and physiological role of native P2X-like currents in single frog proximal tubule cells using the whole-cell patch-clamp technique. Extracellular ATP activated a cation conductance (P2Xf) that was also Ca2+-permeable. The agonist sequence for activation was ATP = αβ-MeATP > BzATP = 2-MeSATP, and P2Xf was inhibited by suramin, PPADS and TNP-ATP. Activation of P2Xf attenuated the rundown of a quinidine-sensitive K+ conductance, suggesting that P2Xf plays a role in K+ channel regulation. In addition, ATP/ADP apyrase and inhibitors of P2Xf inhibited regulatory volume decrease (RVD). These data are consistent with the presence of a P2X receptor that plays a role in the regulation of cell volume and K+ channels in frog renal proximal tubule cells

Multidimensional prognostic indices for use in COPD patient care. A systematic review

Contains fulltext : 98117.pdf (publisher's version ) (Open Access)BACKGROUND: A growing number of prognostic indices for chronic obstructive pulmonary disease (COPD) is developed for clinical use. Our aim is to identify, summarize and compare all published prognostic COPD indices, and to discuss their performance, usefulness and implementation in daily practice. METHODS: We performed a systematic literature search in both Pubmed and Embase up to September 2010. Selection criteria included primary publications of indices developed for stable COPD patients, that predict future outcome by a multidimensional scoring system, developed for and validated with COPD patients only. Two reviewers independently assessed the index quality using a structured screening form for systematically scoring prognostic studies. RESULTS: Of 7,028 articles screened, 13 studies comprising 15 indices were included. Only 1 index had been explored for its application in daily practice. We observed 21 different predictors and 7 prognostic outcomes, the latter reflecting mortality, hospitalization and exacerbation. Consistent strong predictors were FEV1 percentage predicted, age and dyspnoea. The quality of the studies underlying the indices varied between fairly poor and good. Statistical methods to assess the predictive abilities of the indices were heterogenic. They generally revealed moderate to good discrimination, when measured. Limitations: We focused on prognostic indices for stable disease only and, inevitably, quality judgment was prone to subjectivity. CONCLUSIONS: We identified 15 prognostic COPD indices. Although the prognostic performance of some of the indices has been validated, they all lack sufficient evidence for implementation. Whether or not the use of prognostic indices improves COPD disease management or patients' health is currently unknown; impact studies are required to establish this

Identification of a Polycystin-1 Cleavage Product, P100, That Regulates Store Operated Ca2+ Entry through Interactions with STIM1

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a genetic disorder resulting in large kidney cysts and eventual kidney failure. Mutations in either the PKD1 or PKD2/TRPP2 genes and their respective protein products, polycystin-1 (PC1) and polycystin-2 (PC2) result in ADPKD. PC2 is known to function as a non-selective cation channel, but PC1's function and the function of PC1 cleavage products are not well understood. Here we identify an endogenous PC1 cleavage product, P100, a 100 kDa fragment found in both wild type and epitope tagged PKD1 knock-in mice. Expression of full length human PC1 (FL PC1) and the resulting P100 and C-Terminal Fragment (CTF) cleavage products in both MDCK and CHO cells significantly reduces the store operated Ca2+ entry (SOCE) resulting from thapsigargin induced store depletion. Exploration into the roles of P100 and CTF in SOCE inhibition reveal that P100, when expressed in Xenopus laevis oocytes, directly inhibits the SOCE currents but CTF does not, nor does P100 when containing the disease causing R4227X mutation. Interestingly, we also found that in PC1 expressing MDCK cells, translocation of the ER Ca2+ sensor protein STIM1 to the cell periphery was significantly altered. In addition, P100 Co-immunoprecipitates with STIM1 but CTF does not. The expression of P100 in CHO cells recapitulates the STIM1 translocation inhibition seen with FL PC1. These data describe a novel polycystin-1 cleavage product, P100, which functions to reduce SOCE via direct inhibition of STIM1 translocation; a function with consequences for ADPKD

Molecular Variation at the SLC6A3 Locus Predicts Lifetime Risk of PTSD in the Detroit Neighborhood Health Study

Recent work suggests that the 9-repeat (9R) allele located in the 3′UTR VNTR of the SLC6A3 gene increases risk of posttraumatic stress disorder (PTSD). However, no study reporting this association to date has been based on population-based samples. Furthermore, no study of which we are aware has assessed the joint action of genetic and DNA methylation variation at SLC6A3 on risk of PTSD. In this study, we assessed whether molecular variation at SLC6A3 locus influences risk of PTSD. Participants (n = 320; 62 cases/258 controls) were drawn from an urban, community-based sample of predominantly African American Detroit adult residents, and included those who had completed a baseline telephone survey, had provided blood specimens, and had a homozygous genotype for either the 9R or 10R allele or a heterozygous 9R/10R genotype. The influence of DNA methylation variation in the SLC6A3 promoter locus was also assessed in a subset of participants with available methylation data (n = 83; 16 cases/67 controls). In the full analytic sample, 9R allele carriers had almost double the risk of lifetime PTSD compared to 10R/10R genotype carriers (OR = 1.98, 95% CI = 1.02–3.86), controlling for age, sex, race, socioeconomic status, number of traumas, smoking, and lifetime depression. In the subsample of participants with available methylation data, a significant (p = 0.008) interaction was observed whereby 9R allele carriers showed an increased risk of lifetime PTSD only in conjunction with high methylation in the SLC6A3 promoter locus, controlling for the same covariates. Our results confirm previous reports supporting a role for the 9R allele in increasing susceptibility to PTSD. They further extend these findings by providing preliminary evidence that a “double hit” model, including both a putatively reduced-function allele and high methylation in the promoter region, may more accurately capture molecular risk of PTSD at the SLC6A3 locus

Probing the Flexibility of Large Conformational Changes in Protein Structures through Local Perturbations

Protein conformational changes and dynamic behavior are fundamental for such processes as catalysis, regulation, and substrate recognition. Although protein dynamics have been successfully explored in computer simulation, there is an intermediate-scale of motions that has proven difficult to simulate—the motion of individual segments or domains that move independently of the body the protein. Here, we introduce a molecular-dynamics perturbation method, the Rotamerically Induced Perturbation (RIP), which can generate large, coherent motions of structural elements in picoseconds by applying large torsional perturbations to individual sidechains. Despite the large-scale motions, secondary structure elements remain intact without the need for applying backbone positional restraints. Owing to its computational efficiency, RIP can be applied to every residue in a protein, producing a global map of deformability. This map is remarkably sparse, with the dominant sites of deformation generally found on the protein surface. The global map can be used to identify loops and helices that are less tightly bound to the protein and thus are likely sites of dynamic modulation that may have important functional consequences. Additionally, they identify individual residues that have the potential to drive large-scale coherent conformational change. Applying RIP to two well-studied proteins, Dihdydrofolate Reductase and Triosephosphate Isomerase, which possess functionally-relevant mobile loops that fluctuate on the microsecond/millisecond timescale, the RIP deformation map identifies and recapitulates the flexibility of these elements. In contrast, the RIP deformation map of α-lytic protease, a kinetically stable protein, results in a map with no significant deformations. In the N-terminal domain of HSP90, the RIP deformation map clearly identifies the ligand-binding lid as a highly flexible region capable of large conformational changes. In the Estrogen Receptor ligand-binding domain, the RIP deformation map is quite sparse except for one large conformational change involving Helix-12, which is the structural element that allosterically links ligand binding to receptor activation. RIP analysis has the potential to discover sites of functional conformational changes and the linchpin residues critical in determining these conformational states

Predicting volume of distribution with decision tree-based regression methods using predicted tissue:plasma partition coefficients

Background: Volume of distribution is an important pharmacokinetic property that indicates the extent of a drug's distribution in the body tissues. This paper addresses the problem of how to estimate the apparent volume of distribution at steady state (Vss) of chemical compounds in the human body using decision tree-based regression methods from the area of data mining (or machine learning). Hence, the pros and cons of several different types of decision tree-based regression methods have been discussed. The regression methods predict Vss using, as predictive features, both the compounds' molecular descriptors and the compounds' tissue:plasma partition coefficients (Kt:p) - often used in physiologically-based pharmacokinetics. Therefore, this work has assessed whether the data mining-based prediction of Vss can be made more accurate by using as input not only the compounds' molecular descriptors but also (a subset of) their predicted Kt:p values. Results: Comparison of the models that used only molecular descriptors, in particular, the Bagging decision tree (mean fold error of 2.33), with those employing predicted Kt:p values in addition to the molecular descriptors, such as the Bagging decision tree using adipose Kt:p (mean fold error of 2.29), indicated that the use of predicted Kt:p values as descriptors may be beneficial for accurate prediction of Vss using decision trees if prior feature selection is applied. Conclusions: Decision tree based models presented in this work have an accuracy that is reasonable and similar to the accuracy of reported Vss inter-species extrapolations in the literature. The estimation of Vss for new compounds in drug discovery will benefit from methods that are able to integrate large and varied sources of data and flexible non-linear data mining methods such as decision trees, which can produce interpretable models. Figure not available: see fulltext. © 2015 Freitas et al.; licensee Springer

The relationship between various measures of obesity and arterial stiffness in morbidly obese patients

<p>Abstract</p> <p>Background</p> <p>Obesity is associated with increased risk of cardiovascular disease. Arterial stiffness assessed by carotid femoral pulse wave velocity (PWV) is an independent predictor of cardiovascular morbidity and mortality. We aimed to investigate how various measures of body composition affect arterial stiffness.</p> <p>Methods</p> <p>This is an analysis of cross-sectional baseline data from a controlled clinical trial addressing changes in arterial stiffness after either surgery or lifestyle intervention in a population of morbidly obese patients. High-fidelity applanation tonometry (Millar^®, Sphygmocor^®) was used to measure pulse wave velocity (PWV). Carotid femoral PWV is a direct measure of arterial stiffness and is considered to be the gold standard method. The Inbody 720 Body Composition Analyzer was used for bioelectrical impedance analysis (BIA). Spearman's correlation, independent samples <it>t</it>-test, chi-square tests, Fisher's exact test and multiple linear regression analyses were used as statistical methods.</p> <p>Results</p> <p>A total of 133 patients (79 women), with a mean (SD) age of 43 (11) years were included in the study. Men had a significantly higher prevalence of obesity related comorbidities and significantly higher PWV, 9.1 (2.0) m/s vs. 8.1 (1.8) m/s, p = 0.003, than women. In the female group, PWV was positively correlated with WC, WHtR, BMI and visceral fat area. In the male group, PWV was negatively correlated with BMI. Multiple linear regression analysis showed that increasing BMI, WC, WHtR, visceral fat area and fat mass were independently associated with higher PWV in women, but not in men, after adjustment for age, hypertension and type 2 diabetes.</p> <p>Conclusion</p> <p>Most measures of general and abdominal obesity were predictors of arterial stiffness in female morbidly obese patients.</p> <p>Trial registration</p> <p>ClinicalTrials.gov Identifier <a href="http://www.clinicaltrials.gov/ct2/show/NCT00626964">NCT00626964</a></p

Investigating Homology between Proteins using Energetic Profiles

Accumulated experimental observations demonstrate that protein stability is often preserved upon conservative point mutation. In contrast, less is known about the effects of large sequence or structure changes on the stability of a particular fold. Almost completely unknown is the degree to which stability of different regions of a protein is generally preserved throughout evolution. In this work, these questions are addressed through thermodynamic analysis of a large representative sample of protein fold space based on remote, yet accepted, homology. More than 3,000 proteins were computationally analyzed using the structural-thermodynamic algorithm COREX/BEST. Estimated position-specific stability (i.e., local Gibbs free energy of folding) and its component enthalpy and entropy were quantitatively compared between all proteins in the sample according to all-vs.-all pairwise structural alignment. It was discovered that the local stabilities of homologous pairs were significantly more correlated than those of non-homologous pairs, indicating that local stability was indeed generally conserved throughout evolution. However, the position-specific enthalpy and entropy underlying stability were less correlated, suggesting that the overall regional stability of a protein was more important than the thermodynamic mechanism utilized to achieve that stability. Finally, two different types of statistically exceptional evolutionary structure-thermodynamic relationships were noted. First, many homologous proteins contained regions of similar thermodynamics despite localized structure change, suggesting a thermodynamic mechanism enabling evolutionary fold change. Second, some homologous proteins with extremely similar structures nonetheless exhibited different local stabilities, a phenomenon previously observed experimentally in this laboratory. These two observations, in conjunction with the principal conclusion that homologous proteins generally conserved local stability, may provide guidance for a future thermodynamically informed classification of protein homology