Studies of Receptor Tyrosine Kinase Transmembrane Domain Interactions: The EmEx-FRET Method

The energetics of transmembrane (TM) helix dimerization in membranes and the thermodynamic principles behind receptor tyrosine kinase (RTK) TM domain interactions during signal transduction can be studied using Förster resonance energy transfer (FRET). For instance, FRET studies have yielded the stabilities of wild-type fibroblast growth factor receptor 3 (FGFR3) TM domains and two FGFR3 pathogenic mutants, Ala391Glu and Gly380Arg, in the native bilayer environment. To further our understanding of the molecular mechanisms of deregulated FGFR3 signaling underlying different pathologies, we determined the effect of the Gly382Asp FGFR3 mutation, identified in a multiple myeloma cell line, on the energetics of FGFR3 TM domain dimerization. We measured dimerization energetics using a novel FRET acquisition and processing method, termed “emission-excitation FRET (EmEx-FRET),” which improves the precision of thermodynamic measurements of TM helix association. The EmEx-FRET method, verified here by analyzing previously published data for wild-type FGFR3 TM domain, should have broad utility in studies of protein interactions, particularly in cases when the concentrations of fluorophore-tagged molecules cannot be controlled.     

Utility of surface-supported bilayers in studies of transmembrane helix dimerization

This review focuses on the methods that are available to study transmembrane (TM) helix dimerization in membrane-like environments (either bacterial membranes or lipid bilayers, as mimics of the eukaryotic cellular membrane), with an emphasis on the utility of surface-supported bilayers in such studies.     

Characterization of antimicrobial peptide activity by electrochemical impedance spectroscopy

Electrochemical impedance spectroscopy performed on surface-supported bilayer membranes allows for the monitoring of changes in membrane properties, such as thickness, ion permeability, and homogeneity, after exposure to antimicrobial peptides (AMPs). We show that two model cationic peptides, very similar in sequence but different in activity, induce dramatically different changes in membrane properties as probed by impedance spectroscopy. Moreover, the impedance results excluded the “barrel-stave” and the “toroidal pore” models of AMP mode of action, and are more consistent with the “carpet” and the “detergent” models. The impedance data provide important new insights about the kinetics and the scale of the peptide action which currently are not addressed by the “carpet” and the “detergent” models. The method presented not only provides additional information about the mode of action of a particular AMP, but offers a means of characterizing AMP activity in reproducible, well-defined quantitative terms.     

A simple "proximity" correction for Förster resonance energy transfer efficiency determination in membranes using lifetime measurements

Accurate measurements of oligomerization in membranes by Förster resonance energy transfer (FRET) are always compromised by a substantial contribution from random chance colocalization of donors and acceptors. Recently, Li and coworkers demonstrated the use of computer simulation in estimating the contribution of this “proximity” component to correct the FRET efficiency and estimate the free energy of dimer formation of the G380R mutants of fibroblast growth factor receptor 3 (FGFR3) transmembrane domain immersed into lipid bilayer. Because tight dimerization will result in complete energy transfer from donor to acceptor, we have used the same experimental system of fluorescein- and rhodamine-labeled G380R mutants of FGFR3 for the experimental assessment of the proximity FRET corrections using fluorescence lifetime measurements. The experimental proximity FRET correction, based on time-resolved fluorescence measurements, is expected to have general advantages over theoretical correction, especially in the case of nonrandomly distributed monomers.     

The Influence of an Extract from the Marine Brown Alga <Emphasis Type="Italic">Sargassum pallidum</Emphasis> on the Metabolic Reactions in the Liver under Experimental Toxic Hepatitis

This study explores the influence of an extract enriched with polyphenol substances obtained from the brown alga Sargassum pallidum and the effect of the Legalon commercial reference preparation on the biochemical indices of the blood at acute hepatic injury induced in rats with carbon tetrachloride (CTC). Administration of S. pallidum extract exhibited a pronounced antioxidant and hepatoprotective effect revealed in reduction of alanine aminotransferase (AlAT) activity in blood plasm, in normalization of superoxide dismutase activity and reduced glutathione contents, of the level of lipid peroxidation, and the liver antiradical activity. We observed recovery of lipid metabolism and elimination of liver fatty infiltration. The S. pallidum extract was as effective as the reference Legalon hepatoprotector and even surpassed it in its ability to restore the phospholipid metabolism. In our opinion, this hepatoprotective effect of S. pallidum extract is associated with the action of polyphenols contained in brown algae, that is, phlorotannins, which are highly active antioxidants and restore the liver metabolic reactions after toxic hepatic injury with CTC.     

Lipid Composition,Content of Polyphenols,and Antiradical Activity in Some Representatives of Marine Algae

Russian Journal of Plant Physiology - Qualitative and quantitative composition of the lipid complex and total content of polyphenols were compared in aqueous alcoholic extracts from three species...     

Transmembrane helix heterodimerization in lipid bilayers: probing the energetics behind autosomal dominant growth disorders

Here, we show that the energetics of transmembrane helix heterodimer formation can be characterized in liposomes using F?rster resonance energy transfer (FRET). We present the theory and the protocol for measuring the free energy of heterodimerization, and the total (hetero and homo-dimeric) dimer fraction. We use the presented methodology to determine the propensity for heterodimer formation between wild-type fibroblast growth factor receptor 3 (FGFR3) transmembrane domain and the Ala391Glu mutant, linked to Crouzon syndrome with acanthosis nigricans.     

Feeding Patterns in Different Size Larvae of Walleye Pollack, Theragra chalcogramma (Pallas, 1814) on the Shelf of Western Kamchatka

The stomach contents were examined in 373 walleye pollack larvae of different sizes. The diet of pollack larvae included more than 20 plankters of different sizes. Phytoplankton was the major food source of 4- to 6-mm-long larvae. The proportion of phytoplankton in the larval diet decreased as the larvae grew, and the late larval stages shifted entirely to a diet of zooplankton. As the larvae increased in size, the spectrum of food organisms changed from smaller to larger sizes. In the larvae 4 to 31 mm long, the length of prey varied from 0.1 (Coscinodiscus) to 6.0 mm (Neocalanus plumchrus). The average daily repleteness of pollack larvae varied from 32 to 210. The minimum repleteness was registered in 4- to 6-mm-long larvae with mixed feeding (endogenous and exogenous); in 30- to 35-mm-long fish the repleteness was the greatest. The daily food rations calculated for the most abundant size groups of larvae ranged from 4.3 to 6.6% of their body weight.     

Hill Coefficient Analysis of Transmembrane Helix Dimerization

Sertoli cells are responsible for regulating a wide range of processes that lead to the differentiation of male germ cells into spermatozoa. Cytoplasmic pH (pH _i ) has been shown to be an important parameter in cell physiology, regulating namely cell metabolism and differentiation. However, membrane transport mechanisms involved in pH _i regulation mechanisms of Sertoli cells have not yet been elucidated. In this work, pH _i was determined using the pH-sensitive fluorescent probe 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Addition of weak acids resulted in rapid acidification of the intracellular milieu. Sertoli cells then recovered pH _i by a mechanism that was shown to be sensitive to external Na⁺. pH _i recovery was also greatly reduced in the presence of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and amiloride. These results point toward the action of an Na⁺-driven HCO₃⁻/Cl⁻ exchanger and/or an Na⁺/HCO₃⁻ cotransporter and the action of the Na⁺/H⁺ exchanger on pH _i regulation in the experimental conditions used. pH _i recovery was only slightly affected by ouabain, suggesting that the inhibition of Na⁺/K⁺-ATPase affects recovery indirectly, possibly via the shift on the Na⁺ gradient. On the other hand, recovery from the acid load was independent of the presence of concanamycin A, a specific inhibitor of the V-type ATPases, suggesting that these pumps do not have a relevant action on pH _i regulation in bovine Sertoli cells.     

Current Trends in the Condition of Bottom Fish Communities from the West Kamchatka Shelf

Based on the data of two summer surveys carried out in 2002, the biomass of bottom fishes of the West Kamchata Shelf was assessed. The obtained results were considered within the series of similar annual assessments conducted since 1996. It was displayed that against the background of some increase in the total biomass of bottom fishes, the biomass of the plaices prevailing there, which had previously been the two dominating species had decreased. As a result of the quantitative variations that occurred, the present community structure differs considerably from that observed over the past twenty years.