Homology modeling and molecular dynamics simulations of the glycine receptor ligand binding domain

We present a homology based model of the ligand binding domain (LBD) of the homopentameric alpha1 glycine receptor (GlyR). The model is based on multiple sequence alignment with other members of the nicotinicoid ligand gated ion channel superfamily and two homologous acetylcholine binding proteins (AChBP) from the freshwater (Lymnaea stagnalis) and saltwater (Aplysia californica) snails with known high resolution structure. Using two template proteins with known structure to model three dimensional structure of a target protein is especially advantageous for sequences with low homology as in the case presented in this paper. The final model was cross-validated by critical evaluation of experimental and published mutagenesis, functional and other biochemical studies. In addition, a complex structure with strychnine antagonist in the putative binding site is proposed based on docking simulation using Autodock program. Molecular dynamics (MD) simulations with simulated annealing protocol are reported on the proposed LBD of GlyR, which is stable in 5 ns simulation in water, as well as for a deformed LBD structure modeled on the corresponding domain determined in low-resolution cryomicroscopy structure of the alpha subunit of the full-length acetylcholine receptor (AChR). Our simulations demonstrate that the beta-sandwich central core of the protein monomer is fairly rigid in the simulations and resistant to deformations in water.     

RGS6, but Not RGS4, Is the Dominant Regulator of G Protein Signaling (RGS) Modulator of the Parasympathetic Regulation of Mouse Heart Rate

Parasympathetic activity decreases heart rate (HR) by inhibiting pacemaker cells in the sinoatrial node (SAN). Dysregulation of parasympathetic influence has been linked to sinus node dysfunction and arrhythmia. RGS (regulator of G protein signaling) proteins are negative modulators of the parasympathetic regulation of HR and the prototypical M₂ muscarinic receptor (M₂R)-dependent signaling pathway in the SAN that involves the muscarinic-gated atrial K⁺ channel I_KACh. Both RGS4 and RGS6-Gβ5 have been implicated in these processes. Here, we used Rgs4^−/−, Rgs6^−/−, and Rgs4^−/−:Rgs6^−/− mice to compare the relative influence of RGS4 and RGS6 on parasympathetic regulation of HR and M₂R-I_KACh-dependent signaling in the SAN. In retrogradely perfused hearts, ablation of RGS6, but not RGS4, correlated with decreased resting HR, increased heart rate variability, and enhanced sensitivity to the negative chronotropic effects of the muscarinic agonist carbachol. Similarly, loss of RGS6, but not RGS4, correlated with enhanced sensitivity of the M₂R-I_KACh signaling pathway in SAN cells to carbachol and a significant slowing of M₂R-I_KACh deactivation rate. Surprisingly, concurrent genetic ablation of RGS4 partially rescued some deficits observed in Rgs6^−/− mice. These findings, together with those from an acute pharmacologic approach in SAN cells from Rgs6^−/− and Gβ5^−/− mice, suggest that the partial rescue of phenotypes in Rgs4^−/−:Rgs6^−/− mice is attributable to another R7 RGS protein whose influence on M₂R-I_KACh signaling is masked by RGS4. Thus, RGS6-Gβ5, but not RGS4, is the primary RGS modulator of parasympathetic HR regulation and SAN M₂R-I_KACh signaling in mice.     

Action of plasmid ColIb-P9 on the survival after ultraviolet irradiation and on the mutagenesis of the imiC, uvm, recL, uvrE and tif1 sfiA lexA spr mutants of Escherichia coli K-12 cells

To clarify the mechanisms whereby the ColIb-P9 plasmid affects DNA repair processes, its effect was studied in mutant Escherichia coli K-12 cells with altered mutagenesis and DNA repair. The plasmid was shown to protect umuC, uvm, recL and uvrE mutants after UV irradiation. The frequency of UV-induced his+ revertants increased in the presence of the plasmid in umuC, uvm and recL mutant cells. The ColIb-P9 plasmid completely restored the UV mutability and survival of umuC mutants. These results suggest that the ColIb-P9 plasmid may encode a product similar to that of the umuC gene. In the tif1 sfiA lexA spr mutant cells where SOS functions are constitutively expressed, the ColIb-P9 plasmid increased the number of his+ revertants several times. This suggests that the action of ColIb-P9 is probably brought about not via the derepression of the recA gene but at the subsequent stages of the recA+lexA+-dependent DNA error-prone repair.     

The microbial loop in the planktonic communities in lakes with various trophic status

The structure of planktic trophic chains was studied in eight lakes of European Russia and five lakes in Central Asia. The lakes differed in the level of productivity, morphometric parameters, and the type of agitation and mineralization. It is found that the microbial loop of picophototrophic organisms, bacteria, heterotrophic flagellates, infusoria, and viruses constitutes 12.3-64.7% of the total plankton biomass. Positive correlation between the biomass of microbial community and the primary production of phytoplankton is observed, whereas no relation is revealed between the share of microorganisms in the plankton biomass and the trophic status of the water body. The presence of a great number of cladocerans decreased the role of the microbial loop in the structural organization of the planktic community. Heterotrophic flagellates consuming 3-81% of daily bacterial production were the principal cause of bacteria elimination only in some of the studied water bodies.     

Potassium supplements’ effect on potassium balance in athletes during prolonged hypokinetic and ambulatory conditions

Electrolyte supplements may be used to prevent changes in electrolyte balance during hypokinesia (diminished movement). The aim of this study was to measure the effect of potassium (K) supplements on K balance during prolonged hypokinesia (HK). Studies were done during 30 d of a pre-HK period and during 364 d of an HK period. Forty male athletes aged 25.1±4.4 yr were chosen as subjects. They were divided equally into four groups: unsupplemented ambulatory control subjects (UACS), unsupplemented hypokinetic subjects (UHKS), supplemented hypokinetic subjects (SHKS) and supplemented ambulatory control subjects (SACS). The SHKS and UHKS groups were kept under an average walking distance of 0.7 km/d. The SACS and SHKS groups were supplemented daily with 50.0 mg elemental potassium chloride (KCl) per kilogram body weight. The K balance, fecal K excretion, urinary K, sodium (Na), and chloride (Cl) excretion, plasma K, Na, and Cl concentration, plasma renin activity (PRA) and plasma aldosterone (PA) concentration, anthropometric characteristics and peak oxygen uptake were measured. Negative K balance, fecal K excretion, urinary K, Na, and Cl excretion, plasma K, Na, and Cl concentration, and PRA and PA concentration increased significantly (p≤0.01), whereas body weight and peak oxygen uptake decreased significantly in the SHKS and UHKS groups when compared with SACS and UACS groups. However, the measured parameters changed much faster and much more in SHKS group than UHKS group. By contrast, K balance, fecal, urinary, and plasma K, plasma hormones, body weight, and peak oxygen uptake did not change significantly in the SACS and UACS groups when compared with the baseline control values. It was concluded that prolonged HK induces a significant negative K balance associated with increased plasma K concentration and urinary and fecal K excretion. However, negative K balance appeared much faster and was much greater in the SHKS group than UHKS group. Thus, K supplementation was not effective in preventing negative K balance during prolonged HK.     

Recent developments in superhydrophobic surfaces and their relevance to marine fouling: a review

Abstract

In this review, a brief synopsis of superhydrophobicity (i.e. extreme non-wettability) and its implications on marine fouling are presented. A short overview of wettability and recent experimental developments aimed at fabricating superhydrophobic surfaces by tailoring their chemical nature and physical appearance (i.e. substratum texture) are reviewed. The formation of responsive/“smart” surfaces, which adjust their physico-chemical properties to variations in some outside physical stimulus, including light, temperature, electric field, or solvent, is also described. Finally, implications of tailoring the surface chemistry, texture, and responsiveness of surfaces on the design of effective marine fouling coatings are considered and discussed.     

Probing a putative active site of the catalytic subunit of pyruvate dehydrogenase phosphatase 1 (PDP1c) by site-directed mutagenesis

The catalytic subunit of pyruvate dehydrogenase phosphatase 1 (PDP1c) is a magnesium-dependent protein phosphatase that regulates the activity of mammalian pyruvate dehydrogenase complex. Based on the sequence analysis, it was hypothesized that PDP1c is related to the mammalian magnesium-dependent protein phosphatase type 1, with Asp54, Asp347, and Asp445 contributing to the binuclear metal-binding center, and Asn49 contributing to the phosphate-binding sites. In this study, we analyzed the functional significance of these amino acid residues using a site-directed mutagenesis. It was found that substitution of each of these residues had a significant impact on PDP1c activity toward the protein substrate. The activities of Asp54, Asp347, and Asp445 mutants were decreased more than 1000-fold. The activity of Asn49 mutant was 2.5-fold lower than the activity of wild-type PDP1c. The decrease in activity of Asp54 and Asp347 came about, most likely, as a result of impaired magnesium binding. Unexpectedly, it was found that the Asp445 mutant bound Mg(2+) ions similarly to the wild-type enzyme. Accordingly, the Asp445 mutant was found to be active with the artificial substrate p-nitrophenyl phosphate (pNPP). Asp54 and Asp347 mutants did not demonstrate any appreciable activity with pNPP. Together, these observations strongly suggest that Asn49, Asp54, and Asp347 are important for the catalysis of the phosphatase reaction, contributing to the phosphate- and metal-binding centers of PDP1c. In contrast, Asp445 is not required for catalysis. The exact role of Asp445 remains to be established, but indirect evidence suggests that it might be involved in the control of interactions between PDP1c and the protein substrate pyruvate dehydrogenase.     

Termination of tyrphostin AG1478 application results in different recovery of EGF receptor tyrosine residues 1045 and 1173 phosphorylation in A431 cells

Tyrphostin AG1478 is known as a specific and reversible inhibitor of TK (tyrosine kinase) activity of the EGFR [EGF (epidermal growth factor) receptor]. It is attractive as an anticancer agent for cancers with elevated EGFR TK levels. However, post‐application effects of AG1478 are not well studied. We have analysed EGFR phosphorylation after termination of AG1478 application using human epidermoid carcinoma A431 cells. It was found that AG1478 inhibitory action is fast, but not fully reversible: removal of tyrphostin resulted in incomplete restoration of the overall EGFR phosphorylation. Analysing the state of two individual autophosphorylation sites of internalized EGFR, Tyr¹⁰⁴⁵ and Tyr¹¹⁷³, we demonstrated that phosphorylation of Tyr¹¹⁷³ involved in stimulation of the MAPK (mitogen‐activated protein kinase) cascade was restored much more efficiently than that in position 1045, which binds the ubiquitin ligase c‐Cbl and is necessary for targeting the receptor for lysosomal degradation. c‐Cbl association with EGFR abolished by AG1478 was not reestablished after tyrphostin cessation. As a consequence, ubiquitination‐dependent EGFR delivery to lysosomes was blocked, while phosphorylation of ERK1/2 (extracellular‐signal‐regulated kinase 1/2) was even increased. Thus, after termination of AG1478, the intracellular level of the inhibitor can be reached at which mitogenic signalling will be restored, whereas the EGFR negative regulation due to lysosomal degradation will not.     

Membrane Anchor R9AP Potentiates GTPase-accelerating Protein Activity of RGS11��G��5 Complex and Accelerates Inactivation of the mGluR6-Go Signaling

The R7 subfamily of RGS proteins critically regulates neuronal G protein-signaling pathways that are essential for vision, nociception, motor coordination, and reward processing. A member of the R7 RGS family, RGS11, is a GTPase-accelerating protein specifically expressed in retinal ON-bipolar cells where it forms complexes with the atypical G protein β subunit, Gβ₅, and transmembrane protein R9AP. Association with R9AP has been shown to be critical for the proteolytic stability of the complex in the retina. In this study we report that R9AP can in addition stimulate the GTPase-accelerating protein activity of the RGS11·Gβ₅ complex at Gα_o. Single turnover GTPase assays reveal that R9AP co-localizes RGS11·Gβ₅ and Gα_o on the membrane and allosterically potentiates the GTPase-accelerating function of RGS11·Gβ₅. Reconstitution of mGluR6-Gα_o signaling in Xenopus oocytes indicates that RGS11·Gβ₅-mediated GTPase acceleration in this system requires co-expression of R9AP. The results provide new insight into the regulation of mGluR6-Gα_o signaling by the RGS11·Gβ₅·R9AP complex and establish R9AP as a general GTPase-accelerating protein activity regulator of R7 RGS complexes.