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Cakil Y.D.,Institutes of Medical Chemistry | Cakil Y.D.,Medical University of Vienna | Khunweeraphong N.,Institutes of Medical Chemistry | Parveen Z.,Institutes of Medical Chemistry | And 8 more authors.
Molecular Pharmacology | Year: 2014

The multispecific efflux transporter, P-glycoprotein, plays an important role in drug disposition. Substrate translocation occurs along the interface of its transmembrane domains. The rotational C2 symmetry of ATP-binding cassette transporters implies the existence of two symmetry-related sets of substrateinteracting amino acids. These sets are identical in homodimeric transporters, and remain evolutionary related in full transporters, such as P-glycoprotein, in which substrates bind preferentially, but nonexclusively, to one of two binding sites. We explored the role of pore-exposed tyrosines for hydrogen-bonding interactions with propafenone type ligands in their preferred binding site 2. Tyrosine 953 is shown to form hydrogen bonds not only with propafenone analogs, but also with the preferred site 1 substrate rhodamine123. Furthermore, an accessory role of tyrosine 950 for binding of selected propafenone analogs is demonstrated. The present study demonstrates the importance of domain interface tyrosine residues for interaction of small molecules with P-glycoprotein. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Hochberg I.,Institute of Endocrinology | Hochberg I.,University of Michigan | Harvey I.,Physiology | Tran Q.T.,Preventive Medicine | And 5 more authors.
Journal of Molecular Endocrinology | Year: 2015

Glucocorticoids have major effects on adipose tissue metabolism. To study tissue mRNA expression changes induced by chronic elevated endogenous glucocorticoids, we performed RNAsequencingonthe subcutaneousadipose tissue frompatientswithCushing’s disease(n=5) compared to patients with nonfunctioning pituitary adenomas (n=11).We found a higher expression of transcripts involved in several metabolic pathways, including lipogenesis, proteolysis and glucose oxidation as well as a decreased expression of transcripts involved in inflammation and protein synthesis. To further study this in amodel system,we subjectedmice todexamethasone treatment for 12weeks andanalyzedtheir inguinal (subcutaneous) fat pads, which led to similar findings. Additionally, mice treated with dexamethasone showed drastic decreases in lean body mass as well as increased fat mass, further supporting the human transcriptomic data. These data provide insight to transcriptional changes that may be responsible for the comorbidities associated with chronic elevations of glucocorticoids. © 2015 The authors.

PubMed | Physiology and Behavior., Physiology and Membrane Biology and., University of California at Davis and Physiology
Type: Journal Article | Journal: The Journal of biological chemistry | Year: 2015

The plasma membrane (PM) comprises distinct subcellular domains with diverse functions that need to be dynamically coordinated with intracellular events, one of the most impactful being mitosis. The Kv2.1 voltage-gated potassium channel is conditionally localized to large PM clusters that represent specialized PM:endoplasmic reticulum membrane contact sites (PM:ER MCS), and overexpression of Kv2.1 induces more exuberant PM:ER MCS in neurons and in certain heterologous cell types. Localization of Kv2.1 at these contact sites is dynamically regulated by changes in phosphorylation at one or more sites located on its large cytoplasmic C terminus. Here, we show that Kv2.1 expressed in COS-1 cells undergoes dramatic cell cycle-dependent changes in its PM localization, having diffuse localization in interphase cells, and robust clustering during M phase. The mitosis-specific clusters of Kv2.1 are localized to PM:ER MCS, and M phase clustering of Kv2.1 induces more extensive PM:ER MCS. These cell cycle-dependent changes in Kv2.1 localization and the induction of PM:ER MCS are accompanied by increased mitotic Kv2.1 phosphorylation at several C-terminal phosphorylation sites. Phosphorylation of exogenously expressed Kv2.1 is significantly increased upon metaphase arrest in COS-1 and CHO cells, and in a pancreatic cell line that express endogenous Kv2.1. The M phase clustering of Kv2.1 at PM:ER MCS in COS-1 cells requires the same C-terminal targeting motif needed for conditional Kv2.1 clustering in neurons. The cell cycle-dependent changes in localization and phosphorylation of Kv2.1 were not accompanied by changes in the electrophysiological properties of Kv2.1 expressed in CHO cells. Together, these results provide novel insights into the cell cycle-dependent changes in PM protein localization and phosphorylation.

PubMed | Physiology., University of Western Ontario, Shandong University and Shansong University
Type: | Journal: American journal of physiology. Gastrointestinal and liver physiology | Year: 2016

Gamma-aminobutyric acid (GABA) is produced by various cells through the catalytic activity of glutamic acid decarboxylase (GAD). Activation of type-A GABA receptor (GABA

PubMed | Viking Genetics, Physiology and Indonesian Institute of Sciences
Type: Journal Article | Journal: Journal of dairy science | Year: 2014

The present study aimed to evaluate the effect of single-layer centrifugation (SLC) through a species-specific colloid (Androcoll-B; patent pending, J. M. Morrell) on bull sperm quality. Computer-assisted sperm analysis of motility and flow cytometric analysis of sperm viability (SYBR-14/propidium iodide staining), chromatin integrity (acridine orange staining), reactive oxygen species production [Hoechst 33258-hydroethidine-2,7-dichlorodihydrofluorescein diacetate (HO-HE-DCFDA) staining], mitochondrial membrane potential (staining with JC-1 probe), and protein tyrosine phosphorylation (specific antibody staining) were performed on unselected and SLC-selected sperm samples. Single-layer centrifugation of bull spermatozoa resulted in the selection of a sperm population that had high mitochondrial membrane potential, a higher content of phosphorylated protein, and more reactive oxygen species than control samples. Sperm chromatin damage was lower in the SLC samples although sperm viability and motility did not differ between SLC samples and controls. These observations suggest that SLC of bull semen in a soybean-containing extender improved some, but not all, parameters of sperm quality.

PubMed | University of Washington, Pathology and Laboratory Medicine, Physiology, Data Management and Central University of the Caribbean
Type: Journal Article | Journal: The Journal of biological chemistry | Year: 2016

TheKCNJ10gene encoding Kir4.1 contains numerous SNPs whose molecular effects remain unknown. We investigated the functional consequences of uncharacterized SNPs (Q212R, L166Q, and G83V) on homomeric (Kir4.1) and heteromeric (Kir4.1-Kir5.1) channel function. We compared these with previously characterized EAST/SeSAME mutants (G77R and A167V) in kidney-derived tsA201 cells and in glial cell-derived C6 glioma cells. The membrane potentials of tsA201 cells expressing G77R and G83V were significantly depolarized as compared with WTKir4.1, whereas cells expressing Q212R, L166Q, and A167V were less affected. Furthermore, macroscopic currents from cells expressing WTKir4.1 and Q212R channels did not differ, whereas currents from cells expressing L166Q, G83V, G77R, and A167V were reduced. Unexpectedly, L166Q current responses were rescued when co-expressed with Kir5.1. In addition, we observed notable differences in channel activity between C6 glioma cells and tsA201 cells expressing L166Q and A167V, suggesting that there are underlying differences between cell lines in terms of Kir4.1 protein synthesis, stability, or expression at the surface. Finally, we determined spermine (SPM) sensitivity of these uncharacterized SNPs and found that Q212R-containing channels displayed reduced block by 1 mSPM. At 100 mSPM, the block was equal to or greater than WT, suggesting that the greater driving force of SPM allowed achievement of steady state. In contrast, L166Q-Kir5.1 channels achieved a higher block than WT, suggesting a more stable interaction of SPM in the deep pore cavity. Overall, our data suggest that G83V, L166Q, and Q212R residues play a pivotal role in controlling Kir4.1 channel function.

PubMed | Physiology., La Trobe University, Walter and Eliza Hall Institute of Medical Research, Case Western Reserve University and Physiology
Type: Journal Article | Journal: The Journal of biological chemistry | Year: 2014

Crystallographic studies of insulin bound to fragments of the insulin receptor have recently defined the topography of the primary hormone-receptor interface. Here, we have investigated the role of Phe(B24), an invariant aromatic anchor at this interface and site of a human mutation causing diabetes mellitus. An extensive set of B24 substitutions has been constructed and tested for effects on receptor binding. Although aromaticity has long been considered a key requirement at this position, Met(B24) was found to confer essentially native affinity and bioactivity. Molecular modeling suggests that this linear side chain can serve as an alternative hydrophobic anchor at the hormone-receptor interface. These findings motivated further substitution of Phe(B24) by cyclohexanylalanine (Cha), which contains a nonplanar aliphatic ring. Contrary to expectations, [Cha(B24)]insulin likewise exhibited high activity. Furthermore, its resistance to fibrillation and the rapid rate of hexamer disassembly, properties of potential therapeutic advantage, were enhanced. The crystal structure of the Cha(B24) analog, determined as an R6 zinc-stabilized hexamer at a resolution of 1.5 , closely resembles that of wild-type insulin. The nonplanar aliphatic ring exhibits two chair conformations with partial occupancies, each recapitulating the role of Phe(B24) at the dimer interface. Together, these studies have defined structural requirements of an anchor residue within the B24-binding pocket of the insulin receptor; similar molecular principles are likely to pertain to insulin-related growth factors. Our results highlight in particular the utility of nonaromatic side chains as probes of the B24 pocket and suggest that the nonstandard Cha side chain may have therapeutic utility.

PubMed | Mount Sinai School of Medicine, Physiology and New York Medical College
Type: | Journal: The Journal of physiology | Year: 2016

Arrhythmias result from disruptions to cardiac electrical activity, although the factors that control cellular action potentials are incompletely understood. We combined mathematical modelling with experiments in heart cells from guinea pigs to determine how cellular electrical activity is regulated. A mismatch between modelling predictions and the experimental results allowed us to construct an improved, more predictive mathematical model. The balance between two particular potassium currents dictates how heart cells respond to perturbations and their susceptibility to arrhythmias.Imbalances of ionic currents can destabilize the cardiac action potential and potentially trigger lethal cardiac arrhythmias. In the present study, we combined mathematical modelling with information-rich dynamic clamp experiments to determine the regulation of action potential morphology in guinea pig ventricular myocytes. Parameter sensitivity analysis was used to predict how changes in ionic currents alter action potential duration, and these were tested experimentally using dynamic clamp, a technique that allows for multiple perturbations to be tested in each cell. Surprisingly, we found that a leading mathematical model, developed with traditional approaches, systematically underestimated experimental responses to dynamic clamp perturbations. We then re-parameterized the model using a genetic algorithm, which allowed us to estimate ionic current levels in each of the cells studied. This unbiased model adjustment consistently predicted an increase in the rapid delayed rectifier K

PubMed | Bushehr University of Medical Sciences, Occupational Health Engineering, Pediatric Chronic Kidney Disease Research Center, Dr Shariati Hospital and Physiology
Type: Journal Article | Journal: Journal of gastroenterology and hepatology | Year: 2016

Early detection of response to treatment is critically important in gastrointestinal stromal tumors (GIST). Therefore, the present systematic review and meta-analysis assessed the value of (18) f-fluorodeoxyglucose positron emission tomography ((18) FDG-PET) on prediction of therapeutic response of GIST patients to systemic treatments.The literature search was conducted using PubMed, SCOPUS, Cochrane, and Google Scholar databases, and review article references. Eligible articles were defined as studies included confirmed GIST patients who underwent (18) FDG-PET as well as assessing the screening role of it.Finally, 21 relevant articles were included. The analysis showed the pooled sensitivity and specificity of 18FDG-PET in evaluation of response to treatment of GIST patient were 0.90 (95% CI: 0.85-0.94; I(2) =52.59, P=0.001) and 0.62 (95% CI: 0.49-0.75; I(2) =69.7, P=0.001), respectively. In addition, the pooled prognostic odds ratio of (18) FDG-PET for was 14.99 (95% CI, 6.42-34.99; I(2) =100.0, P<0.001). The Meta regression showed that sensitivity of (18) FDG-PET was higher if the sample size of study was equal or more than 30 cases (sensitivity=0.93; 95% CI: 0.89-0.97), when using PET/CT (sensitivity=0.92; 95% CI: 0.89-0.97), and self-design criteria (sensitivity=0.93; 95% CI: 0.87-1.0).The present meta-analysis showed (18) FDG-PET has a significant value in predicting treatment response in GIST patients.

Long E.K.,Physiology | Rosenberger T.A.,Physiology | Picklo Sr. M.J.,University of North Dakota | Picklo Sr. M.J.,U.S. Department of Agriculture
Free Radical Biology and Medicine | Year: 2010

Ethanol withdrawal increases lipid peroxidation of the polyunsaturated fatty acid (PUFA) docosahexaenoate (22:6; n-3) in the CNS. To further define the role of oxidative damage of PUFAs during ethanol withdrawal, we measured the levels of glutathione adducts of 4-hydroxy-2-hexenal (GSHHE) and 4-hydroxy-2-nonenal (GSHNE) as biomarkers of brain lipid peroxidation of n-3 and n-6 PUFAs, respectively. In this study rats received an ethanol-containing diet for 6 weeks followed by withdrawal ranging from 0 to 7 days. GSHHE content was elevated (> 350%) in the cerebral cortex after 2 days of withdrawal with no change in GSHNE. The levels of GSHHE were significantly greater (2- to 20-fold) than those of GSHNE in multiple brain regions. Experiments demonstrated that intoxication and withdrawal did not alter the enzymatic rate of formation of GSHHE or GSHNE, but the rate of formation of GSHHE was higher (∼ 50%) than that of GSHNE. These results indicate that selective oxidative damage to n-3 PUFAs occurs in the cerebral cortex as a result of ethanol withdrawal and that 4-hydroxy-2-hexenal is metabolized to the GSH adduct more efficiently than HNE. © 2009.

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