Biophysics

Molecular tools for fluorescent imaging of cells and their components are vital for understanding the function and activity of cells. Here, we report an imidazole functionalized pentameric oligothiophene, p-HTIm, that can be utilized for fluorescent imaging of cells. p-HTIm fluorescence in normal cells appeared in a peripheral punctate pattern partially co-localized with lysosomes, whereas a one-sided perinuclear Golgi associated localization of the dye was observed in malignant cells. The uptake of p-HTIm was temperature dependent and the intracellular target was reached within 1 h after staining. The ability of p-HTIm to stain cells was reduced when the imidazole side chain was chemically altered, verifying that specific imidazole side-chain functionalities are necessary for achieving the observed cellular staining. Our findings confirm that properly functionalized oligothiophenes can be utilized as fluorescent tools for vital staining of cells and that the selectivity toward distinct intracellular targets are highly dependent on the side-chain functionalities along the conjugated thiophene backbone.

The F-type ATP synthase and V-type ATPase are rotating enzymes in which proton transport across membranes is coupled with ATP synthesis and ATP hydrolysis, respectively. Rotation of the stalk domain is required for continuous catalysis of the both enzymes coupling to transport.

ATP is synthesized by F-type proton-translocating ATPases (F-ATPases) coupled with an electrochemical proton gradient established by an electron transfer chain. This mechanism is ubiquitously found in mitochondria, chloroplasts and bacteria. Vacuolar-type ATPases (V-ATPases) are found in endomembrane organelles, including lysosomes, endosomes, synaptic vesicles, etc., of animal and plant cells. These two physiologically different proton pumps exhibit similarities in subunit assembly, catalysis and the coupling mechanism from chemistry to proton transport through subunit rotation. We mostly discuss our own studies on the two proton pumps over the last three decades, including ones on purification, kinetic analysis, rotational catalysis and the diverse roles of acidic luminal organelles. The diversity of organellar proton pumps and their stochastic fluctuation are the important concepts derived recently from our studies.

We discuss the most recent reports on two proton pumps, F-ATPase (ATP synthase) and V-ATPase (endomembrane proton pump). They are formed from similar extrinsic (F 1 or V 1 ) and intrinsic (Fo or Vo) membrane sectors, and couple chemistry and proton transport through subunit rotation for apparently different physiological roles. Emphasis is placed on the stochastic rotational catalysis of F-ATPase and isoforms of V-ATPase.

In LQT3 patients, SCN5A mutations were found that lead to a small fraction of persistent hNav1.5 current. We explored the effects of such a change on the intracellular ionic homeostasis in a model of guinea-pig cardiac ventricular cell. At steady-state under 1 Hz stimulation, the presence of a persistent Na + current (I Nap ) with g Nap 0.02 ms/cm 2 led to a prolongation of the action potential from 153 ms (control) to 223 ms and an increase of [Na + ] i , diastolic and systolic [Ca 2+ ] i and [Ca 2+ ] SRup by 10 %, 30 %, 40 % and 43 %, respectively. These changes were larger at 3 Hz. Such intracellular Na + and Ca 2+ overload was not found when the action potential prolongation (to 222 ms at 1 Hz) was due to decreased I Kr and I Ks currents. The model with I Nap became arrhythmogenic when [K + ] e was lowered from 5.4 to 5.0 mM, whereas control and low K + current models did not produce arrhythmias even when [K + ] e was 2.5 mM.

> Les canaux ioniques dépendants du voltage (VGIC) représentent une large famille de protéines canalaires assurant le passage d'ions de part et d'autre de la membrane plasmique. Les VGIC ont la particularité d'être dotés d'un domaine senseur de voltage (VSD). Cette unité régule l'activité des VGIC en fonction des variations de potentiel membranaire. Récemment, des mutations décrites dans le VSD ont conduit à la découverte d'une nouvelle voie de perméation directement localisée au sein de ce domaine : le pore oméga. Au vu du grand nombre de protéines présentant un VSD et de leur rôle crucial dans diverses fonctions physiologiques, déchiffrer le potentiel pathogénique du pore oméga s'avère désormais essentiel. < Le mouvement du segment S4 possédant de 3 à 7 charges positives (apportées par des arginines et des lysines) induit un changement de conformation du pore aboutissant à son ouverture, et donc au passage des ions. Récemment, plusieurs études ont montré que des modifications du VSD peuvent générer une autre voie pour le passage des ions appelée pore oméga. L'objectif de cette revue est de présenter le concept de pore oméga, ses propriétés biophysiques ainsi que son potentiel pathologique.

Voltage gated sodium channels (Na v ) are transmembrane proteins responsible for action potential initiation. Mutations mainly located in the voltage sensor domain (VSD) of Na v 1.5, the cardiac sodium channel, have been associated with the development of arrhythmias combined with dilated cardiomyopathy. Gating pore currents have been observed with three unrelated mutations associated with similar clinical phenotypes. However, gating pores have never been associated with mutations outside the first domain of Na v 1.5. The aim of this study was to explore the possibility that gating pore currents might be caused by the Na v 1.5 R225P and R814W mutations (R3, S4 in DI and DII, respectively), which are associated with rhythm disturbances and dilated cardiomyopathy. Na v 1.5 WT and mutant channels were transiently expressed in tsA201 cells. The biophysical properties of the alpha pore currents and the presence of gating pore currents were investigated using the patch-clamp technique. We confirmed the previously reported gain of function of the alpha pores of the mutant channels, which mainly consisted of increased window currents mostly caused by shifts in the voltage dependence of activation. We also observed gating pore currents associated with the R225P and R814W mutations. This novel permeation pathway was open under depolarized conditions and remained temporarily open at hyperpolarized potentials after depolarization periods. Gating pore currents could represent a molecular basis for the development of uncommon electrical abnormalities and changes in cardiac morphology. We propose that this biophysical defect be routinely evaluated in the case of Na v 1.5 mutations on the VSD.

Fast inhibitory synaptic transmission in the brain relies on ionotropic GABAA receptors (GABAAR). Eighteen genes code for GABAAR subunits, but little is known about the ε subunit. Our aim was to identify the synaptic transmission properties displayed by native receptors incorporating ε. Immunogold localization detected ε at synaptic sites on locus coeruleus (LC) neurons. In situ hybridization revealed prominent signals from ε, and θ mRNAs, some low β1 and β3 signals, and no γ signal. Using in vivo extracellular and in vitro patch-clamp recordings in LC, we established that neuron firing rates, GABA-activated currents, and mIPSC charge were insensitive to the benzodiazepine flunitrazepam (FLU), in agreement with the characteristics of recombinant receptors including an ε subunit. Surprisingly, LC provided binding sites for benzodiazepines, and GABA-induced currents were potentiated by diazepam (DZP) in the micromolar range. A number of GABAAR ligands significantly potentiated GABA-in...

Amyloid fibrillation is associated with several human maladies, such as Alzheimer&#39;s, Parkinson&#39;s, Huntington&#39;s diseases, prions, amyotrophic lateral sclerosis, and type 2 diabetes diseases. Gaining insights into the mechanism of amyloid fibril formation and exploring novel approaches to fibrillation inhibition are crucial for preventing amyloid diseases. Here, we hypothesized that ligands capable of stabilizing the native state of query proteins might prevent protein unfolding, which, in turn, may reduce the propensity of proteins to form amyloid fibrils. We demonstrated the efficient inhibition of amyloid formation of the human serum albumin (HSA) (up to 85%) and human insulin (up to 80%) by a nonsteroidal anti-inflammatory drug, ibuprofen (IBFN). IBFN significantly increases the conformational stability of both HSA and insulin, as confirmed by differential scanning calorimetry (DSC). Moreover, increasing concentration of IBFN boosts its amyloid inhibitory propensity in...

Protein aggregation and amyloid fibrillation are responsible for several serious pathological conditions (like type II diabetes, Alzheimer&#39;s and Parkinson&#39;s diseases etc.) and protein drugs ineffectiveness. Therefore, a molecule that can inhibit the amyloid fibrillation and potentially clear amyloid fibrils is of great therapeutic value. In this manuscript, we investigated the antiamyloidogenic, fibril disaggregating, as well as cell protective effect of an anti-tuberculosis drug, Capreomycin (CN). Aggregation kinetics data, as monitored by ThT fluorescence, inferred that CN retards the insulin amyloid fibrillation by primarily targeting the fibril elongation step with little effect on lag time. Increasing the dose of CN boosted its inhibitory potency. Strikingly, CN arrested the growth of fibrils when added during the elongation phase, and disaggregated mature insulin fibrils. Our Circular Dichroism (CD) results showed that, although CN is not able to maintain the alpha hel...

The newly synthesized unfolded polypeptide attains its functional and unique three-dimensional conformation through the process of protein folding for which several models have been proposed. The protein misfolding diseases include Alzheimer&#39;s, Parkinson&#39;s and Cataract which are result of formation of amyloid or amorphous aggregates, respectively. The distinction in morphology shows relation with the melting temperature (Tm). The temperatures near or slightly higher than Tm induces amyloids while much higher or low temperature mediate amorphous aggregation. The aggregation is not always deleterious rather it also performs several important cellular functions essential for survival wide range of organisms called as functional amyloids. Protein gets modulated by several modulators which mediate the aggregation, acceleration, delay, transformations, inhibition and disaggregation of protein aggregates. The exclusive properties of inhibition and disaggregation displayed by variou...

This review article summarises the possible mechanisms of the protein-ligand interaction, folding, misfolding, aggregation and inhibition of protein aggregates. Under certain stressed condition the folding process deviates from its path and results into misfolding and aggregation of proteins. So aggregates have to be inhibited in order to cure the diseases. In some cases of protein-ligand interaction studies we have seen that the interaction of a protein with more than one ligand may show both type of quenching mechanisms i.e. dynamic as well as static quenching rather than single type of quenching mechanism, that result can be entirely different by the result of binding study utilising single ligand. So, likewise it is hypothesized that if the aggregates are inhibited by using more than one inhibitor may give more fruitful results rather than application of single inhibitor in inhibition and disaggregation of the preformed aggregates. Therefore, we have hypothesized mechanisms for the inhibition of protein aggregates that may assist in curing the neurodegenerative diseases. Thus, besides the mechanism of protein-ligand interaction, folding, misfolding and aggregation; the hypothesized mechanisms for the inhibition of protein aggregates may show new route to researchers either directly or indirectly in treating the diseases.

The aggregation phenomenon (amyloid and amorphous) is associated with several pathological complications in human, such as Alzheimer&#39;s, Parkinson&#39;s, Huntington, Cataract diseases and Diabetes mellitus type 2. In the present study we are offering evidence and breaking the general belief with regard to the polyphenols action as protein aggregate inhibitors. Herein we confirm that tannic acid (TA) is not only an amyloid inducer, but also it switches one type of conformation, ultimately morphology, into another. We ascertain based on our findings that aggregates are not rigid structures and the stability can be challenged under certain conditions. This study also confirms that unfolded and amorphous aggregates can serve as precursors of amyloids and TA interactions with unordered aggregates (amorphous) bringing orderliness in the conformation via amyloidosis. The shifting of unordered conformation towards orderliness is governed by the modulation in surface hydrophobic patches i...

In this study we are reporting the conformational modulation as well as the plausible mechanism of structural perturbation in conalbumin (CA). The overall structure of CA is altered by the change in dielectric constant upon addition of fluoroalcohols (TFE and HFIP). We studied the formation of aggregates in CA at pH 7.0 by multiple structural probes in the presence of TFE and HFIP. The protein aggregates maximally in 15% (v/v) TFE and 3% (v/v) HFIP. ANS, ThT binding and transmission electron microscopy data suggest that the aggregates induced by TFE and HFIP have amyloid-like properties. Higher concentrations of TFE and HFIP causes increase in helical propensity. Far-UV circular dichroism, intrinsic fluorescence, ANS and ThT fluorescence data suggests that formation of a partially structured intermediate state precedes the onset of the aggregation process.

Cell membranes are intrinsically heterogeneous, as the local protein and lipid distribution is critical to physiological processes. Even in template systems embedding a single protein type, like purple membranes, there can be a different local response to external stimuli or environmental factors, resulting in heterogeneous conformational changes. Despite the dramatic advances of microspectroscopy techniques, the identification of the conformation heterogeneity is still a challenging task. Tip-enhanced infrared nanospectroscopy is here used to identify conformational changes connected to the hydration state of the transmembrane proteins contained in a 50 nm diameter cell membrane area, without the need for fluorescent labels. In dried purple membrane monolayers, areas with fully hydrated proteins are found among large numbers of molecules with randomly distributed hydration states. Infrared nanospectroscopy results are compared to the spectra obtained with diffraction-limited infrar...

Although silicon nanoparticles dispersed in liquids are used in various applications ranging from bio-labeling to hydrogen production, their reactivities with their solvents and their catalytic properties remain still unexplored. Here, we discovered that, because of their surface structures and mechanical strain, silicon nanoparticles react strongly with their solvents and may act as catalysts for the dehydrogenation, at room temperature, of secondary alcohols (e.g. isopropanol) into ketones and hydrogen. This catalytic reaction was monitored by gas chromatography, pH measurements, mass spectroscopy and solid-state NMR. This discovery provides new understanding of the role played by silicon nanoparticles, and nanosilicon in general, in their reactivity in solvents in general as well as being candidates in catalysis.

Uterine contractions during labor are discretely regulated by rhythmic action potentials (AP) of varying duration and form that serve to determine calcium-dependent force production. We have employed a computational biology approach to develop a fuller understanding of the complexity of excitation-contraction (E-C) coupling of uterine smooth muscle cells (USMC). Our overall aim is to establish a mathematical platform of sufficient biophysical detail to quantitatively describe known uterine E-C coupling parameters and thereby inform future empirical investigations of physiological and pathophysiological mechanisms governing normal and dysfunctional labors. From published and unpublished data we construct mathematical models for fourteen ionic currents of USMCs: Ca 2z currents (L-and T-type), Na z current, an hyperpolarization-activated current, three voltage-gated K z currents, two Ca 2z -activated K z current, Ca 2z -activated Cl current, non-specific cation current, Na z -Ca 2z exchanger, Na z -K z pump and background current. The magnitudes and kinetics of each current system in a spindle shaped single cell with a specified surface area:volume ratio is described by differential equations, in terms of maximal conductances, electrochemical gradient, voltage-dependent activation/ inactivation gating variables and temporal changes in intracellular Ca 2z computed from known Ca 2z fluxes. These quantifications are validated by the reconstruction of the individual experimental ionic currents obtained under voltageclamp. Phasic contraction is modeled in relation to the time constant of changing ½Ca 2z i . This integrated model is validated by its reconstruction of the different USMC AP configurations (spikes, plateau and bursts of spikes), the change from bursting to plateau type AP produced by estradiol and of simultaneous experimental recordings of spontaneous AP, ½Ca 2z i and phasic force. In summary, our advanced mathematical model provides a powerful tool to investigate the physiological ionic mechanisms underlying the genesis of uterine electrical E-C coupling of labor and parturition. This will furnish the evolution of descriptive and predictive quantitative models of myometrial electrogenesis at the whole cell and tissue levels.

Changes in action potentials of atrial myocytes and various ionic channels induced by chronic atrial fibrillation (AF) have been described in the human. The mechanisms underlying the AF-induced action potential duration (APD) shortening have not been clearly identified. In this study we modify two different computational models of electrical activity of human atrial myocytes by incorporating chronic AF induced changes in several of the ionic channels systems found in myocytes. We examine the ionic mechanisms underlying the AF induced APD reduction and the relative roles of different remodeled ionic channels in producing the APD reduction. In both models we have found that AF induced changes in the ionic channel conductances and kinetics are able to reproduce the APD reduction seen experimentally. AF-induced down regulation of L-type Ca current is insufficient to account for the observed APD reduction, but up regulation of I/sub Kl/ has a much greater influence.

Uterine contractions during labor are discretely regulated by rhythmic action potentials (AP) of varying duration and form that serve to determine calcium-dependent force production. We have employed a computational biology approach to develop a fuller understanding of the complexity of excitation-contraction (E-C) coupling of uterine smooth muscle cells (USMC). Our overall aim is to establish a mathematical platform of sufficient biophysical detail to quantitatively describe known uterine E-C coupling parameters and thereby inform future empirical investigations of physiological and pathophysiological mechanisms governing normal and dysfunctional labors. From published and unpublished data we construct mathematical models for fourteen ionic currents of USMCs: Ca 2z currents (L-and T-type), Na z current, an hyperpolarization-activated current, three voltage-gated K z currents, two Ca 2z -activated K z current, Ca 2z -activated Cl current, non-specific cation current, Na z -Ca 2z exchanger, Na z -K z pump and background current. The magnitudes and kinetics of each current system in a spindle shaped single cell with a specified surface area:volume ratio is described by differential equations, in terms of maximal conductances, electrochemical gradient, voltage-dependent activation/ inactivation gating variables and temporal changes in intracellular Ca 2z computed from known Ca 2z fluxes. These quantifications are validated by the reconstruction of the individual experimental ionic currents obtained under voltageclamp. Phasic contraction is modeled in relation to the time constant of changing ½Ca 2z i . This integrated model is validated by its reconstruction of the different USMC AP configurations (spikes, plateau and bursts of spikes), the change from bursting to plateau type AP produced by estradiol and of simultaneous experimental recordings of spontaneous AP, ½Ca 2z i and phasic force. In summary, our advanced mathematical model provides a powerful tool to investigate the physiological ionic mechanisms underlying the genesis of uterine electrical E-C coupling of labor and parturition. This will furnish the evolution of descriptive and predictive quantitative models of myometrial electrogenesis at the whole cell and tissue levels.

Uterine contractions during labor are discretely regulated by rhythmic action potentials (AP) of varying duration and form that serve to determine calcium-dependent force production. We have employed a computational biology approach to develop a fuller understanding of the complexity of excitation-contraction (E-C) coupling of uterine smooth muscle cells (USMC). Our overall aim is to establish a mathematical platform of sufficient biophysical detail to quantitatively describe known uterine E-C coupling parameters and thereby inform future empirical investigations of physiological and pathophysiological mechanisms governing normal and dysfunctional labors. From published and unpublished data we construct mathematical models for fourteen ionic currents of USMCs: Ca 2z currents (L-and T-type), Na z current, an hyperpolarization-activated current, three voltage-gated K z currents, two Ca 2z -activated K z current, Ca 2z -activated Cl current, non-specific cation current, Na z -Ca 2z exchanger, Na z -K z pump and background current. The magnitudes and kinetics of each current system in a spindle shaped single cell with a specified surface area:volume ratio is described by differential equations, in terms of maximal conductances, electrochemical gradient, voltage-dependent activation/ inactivation gating variables and temporal changes in intracellular Ca 2z computed from known Ca 2z fluxes. These quantifications are validated by the reconstruction of the individual experimental ionic currents obtained under voltageclamp. Phasic contraction is modeled in relation to the time constant of changing ½Ca 2z i . This integrated model is validated by its reconstruction of the different USMC AP configurations (spikes, plateau and bursts of spikes), the change from bursting to plateau type AP produced by estradiol and of simultaneous experimental recordings of spontaneous AP, ½Ca 2z i and phasic force. In summary, our advanced mathematical model provides a powerful tool to investigate the physiological ionic mechanisms underlying the genesis of uterine electrical E-C coupling of labor and parturition. This will furnish the evolution of descriptive and predictive quantitative models of myometrial electrogenesis at the whole cell and tissue levels.

The dynamics of electron injection have been investigated in intramolecular i-motif conjugates possessing stilbenediether (Sd) and perylenediimide (PDI) chromophores separated by either four or six hemiprotonated cytosine C-C base pairs assembled with synthetic loops. Circular dichroism spectra are consistent with the formation of i-motif structures in the absence or presence of Sd and PDI chromophores. The fluorescence of the Sd chromophore is essentially completely quenched by neighboring C-C base pairs, consistent with their function as an electron donor and electron acceptor, respectively. However, the fluorescence of the PDI chromophore is only partially quenched. The dynamics of electron injection from singlet Sd into the i-motif and subsequent charge recombination has been determined by femtosecond transient absorption (fsTA) spectroscopy and compared with the results for electron injection and charge recombination in Sd-linked hairpins possessing cytosine-guanine (C-G) or 5-...

The dynamics and efficiency of photoinduced charge transport has been investigated in DNA capped hairpins possessing a stilbenedicarboxamide (Sa) hole donor and stilbenediether (Sd) hole acceptor separated by DNA G-quadruplex structures possessing 2-to-4 tetrads by means of femtosecond and nanosecond transient absorption spectroscopy with global analysis. The results for the quadruplex structures are compared with those for the corresponding duplex structures having G-C base pairs in place of the G-tetrads. Following photoinduced charge separation to form a contact radical ion pair, hole transport to form the Sa(-•)/Sd(+•) charge-separated state is slower but more efficient for the quadruplex vs duplex structures. Thus, the G-quadruplex serves as an effective conduit for positive charge rather than as a hole trap when inserted into a duplex, as previously postulated.

DNA-based molecular electronics will require charges to be transported from one site within a 2D or 3D architecture to another. While this has been shown previously in linear, π-stacked DNA sequences, the dynamics and efficiency of charge transport across a DNA three-way junction (3WJ) have yet to be determined. Here, we present an investigation of hole transport and trapping across a DNA-based three-way junction systems by a combination of femtosecond transient absorption spectroscopy and molecular dynamics simulations. Hole transport across the junction is proposed to be gated by conformational fluctuations in the ground state which bring the transiently-populated hole carrier nucleobases into better aligned geometries on the nanosecond timescale, thus modulating the - electronic coupling along the base pair sequence.

Materials. Aq 1 and PDI 2 chromophores were synthesized, purified, and characterized as previously described. Oligonucleotide synthesis was carried out using phosphoramidite chemistry on an Expedite 8909 synthesizer. For Aq and PDI monomers the coupling time was extending to 15 minutes. For PDI an additional wash cycle using dichloromethane was added to the protocol. The oligonucleotides were deprotected by incubating in 30% ammonium hydroxide at room temperature for 24 h, purified by RP-HPLC and characterized using MALDI-TOF spectroscopy. Thermal dissociation profiles were recorded on a Perkin Elmer Lambda 2 UV/VIS spectrometer with a Peltier Temperature Programmer. Circular dichroism (CD) spectra were recorded on a Jasco-J-815 CD spectrometer.

Chemicals. The synthesis of the dumbbell and hairpin was reported earlier. 1 All DNA samples and conjugates were dissolved in 10 mM phosphate buffer (pH=7.2) with 0.1 M NaCl. Concentrations were such that the absorbance varied between 0.3 and 1.0 at the pump wavelength of 265 nm. A melting temperature of 55 °C was determined for the hairpin from the first derivative of the temperature-dependent absorbance at 250 nm (Figure ). This value is slightly higher than the melting temperature of 52°C measured by McCullagh et al. 1 for a similar hairpin with 8 AT base pairs. The melting temperature of the dumbbell is much higher than for the hairpin and could not be precisely determined from our measurements (Figure ). Earlier, McCullagh et al. reported a melting temperature of 78°C for this dumbbell.

We report here the effect of replacing one or both of the purine or pyrimidine blocks of a diblock stilbene donor-acceptor capped hairpin with locked nucleic acid (LNA) bases on the dynamics and efficiency of hole transport. The structures of the DNA and LNA:DNA hybrids are tentatively assigned to B-or A-type structures on the basis of their circular dichroism spectra. Replacing the bases in either the A-block or the G-block of the diblock DNA hairpin with LNA bases results in a modest decrease in the base-to-base hopping rate constant and quantum yield for charge separation. Somewhat larger decreases are observed when all of the purine or pyrimidine bases are replaced by LNA bases.

Plasmonic nanoantennas are key elements in nanophotonics capable of directing radiation or enhancing the transition rate of a quantum emitter. Slot-type magnetic-dipole nanoantennas, which are complementary structures of typical electric-dipole-type antennas, have received little attention, leaving their antenna properties largely unexplored. Here we present a novel magneticdipole-fed multi-slot optical Yagi-Uda antenna. By engineering the relative phase of the interacting surface plasmon polaritons between the slot elements, we demonstrate that the optical antenna exhibits highly unidirectional radiation to free space. The unique features of the slotbased magnetic nanoantenna provide a new possibility of achieving integrated features such as energy transfer from one waveguide to another by working as a future "optical via.

The first steps of vision take place within a stack of tightly packed disc-shaped membranes, or "discs", located in the outer segment compartment of photoreceptor cells. In rod photoreceptors, discs are enclosed inside the outer segment and contain deep indentations in their rims called "incisures". The presence of incisures has been documented in a variety of species, yet their role remains elusive. In this study, we combined traditional electron microscopy with threedimensional electron tomography to demonstrate that incisures are formed only after discs become completely enclosed. We also observed that, at the earliest stage of their formation, discs are not round as typically depicted but rather are highly irregular in shape and resemble expanding lamellipodia. Using genetically manipulated mice and frogs and measuring outer segment protein abundances by quantitative mass spectrometry, we further found that incisure size is determined by the molar ratio between peripherin-2, a disc rim protein critical for the process of disc enclosure, and rhodopsin, the major structural component of disc membranes. While a high perpherin-2 to rhodopsin ratio causes an increase in incisure size and structural complexity, a low ratio precludes incisure formation. Based on these data, we propose a model whereby normal rods express a modest excess of peripherin-2 over the amount required for complete disc enclosure in order to ensure that this important step of disc formation is accomplished. Once the disc is enclosed, the excess peripherin-2 incorporates into the rim to form an incisure.

In the cochlea, Reissner&#39;s membrane separates the scala media endolymphatic compartment that sustains the positive endocochlear potential and ion composition necessary for sound transduction, from the scala vestibuli perilymphatic compartment. It is known that with sustained elevated sound levels, adenosine 5&#39;-triphosphate (ATP) is released into the endolymph and ATP-gated ion channels on the epithelial cells lining the endolymphatic compartment shunt the electrochemical driving force, contributing to protective purinergic hearing adaptation. This study characterises the properties of epithelial cell P2X2-type ATP-activated membrane conductance in the mouse Reissner&#39;s membrane, which forms a substantial fraction of the scale media surface. The cells were found to express two isoforms (a and b) of the P2X2 subunit arising from alternative splicing of the messenger RNA (mRNA) transcript that could contribute to the trimeric subunit assembly. The ATP-activated conductance d...

Active transport by renal proximal tubules plays a significant role in drug disposition. During drug development, estimates of renal excretion are essential to dose determination. Kidney bioreactors that reproduce physiologic cues in the kidney, such as flowinduced shear stress, may better predict in vivo drug behavior than do current in vitro models. In this study, we investigated the role of shear stress on active transport of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP1) by Madin-Darby canine kidney cells exogenously expressing the human organic cation transporters organic cation transporter 2 (OCT2) and multidrug and toxin extrusion protein 1 (MATE1). Cells cultured in a parallel plate under continuous media perfusion formed a tight monolayer with a high barrier to inulin. In response to increasing levels of shear stress (0.2-2 dynes/cm 2 ), cells showed a corresponding increase in transport of ASP1, reaching a maximal 4.2-fold increase at 2 dynes/cm 2 compared with cells cultured under static conditions. This transport was inhibited with imipramine, indicating active transport was present under shear stress conditions. Cells exposed to shear stress of 2 dynes/cm 2 also showed an increase in RNA expression of both transfected human and endogenous OCT2 (3.7-and 2.0-fold, respectively). Removal of cilia by ammonium sulfate eliminated the effects of shear on ASP1 transport at 0.5 dynes/cm 2 with no effect on ASP1 transport under static conditions. These results indicate that shear stress affects active transport of organic cations in renal tubular epithelial cells in a cilia-dependent manner.

Hemodialysis using hollow-fiber membranes provides life-sustaining treatment for nearly 2 million patients worldwide with end stage renal disease (ESRD). However, patients on hemodialysis have worse long-term outcomes compared to kidney transplant or other chronic illnesses. Additionally, the underlying membrane technology of polymer hollow-fiber membranes has not fundamentally changed in over four decades. Therefore, we have proposed a fundamentally different approach using microelectromechanical systems (MEMS) fabrication techniques to create thin-flat sheets of silicon-based membranes for implantable or portable hemodialysis applications. The silicon nanopore membranes (SNM) have biomimetic slit-pore geometry and uniform pores size distribution that allow for exceptional permeability and selectivity. A quantitative diffusion model identified structural limits to diffusive solute transport and motivated a new microfabrication technique to create SNM with enhanced diffusive transpo...

The role electrical charge plays in determining glomerular permeability to macromolecules remains unclear. If the glomerular basement membrane (GBM) has any significant role in permselectivity, physical principles would suggest a negatively charged GBM would reject similarly charged more than neutral species. However, recent in vivo studies with negative and neutral glomerular probes showed the opposite. Whether this observation is due to unique characteristics of the probes used or is a general physiological phenomenon remains to be seen. The goal of this study was to use the basement membrane deposited by Madin-Darby canine kidney epithelial cells as a simple model of a biologically derived, negatively charged filter to evaluate size- and charge-based sieving properties. Fluorescein isothiocyanate-labeled carboxymethylated Ficoll 400 (FITC-CM Ficoll 400) and amino-4-methyl-coumarin-labeled Ficoll 400 (AMC Ficoll 400) were used as negatively charged and neutral tracer molecules, re...

Vascular endothelial growth factor A (VEGF-A) is a promoter of neovascularization and thus a popular therapeutic target for diseases involving excessive growth of blood vessels. In this study, we explored the potential of the disaccharide sucrose octasulfate (SOS) to alter VEGF165 diffusion through Descemet's membrane. Descemet's membranes were isolated from bovine eyes and used as a barrier between two chambers of a diffusion apparatus to measure VEGF transport. Diffusion studies revealed a dramatic increase in VEGF165 transport in the presence of SOS, with little diffusion of VEGF165 across the membrane over a 10-h time course in the absence of SOS. Diffusion studies with VEGF121, a non-heparin binding variant of VEGF, showed robust diffusion with or without SOS. To determine a possible mechanism, we measured the ability of SOS to inhibit VEGF interactions with extracellular matrix (ECM), using cell-free and cell surface binding assays. Binding studies showed SOS had no effect on VEGF165 binding to either heparin-coated plates or endothelial cell surfaces at less than mg/ml concentrations. In contrast, we show that SOS inhibited VEGF165 binding to fibronectin in a dose dependent manner and dramatically accelerated the rate of release of VEGF165 from fibronectin. SOS also inhibited the binding of VEGF165 to fibronectin-rich ECM deposited by vascular smooth muscle cells. These results suggest that fibronectin-rich extracellular matrices serve as barriers to VEGF165 diffusion by providing a network of binding sites that can trap and sequester the protein. Since the content of Descemet's membrane is typical of many basement membranes it is possible that they serve throughout the body as formidable barriers to VEGF165 diffusion and tightly regulate its bioavailability and distribution within tissues.

We examine theoretically the ability of diffusible cytoplasmic carriers to facilitate delivery of substrate to sites within biological membranes. Two processes are analyzed. In one, delivery of Ca to a site on a membrane surface is examined. As a contrasting example, chelator-supported diffusion to a uniform absorbing surface is considered.

We examine theoretically the ability of diffusible cytoplasmic carriers to facilitate delivery of substrate to sites within biological membranes. Two processes are analyzed. In one, delivery of Ca to a site on a membrane surface is examined. As a contrasting example, chelator-supported diffusion to a uniform absorbing surface is considered.

It has been proposed that the concentration of proteins in the cytoplasm maximizes the speed of important biochemical reactions. Here we have used the Xenopus extract system, which can be diluted or concentrated to yield a range of cytoplasmic protein concentrations, to test the effect of cytoplasmic concentration on mRNA translation and protein degradation. We found that protein synthesis rates are maximal in ~1x cytoplasm, whereas protein degradation continues to rise to an optimal concentration of ~1.8x. This can be attributed to the greater sensitivity of translation to cytoplasmic viscosity, perhaps because it involves unusually large macromolecular complexes like polyribosomes. The different concentration optima sets up a negative feedback homeostatic system, where increasing the cytoplasmic protein concentration above the 1x physiological level increases the viscosity of the cytoplasm, which selectively inhibits translation and drives the system back toward the 1x set point.

Cellular adhesion plays an important role in numerous fundamental physiological and pathological processes. Its measurement is relatively complex, requires sophisticated equipment, and, in most cases, cannot be carried out without breaking the links between the studied cell and its target. In this contribution, we propose a novel, nanomotion-based, technique that overcomes these drawbacks. The applied force is generated by the studied cell itself (nanomotion), whereas cellular movements are detected by traditional optical microscopy and dedicated software. The measurement is non-destructive, single-cell sensitive, and permits following the evolution of the adhesion as a function of time. We applied the technique on different strains of the fungal pathogen Candida albicans on a fibronectin-coated surface. We demonstrated that this novel approach can significantly simplify, accelerate, and make more affordable living cells–substrate adhesion measurements.

The use of the Individual Ventilatory Threshold (IVT), as parameter to prescribe exercise intensity in individuals with obesity, has become more frequent during the last years. This study aimed to evaluate the relationship between IVT and Maximal Fat Oxidation (MFO) in women with obesity. Fifty-two obese female adults (age = 43.6±10.9 years; BMI = 38.5±5.2 kg/m 2 ) were included in this study. According to the BMI classification, subjects were divided into three groups: Obese Class I (OBI, n = 16); Obese Class II (OBII, n = 20) and Obese Class III (OBIII, n = 16). All subjects performed an incremental graded exercise test to evaluate peak oxygen uptake (VO 2peak ), IVT and MFO. MFO was evaluated using a stoichiometric equation. Fat max zone was determined for each subject within 10% of fat oxidation rates at MFO. For each HR, %HR max , VO 2 and %VO 2peak variable, Pearson's correlation test was done between IVT and MFO exercise intensity. When statistical correlation was found we used a comparative statistical analysis to assess differences between IVT and MFO. Statistical significance was set at P � 0.05. For each HR, %HR max , VO 2 and %VO 2peak variable there was a positive significant correlation (P<0.01) between IVT and MFO. No significant differences were found for HR, % HR max , and VO 2 between IVT and MFO. %VO 2peak was significantly higher at IVT than at MFO (P = 0.03).

Biodiversity-based approaches are at the forefront of creating new varieties in soybean production. In this paper, we analyzed the natural variability in the population of cultivated soybean varieties used in breeding in the Russian Federation using genome-wide association studies. Genome regions that control the protein and oil contents in seeds, as well as productivity, have been identified, many of which have not been described previously. These results open the way to the design of new markers for marker selection of this crop.

The modulation of large conductance Ca2+‐activated K+ (BKCa) channels by the nitric oxide (NO·) donor, S‐nitroso‐L‐cysteine (NOCys) and three sulfhydryl‐specific methanethiosulfonate (MTS) reagents, positively charged 2‐aminoethyl MTS hydrobromide (MTSEA C3H9NO2S2HBr) and [2‐(trimethylammonium) ethyl MTS bromide (MTSET C6H16NO2S2Br), and negatively charged sodium (2‐sulfonatoethyl) MTS (MTSES C3H7O5S3Na) were compared in excised inside‐out membrane patches of the guinea‐pig taenia caeca. In membrane patches bathed in a low Ca2+ (15 nM) high K+ physiological salt solution, 1–3 BKCa channels opened with a low probability (N.Po) of 0.019±0.011 at 0 mV. N.Po readily increased with membrane depolarization, raised Ca2+ concentration or upon the addition of NOCys (10 μM for 2–5 min) such that 5–15 open BKCa channels were evident. MTSEA (2.5 mM) decreased, while MTSES (2.5 mM) increased N.Po (at 0 mV) and the number of open BKCa channels at positive potentials. These changes in channel acti...

Apd1, a cytosolic yeast protein, and Aim32, its counterpart in the mitochondrial matrix, have a C-terminal thioredoxinlike ferredoxin domain and a widely divergent N-terminal domain. These proteins are found in bacteria, plants, fungi and unicellular pathogenic eukaryotes, but not in Metazoa. Our chemogenetic experiments demonstrate that the highly conserved cysteine and histidine residues within the C-X8-C-X24-75-H-X-G-G-H motif of the TLF domain of Apd1 and Aim32 proteins are essential for viability upon treatment of yeast cells with the redox potentiators gallobenzophenone or pyrogallol, respectively. UV-Vis, EPR and Mössbauer spectroscopy of purified wild type Apd1 and three His to Cys variants demonstrated that Cys207 and Cys216 are the ligands of the ferric ion and His255 and His259 are the ligands of the reducible iron ion of the [2Fe-2S]2+/1+ cluster. The [2Fe-2S] center of Apd1 (Em,7 = -164±5 mV, pKox1,2=7.9±0.1 and 9.7±0.1) differs from both dioxygenase (Em,7 ≈ -150 mV, pK...

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins 1-4 . In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N 2 ; <2 ppm O 2 ) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.

Semiconductor photoelectrodes for photoelectrochemical (PEC) water splitting require efficient carrier generation, separation and transport at and beyond the space charge region (SCR) formed at the aqueous interface. The trade-off between photon collection and minority carrier delivery governs the photoelectrode design and implies maximum water splitting efficiency at an electrode thickness equivalent to the light absorption depth. Here, using planar ZnO thin films as a model system, we identify the photocarriers beyond the SCR as another significant source to substantially enhance the PEC performance. The high-quality ZnO films synthesized by pulsed laser deposition feature very few deep trap states and support a long photocarrier lifetime. Combined with photoelectrochemical characterization, ultrafast spectroscopy and numerical calculations, it is revealed that engineering the exciton concentration gradient by film thickness facilitates the inward diffusion of photocarriers from the neighboring illuminated region to the SCR, and therefore achieves a record high quantum efficiency over 80% at a thickness far beyond its light absorption depth and the SCR width. These results elucidate the important role of the photocarriers beyond SCR for PEC process and provide new insight to explore the full potential for efficient photoelectrode materials with large exciton diffusivity.

The authors state that there was a minor error in Fig. (a) of the original article 1 where the images shown under DU145 panel for both EpCAM and nucleolin functionalized devices are same as those under Caco-2. The first author apologizes for this unintentional human error and states that this does not change the scientific conclusions of the article in any way. The corrected Fig. is given below.

Understanding the functional relevance of G protein-coupled receptor (GPCR) homodimerization has been limited by the insufficient tools to assess asymmetric signaling occurring within dimers comprised of the same receptor type. We present unmatched bivalent ligands (UmBLs) to study the asymmetric function of melanocortin homodimers. UmBLs contain one agonist and one antagonist pharmacophore designed to target a melanocortin homodimer such that one receptor is occupied by an agonist and the other receptor by an antagonist pharmacophore. First-in-class biased UmBLs (BUmBLs) targeting the human melanocortin-4 receptor (hMC4R) were discovered. The BUmBLs displayed biased agonism by potently stimulating cAMP signaling *

Pharmacological probes for the melanocortin receptors have been utilized for studying various disease states including cancer, sexual function disorders, Alzheimer's disease, social disorders, cachexia, and obesity. This study focused on the design and synthesis of bivalent ligands to target melanocortin receptor homodimers. Lead ligands increased binding affinity by 14-to 25-fold and increased cAMP signaling potency by 3-to 5-fold compared to their monovalent counterparts. Unexpectedly, different bivalent ligands showed preferences for particular melanocortin receptor subtypes depending on the linker that connected the binding scaffolds suggesting structural differences between the various dimer subtypes. Homobivalent compound 12 (CJL-1-140) possessed a functional profile that was unique from its monovalent counterparts providing evidence of the discrete effects of bivalent ligands. Lead compound 7 (CJL-1-87) significantly decreased feeding in mice after intracerebroventricular administration. To the best of our knowledge, this is the first report of a melanocortin bivalent ligand's in vivo physiological effects.

The melanocortin-3 (MC3R) and melanocortin-4 (MC4R) receptors are expressed in the brain and are implicated in the regulation of food intake and energy homeostasis. The endogenous agonist ligands for these receptors (α-, β-, γ-MSH and ACTH) are linear peptides with limited receptor subtype selectivity and metabolic stability, thus minimizing their use as probes to characterize the overlapping pharmacological and physiological functions of the melanocortin receptor subtypes. In the present study, an engineered template, in which the peptide backbone was modified by a heterocyclic reverse turn mimetic at the Trp 7 residue, was synthesized using solid phase peptide synthesis and characterized by a β-galactosidase cAMP based reporter gene assay. The functional assay identified a ∼5 nM mouse MC4R agonist (AST3-88) with more than 50-fold selectivity over the mMC3R. Biophysical studies (2D 1 H NMR spectroscopy and molecular dynamics) of AST3-88 identified a type VIII β-turn secondary structure spanning the pharmacophore domain stabilized by the intramolecular interactions between the side chains of the His and Trp residues. Enzymatic studies of AST3-88 revealed enhanced stability of AST3-88 over the α-MSH endogenous peptide in rat serum. Upon central administration of AST3-88 into rats, a decreased food intake response was observed. This is the first study to probe the in vivo physiological activity of this engineered peptide-heterocycle template. These findings advance the present knowledge of pharmacophore design for potent, selective, and metabolically stable melanocortin ligands.