Fluorescence recovery after photobleaching (FRAP) enables characterization of quantitative dynamic properties such as diffusion coefficients of fluorescent molecules in living cells by analyzing the recovery of fluorescence intensity after photobleaching in a specific cellular compartment or area. To quantitatively determine high intracellular diffusion coefficients, a suitable optical system as well as an appropriate model for fast diffusion analysis is necessary. Here, we propose a procedure to quantify the diffusion coefficient of rapidly-diffusing fluorescent molecules that makes use of an epi-fluorescence microscope with a photobleaching laser in combination with established models for diffusion analysis. Analysis for the diffusion coefficients of tandemly oligomerized green flurescent proteins (GFPs) in living cells when changing the photobleaching times showed that photobleaching with shorter times than the diffusion speed indicated not the only way to obtain appropriate diffusion coefficients of fast-moving molecules. Our results also showed that the apparent spreading of the effective radius of the photobleached area works as a correction factor for determining appropriate diffusion coefficients of fast-moving molecules like monomeric GFPs. Our procedure provides a useful approach for quantitative measurement of diffusion coefficients in living cells. This procedure is relevant for characterizing dynamic molecular interactions, especially of fast-moving molecules, and is relevant for studies in many biological fields.
Aureochrome1 is a blue-light-receptor protein identified in a stramenopile alga, Vaucheria frigida. Photozipper (PZ) is an N-terminally truncated, monomeric, V. frigida aureochrome1 fragment containing a basic leucine zipper (bZIP) domain and a light–oxygen–voltage (LOV)-sensing domain. PZ dimerizes upon photoexcitation and consequently increases its affinity for the target sequence. In the present study, to understand the equilibria among DNA complexes of PZ, DNA binding by PZ and mutational variants was quantitatively investigated by electrophoretic-mobility-shift assay and fluorescence-correlation spectroscopy in the dark and light states. DNA binding by PZ was sequence-specific and light-dependent. The half-maximal effective concentration of PZ for binding to the target DNA sequence was ~40 nM in the light, which was >10-fold less than the value in the dark. By contrast, the dimeric PZ-S2C variant (with intermolecular disulfide bonds) had higher affinity for the target sequence, with dissociation constants of ~4 nM, irrespective of the light conditions. Substitutions of Glu159 and Lys164 in the leucine zipper region decreased the affinity of PZ for the target sequence, especially in the light, suggesting that these residues form inter-helical salt bridges between leucine zipper regions, stabilizing the dimer–DNA complex. Our quantitative analyses of the equilibria in PZ–DNA-complex formation suggest that the blue-light-induced dimerization of LOV domains and coiled-coil formation by leucine zipper regions are the primary determinants of the affinity of PZ for the target sequence.
Photolyases (PHRs) and cryptochromes (CRYs) belong to the same family known as blue-light photoreceptors. Although their amino acid sequences and corresponding structures are similar to each other, they exert different functions. PHRs function as an enzyme to repair UV-induced deoxyribonucleic acid (DNA) lesions such as a cyclobutane pyrimidine dimer (CPD) and a (6-4) photoproduct ((6-4)pp), whereas CRYs are a circadian photoreceptor in plants and animals and at the same time they control the photoperiodic induction of flowering in plants. When a new type cryptochrome was identified, it was assumed that another type of CRYs, cryptochrome-DASH (CRY-DASH), which is categorized as a subfamily of photolyase/cryptochrome family, would possess the DNA photolyase activity. However, CRY-DASH had a weak DNA photolyase activity, but the reason for this is still unclear. To clarify the reason, we performed molecular dynamics (MD) simulations for a complex of CPD-PHR or CRY-DASH with damaged double-stranded DNA (dsDNA) and estimated the binding free energy, ΔGbind, between the protein and the damaged dsDNA by using a molecular mechanics/Poisson–Boltzmann surface area (MM/PBSA) method. ΔGbind for both proteins were –35 and 57 kcal mol–1, respectively, indicating that the structural stability of CRY-DASH was lower than that of CPD-PHR upon the damaged dsDNA binding. In particular, the number of amino acid residues relevant to the damaged dsDNA binding on the CRY-DASH surface was smaller than that on CPD-PHR. Therefore, the present result suggests that CRY-DASH has a weak DNA photolyase activity because it has a lower binding affinity than CPD-PHR.
The bacterial flagellar hook is a short, highly curved tubular structure connecting the basal body as a rotary motor and the filament as a helical propeller to function as a universal joint to transmit motor torque to the filament regardless of its orientation. This highly curved form is known to be part of a supercoil as observed in the polyhook structure. The subunit packing interactions in the Salmonella hook structure solved in the straight form gave clear insights into the mechanisms of its bending flexibility and twisting rigidity. Salmonella FlgE consists of four domains, D0, Dc, D1 and D2, arranged from inside to outside of the tube, and an atomic model of the supercoiled hook built to simulate the hook shape observed in the native flagellum suggested that the supercoiled form is stabilized by near-axial interactions of the D2 domains on the inner surface of the supercoil. Here we show that the deletion of domain D2 from FlgE makes the hook straight, providing evidence to support the proposed hook supercoiling mechanism that it is the near-axial interactions between the D2 domains that stabilize the highly curved hook structure.
Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that belongs to the ATP binding cassette protein superfamily. Deletion of phenylalanine at position 508 (ΔF508) is the most common CF-associated mutation and is present in nearly 90% of CF patients. Currently, atomistic level studies are insufficient for understanding the mechanism by which the deletion of a single amino acid causes greatly reduced folding as well as trafficking and gating defects. To clarify this mechanism, we first constructed an atomic model of the inward-facing ΔF508-CFTR and performed all-atom molecular dynamics (MD) simulations of the protein in a membrane environment. All of the computational methodologies used are based on those developed in our previous study for wild-type CFTR. Two important findings were obtained. First, consistent with several previous computational results, the deletion of F508 causes a disruption of a hydrophobic cluster located at the interface between the nucleotide binding domain 1 (NBD1) and intracellular loop 4 (ICL4). This exerts unfavorable influences on the correlated motion between ICLs and transmembrane domains (TMDs), likely resulting in gating defects. Second, the F508 deletion affected the NBD1–NBD2 interface via allosteric communication originating from the correlated motion between NBDs and ICLs. As a result, several unusual inter-residue interactions are caused at the NBD1–NBD2 interface. In other words, their correct dimerization is impaired. This study provided insight into the atomic-level details of structural and dynamics changes caused by the ΔF508 mutation and thus provides good insight for drug design.
The electronic structure of manganese (Mn) cluster in photosystem II was investigated by electron paramagnetic resonance (EPR) spectroscopy. In order to determine the spin density distribution in magnetically coupled Mn in the S2 state Mn cluster, pulsed electron–electron double resonance (PELDOR) measurement was performed. The local environment of the Mn cluster was investigated by electron-nuclear double resonance (ENDOR). Using spin projections determined by PELDOR, ENDOR signals were assigned to the water molecules ligated to the Mn cluster. The location of a high-affinity Mn2+ site in apo-photosystem II, which is the initial site of photoactivation of the Mn cluster, was determined by PELDOR.
Self-propelled rods, which propel by themselves in the direction from the tail to the head and align nematically through collision, have been well-investigated theoretically. Various phenomena including true long-range ordered phase with the Giant number fluctuations, and the collective motion composed of many vorices were predicted using the minimal mathematical models of self-propelled rods. Using filamentous bacteria and running microtubules, we found that the predicted phenomena by the minimal models occur in the real world. This strongly indicates that there exists the unified description of self-propelled rods independent of the details of the systems. The theoretically predicted phenomena and the experimental results concerning the phenomena are reviewed.
We propose methods for directly analyzing structural compliance (SC) properties of elastic network models of proteins, and we also propose methods for extracting information about motion properties from the SC properties. The analysis of SC properties involves describing the relationships between the applied forces and the deformations. When decomposing the motion according to the magnitude of SC (SC mode decomposition), we can obtain information about the motion properties under the assumption that the lower SC mode motions or the softer motions occur easily. For practical applications, the methods are formulated in a general form. The parts where forces are applied and those where deformations are evaluated are separated from each other for enabling the analyses of allosteric interactions between the specified parts. The parts are specified not only by the points but also by the groups of points (the groups are treated as flexible bodies). In addition, we propose methods for quantitatively evaluating the properties based on the screw theory and the considerations of the algebraic structures of the basic equations expressing the SC properties. These methods enable quantitative discussions about the relationships between the SC mode motions and the motions estimated from two different conformations; they also help identify the key parts that play important roles for the motions by comparing the SC properties with those of partially constrained models. As application examples, lactoferrin and ATCase are analyzed. The results show that we can understand their motion properties through their lower SC mode motions or the softer motions.
Organisms generally display two contrasting properties: large biodiversity and a uniform state of “life”. In this study, we focused on the question of how genome sequences describe “life” where a large number of biomolecules are harmonized. We analyzed the whole genome sequence of 2664 organisms, paying attention to the nucleotide composition which is an intensive parameter from the genome sequence. The results showed that all organisms were plotted in narrow regions of the nucleotide composition space of the first and second letters of the codon. Since all genome sequences overlap irrespective of the living environment, it can be called a “habitable zone”. The habitable zone deviates by 500 times the standard deviation from the nucleotide composition expected from the random sequence, indicating that unexpectedly rare sequences are realized. Furthermore, we found that the habitable zones at the first and second letters of the codon serve as the background mechanisms for the functional network of biological systems. The habitable zone at the second letter of the codon controls the formation of transmembrane regions and the habitable zone at the first letter controls the formation of molecular recognition unit. These analyses showed that the habitable zone of the nucleotide composition space and the exquisite arrangement of amino acids in the codon table are conjugated to form biological systems. Finally, we discussed the evolution of the higher order of genome sequences.
We have developed a three-dimensional structure database of natural metabolites (3DMET). Early development of the 3DMET database relied on content auto-generated from 2D-structures of other chemical databases. From 2009, we began manual curation, obtaining new compounds from published works. In the process of curation, problems of digitizing 3D-structures from structure drawings of documents were accumulated. As the same as auto-generation, structure drawings should be also payed attention about stereochemistry. Our experiences in manual curation of 3DMET, as described herein, may be useful to others in this field of research and for the development of supporting systems of a chemical structure database. Manual curation is still necessary for proper database entry of the 3D-configurations of chiral atoms, a problem encountered frequently among natural products.
Membrane transporter proteins play important roles in transport of nutrients into the cell, in transport of waste out of the cell, in maintenance of homeostasis, and in signal transduction. Solute carrier (SLC) transporter is the superfamily, which has the largest number of genes (>400 in humans) in membrane transporter and consists of 52 families. SLC transporters carry a wide variety of substrates such as amino acids, peptides, saccharides, ions, neurotransmitters, lipids, hormones and related materials. Despite the apparent importance for the substrate transport, the information of sequence variation and three-dimensional structures have not been integrated to the level of providing new knowledge on the relationship to, for instance, diseases. We, therefore, built a new database named iMusta4SLC, which is available at http://cib.cf.ocha.ac.jp/slc/, that connected the data of structural properties and of pathogenic mutations on human SLC transporters. iMusta4SLC helps to investigate the structural features of pathogenic mutations on SLC transporters. With this database, we found that the mutations at the conserved arginine were frequently involved in diseases, and were located at a border between the membrane and the cytoplasm. Especially in SLC families 2 and 22, the conserved residues formed a large cluster at the border. In SLC2A1, one third of the reported pathogenic missense mutations were found in this conserved cluster.
We report the development of the SEVENS database, which contains information on G-protein coupled receptor (GPCR) genes that are identified with high confidence levels (A, B, C, and D) from various eukaryotic genomes, by using a pipeline comprising bioinformatics softwares, including a gene finder, a sequence alignment tool, a motif and domain assignment tool, and a transmembrane helix predictor.
SEVENS compiles detailed information on GPCR genes, such as chromosomal mapping position, phylogenetic tree, sequence similarity to known genes, and protein function described by motif/domain and transmembrane helices. They are presented in a user-friendly interface. Because of the comprehensive gene findings from genomes, SEVENS contains a larger data set than that of previous databases and enables the performance of a genome-scale overview of all the GPCR genes. We surveyed the complete genomes of 68 eukaryotes, and found that there were between 6 and 3,470 GPCR genes for each genome (Level A data). Within these genes, the number of receptors for various molecules, including biological amines, peptides, and lipids, were conserved in mammals, birds, and fishes, whereas the numbers of odorant receptors and pheromone receptors were highly diverse in mammals. SEVENS is freely available at http://sevens.cbrc.jp or http://sevens.chem.aoyama.ac.jp.
We studied the effect of myosin inhibitors, N-benzyl-p-toluenesulfonamide (BTS), blebbistatin, and butanedione monoxime (BDM) on X-ray diffraction patterns from rabbit psoas fibers under relaxing and contracting conditions. The first two inhibitors suppressed the contractile force almost completely at a 100 μM concentration, and a similar effect was obtained at 50 mM for BDM. However, still substantial changes were observed in the diffraction patterns upon calcium-activation of inhibited muscle fibers. (1) The 2nd actin layer-line reflection was enhanced normally, indicating that calcium binding to troponin and the subsequent movement of tropomyosin are not inhibited, (2) the myosin layer-line reflections became much weaker, and (3) the 1,1/1,0 intensity ratio of the equatorial reflections was increased. The observations (2) and (3) indicate that, even in the presence of the inhibitors at a saturating concentration, myosin heads leave the helix on the thick filaments and approach the thin filaments. Interestingly, the d1,0 spacing of the filament lattice remained unchanged upon activation of inhibited fibers, in contrast to the case of normal activation in which the spacing is decreased. This suggests that the normal activated myosin heads exert a pull in both axial and radial directions, but in the presence of the inhibitors, the pull is suppressed, and as a result, the heads simply bind to actin without exerting any force. The results support the idea that the inhibitors do not block the myosin binding to actin, but block the step of force-producing transition of the bound actomyosin complex.
In many microorganisms helical structures are important for motility, e.g., bacterial flagella and kink propagation in Spiroplasma eriocheiris. Motile archaea also form a helical-shaped filament called the ‘archaellum’ that is functionally equivalent to the bacterial flagellum, but structurally resembles type IV pili. The archaellum motor consists of 6–8 proteins called fla accessory genes, and the filament assembly is driven by ATP hydrolysis at catalytic sites in FlaI. Remarkably, previous research using a dark-field microscopy showed that right-handed filaments propelled archaeal cells forwards or backwards by clockwise or counterclockwise rotation, respectively. However, the shape and rotational rate of the archaellum during swimming remained unclear, due to the low signal and lack of temporal resolution. Additionally, the structure and the motor properties of the archaellum and bacterial flagellum have not been precisely determined during swimming because they move freely in three-dimensional space. Recently, we developed an advanced method called “cross-kymography analysis”, which enables us to be a long-term observation and simultaneously quantify the function and morphology of helical structures using a total internal reflection fluorescence microscope. In this review, we introduce the basic idea of this analysis, and summarize the latest information in structural and functional characterization of the archaellum motor.
To find a causal mechanism of learning and memory is a heuristically important topic in neuroscience. In the pond snail Lymnaea stagnalis, the following experimental facts have accrued regarding a classical conditioning procedure known as conditioned taste aversion (CTA): (1) one-day food-deprived Dutch snails have superior CTA memory formation; (2) the one-day food-deprived snails have a low monoamine content (e.g., serotonin, dopamine, octopamine) in their central nervous system (CNS); (3) fed or five-day food-deprived snails have poorer CTA memory and a higher monoamine content; (4) the Dutch snails form better CTA memory than the Canadian TC1 strain; and, (5) the F1 cross snails between the Dutch and Canadian TC1 strains also form poor CTA memory. Here, in one-day food-deprived snails, we measured the monoamine content in the CNSs of the 3 populations. In most instances, the monoamine content of the Dutch strain was lower than in the other two populations. The F1 cross snails had the highest monoamine content. A lower monoamine content is correlated with the better CTA memory formation.
When three cyanobacterial proteins, KaiA, KaiB, and KaiC, are incubated with ATP in vitro, the phosphorylation level of KaiC hexamers shows stable oscillation with approximately 24 h period. In order to understand this KaiABC clockwork, we need to analyze both the macroscopic synchronization of a large number of KaiC hexamers and the microscopic reactions and structural changes in individual KaiC molecules. In the present paper, we explain two coarse-grained theoretical models, the many-molecule (MM) model and the single-molecule (SM) model, to bridge the gap between macroscopic and microscopic understandings. In the simulation results with these models, ATP hydrolysis in the CI domain of KaiC hexamers drives oscillation of individual KaiC hexamers and the ATP hydrolysis is necessary for synchronizing oscillations of a large number of KaiC hexamers. Sensitive temperature dependence of the lifetime of the ADP bound state in the CI domain makes the oscillation period temperature insensitive. ATPase activity is correlated to the frequency of phosphorylation oscillation in the single molecule of KaiC hexamer, which should be the origin of the observed ensemble-level correlation between the ATPase activity and the frequency of phosphorylation oscillation. Thus, the simulation results with the MM and SM models suggest that ATP hydrolysis stochastically occurring in each CI domain of individual KaiC hexamers is a key process for oscillatory behaviors of the ensemble of many KaiC hexamers.
An enormous amount of research has been performed to characterize actin dynamics. Structural biology investigations have determined the localization of main chains and their changes coupled with G (Globular)-F (Filamentous) transformation of actin, whereas local thermal fluctuations that may be caused by free rotations of the tips of side chains are not yet fully investigated. This paper argues if the entropy change of actin accompanied by the G-F transformation is simply attributable to the changes in hydration. It took almost 10 years to understand that the actin filament is semi-flexible. This flexibility was visually confirmed as the development of optical microscope techniques, and the direct observation of actin severing events in the presence of actin binding proteins became possible. Finally, I expect the deep understanding of actin dynamics will lead to the elucidation of self-assembly mechanisms of the living creature.
An individual’s personality develops through a combination of experiences and parental inheritance. When faced with a conflict, will an individual take an innate behavior or a learned one? In such situations, individuality will manifest itself. Here, we focused on turn alternation behavior, which is a habitual tendency to turn in the direction opposite the preceding turn, in earthworms (Eisenia fetida) and examined how this behavior is affected by an aversive stimulus. Of 10 earthworms, 3 were affected by the stimulus. Turn alternation deteriorated in two worms, one of which showed anti-turn alternation behavior, whereas the remaining worm showed an enhanced tendency toward turn alternation. Earthworms have a relatively simple nervous system. This study opens the door to investigate the neuronal basis for individuality that emerges between nature and nurture.
The replica-exchange molecular dynamics (REMD) method has been used for conformational sampling of various biomolecular systems. To maximize sampling efficiency, some adjustable parameters must be optimized. Although it is agreed that shorter intervals between the replica-exchange attempts enhance traversals in the temperature space, details regarding the artifacts caused by these short intervals are controversial. In this study, we revisit this problem by performing REMD simulations on an alanine octapeptide in an implicit solvent. Fifty different sets of conditions, which are a combination of five replica-exchange periods, five different numbers of replicas, and two thermostat coupling time constants, were investigated. As a result, although short replica-exchange intervals enhanced the traversals in the temperature space, they led to artifacts in the ensemble average of the temperature, potential energy, and helix content. With extremely short replica-exchange intervals, i.e., attempted at every time step, the ensemble average of the temperature deviated from the thermostat temperature by ca. 7 K. Differences in the ensembles were observed even for larger replica-exchange intervals (between 100 and 1,000 steps). In addition, the shorter thermostat coupling time constant reduced the artifacts found when short replica-exchange intervals were used, implying that these artifacts are caused by insufficient thermal relaxation between the replica-exchange events. Our results will be useful to reduce the artifacts found in REMD simulations by adjusting some key parameters.
Type III secretion system (T3SS) is a protein translocator complex family including pathogenic injectisome or bacterial flagellum. The inejectisomal T3SS serves to deliver virulence proteins into host cell and the flagellar T3SS constructs the flagellar axial structure. Although earlier studies have provided many findings on the molecular mechanism of the Type III protein export, they were not sufficient to reveal energy transduction mechanism due to difficulties in controlling measurement conditions in vivo. Recently, we developed an in vitro flagellar Type III protein transport assay system using inverted membrane vesicles (IMVs), and analyzed protein export by using the in vitro method. We reproduced protein export of the flagellar T3SS, hook assembly and substrate specificity switch in IMV to a similar extent to what is seen in living cell. Furthermore, we demonstrated that ATP-hydrolysis energy can drive protein transport even in the absence of proton-motive force (PMF). In this mini-review, we will summarize our new in vitro Type III transport assay method and our findings on the molecular mechanism of Type III protein export.
Anion channelrhodopsin-2 (ACR2), a light-gated channel recently identified from the cryptophyte alga Guillardia theta, exhibits anion channel activity with exclusive selectivity. In addition to its novel function, ACR2 has become a focus of interest as a powerful tool for optogenetics. Here we combined experimental and computational approaches to investigate the roles of conserved carboxylates on the anion transport activity of ACR2 in Escherichia coli membrane. First, we replaced six conserved carboxylates with a neutral residue (i.e. E9Q, E56Q, E64Q, E159Q, E219Q and D230N), and measured anion transport activity using E. coli expression system. E159Q and D230N exhibited significantly lower anion transport activity compared with wild-type ACR2 (1/12~1/3.4), which suggests that E159 and D230 play important roles in the anion transport. Second, to explain its molecular aspects, we constructed a homology model of ACR2 based on the crystal structure of a cation channelrhodopsin (ChR). The model structure showed a cavity formed by four transmembrane helices (TM1, TM2, TM3 and TM7) similar to ChRs, as a putative anion conducting pathway. Although E159 is not located in the putative pathway, the model structure showed hydrogen bonds between E159 and R129 with a water molecule. D230 is located in the pathway near the protonated Schiff base (PSB) of the chromophore retinal, which suggests that there is an interaction between D230 and the PSB. Thus, we demonstrated the functional importance and the hypothetical roles of two conserved carboxylates, E159 and D230, in the anion transport activity of ACR2 in E. coli membrane.
The nucleus in eukaryotic cells is the site for genomic functions such as RNA transcription, DNA replication, and DNA repair/recombination. However, the nucleus is subjected to various mechanical forces associated with diverse cellular activities, including contraction, migration, and adhesion. Although it has long been assumed that the lamina structure, underlying filamentous meshwork of the nuclear envelope, plays an important role in resisting mechanical forces, the involvement of compact chromatin in mechanical resistance has also recently been suggested. However, it is still unclear how chromatin functions to cope with the stresses. To address this issue, we studied the mechanical responses of human cell nuclei by combining a force measurement microscopy setup with controlled biochemical manipulation of chromatin. We found that nuclei with condensed chromatin possess significant elastic rigidity, whereas the nuclei with a decondensed chromatin are considerably soft. Further analyses revealed that the linker DNA and nucleosome-nucleosome interactions via histone tails in the chromatin act together to generate a spring-like restoring force that resists nuclear deformation. The elastic restoring force is likely to be generated by condensed chromatin domains, consisting of interdigitated or “melted” 10-nm nucleosome fibers. Together with other recent studies, it is suggested that chromatin functions not only as a “memory device” to store, replicate, and express the genetic information for various cellular functions but also as a “nuclear spring” to resist and respond to mechanical forces.
Spiral waves are observed in wide variety of reaction-diffusion systems. Those observed in cardiac tissues are important since they are related to serious disease that threatens human lives, such as atrial or ventricular fibrillation. We consider the unpinning of spiral waves anchored to a circular obstacle on excitable media using high-frequency pacing. Here, we consider two types of the obstacle; i.e., that without any diffusive interaction with the environment, and that with diffusive interaction. We found that the threshold frequency for success in unpinning is lower for the obstacle with diffusive interaction than for the one without it. We discuss the threshold frequency based on the angular velocity of a chemical wave anchoring the obstacle.
This investigation understands the interaction between lyophilized crude Viper snake venom (Doboia russellie) and Silver nanoparticles (SNPs) using biophysical and biochemical approaches. SNPs were synthesized by chemical reduction method and characterized using UV-Visible spectroscopy, Dynamic Light Scattering (DLS) and Transmission electron microscope (TEM). The average hydrodynamic size of SNPs was found to be 52 nm with 0.261 PDI. TEM image revealed the spherical shape of SNP. Interaction of SNPs and viper venom was resulted in the formation of complex which was confirmed by using DLS technique. Spectroscopic results showed an increase in absorbance intensity of venom upon interaction with SNPs which indicated interaction with venom proteins. Fluorescence spectroscopic data revealed the quenching in the fluorescence intensity of viper venom upon incubation with varying concentration of SNPs. The results obtained by biochemical assays (Protease and whole blood clotting test) revealed the inhibition of venom action due to presence of silver nanoparticles. The activity of protease enzyme was found to be decreased (10–13% reduction) in presence of silver nanoparticles. Prolonged clotting time (two fold) of viper venom upon interaction with SNPs compared to native crude viper venom was observed. The overall results confirmed the inhibition action of silver nanoparticles against viper venom.
Both sensory neurons and motor neurons transfer signals rapidly through long pathways. Such signals propagate as action potentials through neurons. In myelinated neurons, high conduction velocities of 120 m/s have been reported, even for axons of just 20 µm in diameter. Such a high conduction velocity is enabled by the characteristic morphology of a myelinated axon: repeated regions encased by long uniform myelin sheaths alternating with extremely short exposed regions of the axon called nodes of Ranvier, which generate extremely sharp action potentials. Although the need for the action potential to cross many nodes increases the relay time, it is still able to propagate rapidly. This phenomenon motivated us to derive a new mechanism of the action potential propagation. First, the dielectric effect of the axonal fluid was considered, and it was investigated whether the combination of the characteristic axonal morphology and the dielectric constant of the axonal fluid contributes significantly to the realization of high conduction velocities even with the inclusion of a large loss in the relay time. To this end, we propose a new axon equivalent circuit that incorporates the effect of the dielectric characteristics of the axonal fluid. It was confirmed that a realistically high conduction velocity could be calculated using the proposed circuit and that the dielectric constant calculated using the proposed circuit was in agreement with that of an ionic fluid similar to axonal fluid. Moreover, the contribution of the combination of the axonal morphology and axonal fluid to the conduction velocity was confirmed.
Thermometers play an important role to study the biological significance of temperature. Fluorescent nanodiamonds (FNDs) with negatively-charged nitrogen-vacancy centers, a novel type of fluorescence-based temperature sensor, have physicochemical inertness, low cytotoxicity, extremely stable fluorescence, and unique magneto-optical properties that allow us to measure the temperature at the nanoscale level inside single cells. Here, we demonstrate that the thermosensing ability of FNDs is hardly influenced by environmental factors, such as pH, ion concentration, viscosity, molecular interaction, and organic solvent. This robustness renders FNDs reliable thermometers even under complex biological cellular environment. Moreover, the simple protocol developed here for measuring the absolute temperature inside a single cell using a single FND enables successful temperature measurement in a cell with an accuracy better than ±1°C.
A wild plant called Skunk Cabbage is known to heat itself and keep its body warm before spring. We study its homothermal maintenance mechanism from a mesoscopic point of view. We take the increment process of the temperature time series and consider it as ‘elastic’ force that always tries to backlash its temperature to an intrinsic target temperature. We then propose a kind of extended Poisson distribution for the model of the ‘elastic’ force. The hypothesis testing result by Kolmogorov-Smirnov test suggests that the proposed distribution is a plausible candidate of the model for the ‘elastic’ force, on the temperature range in which the system is in equillibrium. In addition, it turns out that the parameters in the model captures well the linear behaviour of the expectations of the ‘elastic’ force at each of the present temperatures and similarly, the constancy of the variances of the force. Especially, the linearity of the expected increments over displacements of tempertures indicates that the backlash might be considered to be like the elastic force of a spring as described by Fuch’s law.
Recent evidence has suggested that chromatin is not simply repeats of the canonical nucleosome, called the “octasome”, but may include diverse repertoires of basic structural units. During the transcription process, a nucleosome is repositioned by a chromatin remodeler and collides with a neighboring nucleosome, thus creating an unusual nucleosome substructure termed the “overlapping dinucleosome”. We previously developed a method for the large-scale preparation of the overlapping dinucleosome. This method enabled us to solve the crystal structure of the overlapping dinucleosome, which revealed an unexpected structure composed of an octameric histone core associated with a hexameric histone core lacking one H2A-H2B dimer.
Aquaporins (AQPs) are protein channels located across the cell membrane which conduct the water permeation through the cell membrane. Different types of AQPs exist in human organs and play vital roles, as the malfunction of such protein membranes can lead to life-threatening conditions. A specific type of AQP, identified as AQP5, is particularly essential to the generation of saliva, tears and pulmonary secretions. We have adopted Molecular Dynamics (MD) simulation to analyze the water permeation and diffusion in AQP5 structure in a 0.5 microsecond simulation time window. The MD numerical simulation shows the water permeability of the human AQP5 is in the nominal range for other members of human aquaporins family. In addition, we have considered the effect of the osmotic water diffusion and the diffusion occurred by pressure gradient on the protein membrane. The water permeability grows monotonically as the applied pressure on the solvent increases. Furthermore, the forced diffusion increases the minimum radius of Selectivity Filter (SF) region of region AQP5 up to 20% and consequently the permeability coefficients enhance enormously compared to osmotic self-diffusion in AQP5 tetramer. Finally, it is revealed that the MD simulation of human AQP5 provides useful insights into the mechanisms of water regulation through alveolar cells under the different physical conditions; osmotic self-diffusion and forced diffusion condition.