Electric field allowed molecular transitions for one and two photon excitation microscopy

We propose an excitation technique for observing single and two photon excitation in those molecules for which such transitions are forbidden by the selection rules. This is possible by the application of an external electric field that perturbs the molecular orbitals, thereby resulting in a significant shift of energy levels. Such a shift of energy levels may bring those levels in resonance with the radiation field which is normally forbidden by selection rules. Further, parity of the these states may significantly improve the emission process. The external electric field results in the mixing of excited (short lifetime) and metastable states (long lifetime), thus reducing the lifetime of metastable (or near metastable) states. This may provide an effective channel for allowing transition from the metastable states. An application of electric field may result in the excitation of poorly excitable biomolecules. This excitation technique may find applications in single- and multi-photon fluorescence microscopy, bioimaging and optical devices.     

A mechanism for action of oscillating electric fields on cells

The biological effects of electromagnetic fields have seriously concerned the scientific community and the public as well in the past decades as more and more evidence has accumulated about the hazardous consequences of so-called "electromagnetic pollution." This theoretical model is based on the simple hypothesis that an oscillating external electric field will exert an oscillating force to each of the free ions that exist on both sides of all plasma membranes and that can move across the membranes through transmembrane proteins. This external oscillating force will cause a forced vibration of each free ion. When the amplitude of the ions' forced vibration transcends some critical value, the oscillating ions can give a false signal for opening or closing channels that are voltage gated (or even mechanically gated), in this way disordering the electrochemical balance of the plasma membrane and consequently the whole cell function.     

Oscillating viscometer--evaluation of a new bedside test

A viscometer for bedside blood measurements was developed, consisting of an oscillating resonator probe mounted directly into a disposable vacutainer tube for blood withdrawal. It was tested in vitro on blood samples with variable hematocrits (20-60%), increasing fibrinogen concentrations (0-20 g/l), increasing concentrations of an admixed radiographic contrast medium and erythrocyte suspensions in dextran 40 and dextran 70. Results were compared with those obtained with a conventional Couette viscometer. Oscillating viscometry yielded generally higher values than Couette viscometry, and had a good sensitivity for changes in hematocrit with a good correlation between the two methods (r=0.96, p<0.0001). Oscillating viscosity depended on the resonator frequency, it was higher at 3900 Hz than at 215 Hz, suggesting a viscoelastic behavior of blood. Erythrocyte aggregation, induced by increasing fibrinogen concentrations or dextran 70, affected oscillating viscometry. At a high frequency, i.e. a smaller penetration depth of the shear wave, oscillating viscosity tended to decrease, which suggests a depletion of the boundary layer from erythrocytes when they aggregate. At low frequency with a deeper shear wave penetration (about 50 microm), erythrocyte aggregation increased oscillating viscosity. Bedside tests in 17 patients with coronary heart disease and 10 controls confirmed the easy practicability of the test and showed lower oscillating viscosity in these patients despite higher fibrinogen concentrations presumably due to increased erythrocyte aggregation. We conclude that oscillating viscometry is an interesting bedside test, which is capable of providing new information on the biorheology of the erythrocyte-poor boundary layer near the vessel wall.     

Oscillating reactions in open systems

The probable existence of oscillating chemical reactions has been attracting some interest in recent years for their possible role in explaining certain biological phenomena. Perhaps the simplest model of oscillating reactions is that of Lotka (1910), which consists of a chain of autocatalytic reactions. Two “reactor systems” in which such a chain of reactions could take place are considered in this work and are called homogeneous and compartmental models, respectively. The differential equations governing the temporal behavior of the reacting species are solved on an analog computer, and the conditions under which sustained oscillations occur are obtained and discussed. Comparisons of the solution obtained in the two models are discussed.     

Locally disordered conformer of the hamster prion protein: a crucial intermediate to PrPSc?

A crucial step for transformation of the normal cellular isoform of the prion protein (PrP(C)) to the infectious prion protein (PrP(Sc)) is thought to entail a previously uncharacterized intermediate conformer, PrP*, which interacts with a template PrP(Sc) molecule in the conversion process. By carrying out (15)N-(1)H two-dimensional NMR measurements under variable pressure on Syrian hamster prion protein rPrP(90-231), we found a metastable conformer of PrP(C) coexisting at a population of approximately 1% at pH 5.2 and 30 degrees C, in which helices B and C are preferentially disordered. While the identity is still unproven, this observed metastable conformer is most logically PrP* or a closely related precursor. The structural characteristics of this metastable conformer are consistent with available immunological and pathological information about the prion protein.     

Effects of diurnally oscillating pCO2 on the calcification and survival of coral recruits

Manipulative studies have demonstrated that ocean acidification (OA) is a threat to coral reefs, yet no experiments have employed diurnal variations in pCO(2) that are ecologically relevant to many shallow reefs. Two experiments were conducted to test the response of coral recruits (less than 6 days old) to diurnally oscillating pCO(2); one exposing recruits for 3 days to ambient (440 μatm), high (663 μatm) and diurnally oscillating pCO(2) on a natural phase (420-596 μatm), and another exposing recruits for 6 days to ambient (456 μatm), high (837 μatm) and diurnally oscillating pCO(2) on either a natural or a reverse phase (448-845 μatm). In experiment I, recruits exposed to natural-phased diurnally oscillating pCO(2) grew 6-19% larger than those in ambient or high pCO(2). In experiment II, recruits in both high and natural-phased diurnally oscillating pCO(2) grew 16 per cent larger than those at ambient pCO(2), and this was accompanied by 13-18% higher survivorship; the stimulatory effect on growth of oscillatory pCO(2) was diminished by administering high pCO(2) during the day (i.e. reverse-phased). These results demonstrate that coral recruits can benefit from ecologically relevant fluctuations in pCO(2) and we hypothesize that the mechanism underlying this response is highly pCO(2)-mediated, night-time storage of dissolved inorganic carbon that fuels daytime calcification.     

Structure and thermal history dependent phase behavior of hydrated synthetic sphingomyelin analogue: 1,2-dimyristamido-1,2-deoxyphosphatidylcholine

The physical properties of hydrated multilamellar sample of 1,2-dimyristamido-1,2-deoxyphosphatidylcholine (DDPC) were investigated by means of differential scanning calorimetry (DSC), static X-ray diffraction, and simultaneous DSC and X-ray diffraction. The DDPC is a synthetic sphingomyelin analogue and has two amide bonds in its hydrophobic parts. This paper reports on metastable phase behavior of the hydrated DDPC sample. By cooling from a chain-melted state at the rates of greater than 4 degrees C min(-1), hydrated DDPC bilayers form a metastable gel phase. In the gel phase, the hydrophobic chains are tilted with respect to the bilayer normal, as like the gel phase of glycero-phosphatidylcholines. By heating, the metastable gel phase is transformed in to a stable phase associated with an exothermic heat event at 18.3 degrees C (DeltaH=14.6 kJ mol(-1)) and then the stable phase is transformed into a liquid-crystalline phase at 25.6 degrees C (DeltaH=42 kJ mol(-1)). The incubation at 17 degrees C for more than 1 h also induces the formation of the stable phase. In the stable phase, the hydrophobic chains are packed into highly ordered crystal-like structure. However, the X-ray diffraction pattern of the stable phase suggested that the entire DDPC molecules do not form a two-dimensional molecular ordered lattice, differing from normal subgel phase of glycero-phosphatidylcholines. The structure and phase behavior of DDPC revealed by the present study are discussed from the viewpoint of hydrogen bonds.     

The “mechanostat” principle in cell differentiation. The osteochondroprogenitor paradigm

The “mechanostat” principle may be depicted as an oscillating signal of a signaling molecule, in which the amplitude, frequency, cumulative level, delay, and duration of the curve encode the information for concrete cellular responses and biological activities. When the oscillating signal is kept sustained (present delay), cell exit may be performed, whereas when the oscillating signal remains robust, cell proliferation may take place. B-catenin–Wnt signaling pathway has a key role in the differentiation of osteochondroprogenitor cells. Sustained downregulation of the β-catenin–Wnt pathway forces osteochondroprogenitors to a chondrogenic fate instead of an osteoblastic one. Other signaling, for example, bone morphogenetic protein and Notch signaling pathways interact with the Wnt pathway. The crosstalk between biochemical and mechanical stimuli produces the final information that leads to the final cell fate decisions, through the “mechanostat” principle.     

Damped oscillating processes in biological and biochemical systems

Damped nonlinear oscillations in biological and biochemical systems are investigated by the extended Krylov-Bogoliubov-Mitropolskii (KBM) method. A review on the extension made by Popov to the KBM method is given and also further improvements are presented. Applications are made to models of oscillating chemical reactions (Lefever and Nicolis, 1971), FitzHugh (1961) equations, and population dynamics (Gatto and Rinaldi, 1977). Comparison to damped oscillating physical and engineering systems is made.     

Oscillating fluid flow regulates cytosolic calcium concentration in bovine articular chondrocytes.

Mechanical loading is a well-known regulator of cartilage metabolism. This suggests that a loading-induced physical signal regulates chondrocyte behavior. Previous studies have focused on the effects of steady fluid flow on chondrocytes. In contrast to steady flow, loading induced fluid flow occurs in an oscillatory pattern and includes a reversal of flow direction with each loading event. In this study we examined the hypothesis that oscillating fluid flow increases cytosolic Ca2+ concentration ([Ca2+]i) in bovine articular chondrocytes (BAC) in a frequency-dependent manner and that the presence of serum affects this response. The aims of our study were to examine (1) whether BAC respond to physiologic oscillating fluid flow in vitro and compare these results to steady fluid flow, (2) the effect of fetal bovine serum on fluid flow responsiveness of BAC and (3) whether the response of BAC to fluid flow is flow rate and/or frequency dependent. [Ca2+]i was quantified using the fluorescent dye fura-2. BAC were exposed to steady, 0.5, 1, or 5 Hz sinusoidal oscillating fluid flow at five different flow rates in a parallel plate flow chamber. Our findings demonstrate that BAC respond to oscillating fluid flow with an increase in [Ca2+]i (p > 0.05), and furthermore, chondrocyte responsiveness to fluid flow increases with peak flow rate (p < 0.0001) and decreases with increasing frequencies (p < 0.0001). Finally, the presence of serum in the media potentiated the responsiveness of BAC to fluid flow (p < 0.0001). Our results suggest an important role for mechanical load-induced oscillating fluid flow in chondrocyte mechanotransduction.     

Post-translational modification detection using metastable ions in reflector matrix-assisted laser desorption/ionization-time of flight mass spectrometry

In addition to protein identification, characterization of post-translational modifications (PTMs) is an essential task in proteomics. PTMs represent the major reason for the variety of protein isoforms and they can influence protein structure and function. Upon matrix-assisted laser desorption/ionization (MALDI) most post-translationally modified peptides form a fraction of labile molecular ions, which lose PTM-specific residues only after acceleration. Compared to fully accelerated ions these fragment ions are defocused and show in reflector mass spectra reduced resolution. A short time Fourier transform using a Hanning window function now uses this difference in resolution to detect the metastable fragments. Its application over the whole mass range yields frequency distributions and amplitudes as a function of mass, where an increased low frequency proportion is highly indicative for metastable fragments. Applications on the detection of metastable losses originating from carboxamidomethylated cysteines, oxidized methionines, phosphorylated and glycosylated amino acid residues are presented. The metastable loss of mercaptoacetamide detected with this procedure represents a new feature and its integration in search algorithms will improve the specificity of MALDI peptide mass fingerprinting.     

未折叠蛋白质的普遍初始热力学亚稳态

探索和理解蛋白质折叠问题一直是分子生物学、结构生物学和生物物理学的终极挑战.未折叠的蛋白质应该存在一种普遍初始热力学亚稳态,否则无法解释蛋白质是如何在剧烈的热振动干扰下完成快速精确折叠的.本文通过分析水溶液环境和蛋白质折叠的相关性,揭示了一种由水分子屏蔽效应引起的未折叠蛋白质的普遍初始热力学亚稳态,该亚稳态的存在是水溶液环境中水分子的物理性质决定,并赋予未折叠蛋白质抵抗热扰动和避免错误折叠的能力.我们通过研究已发表的实验数据和建立分子模型,找到了该初始热力学亚稳态存在的相关证据,并推测了该亚稳态导致蛋白质精确折叠的相关物理学机制.     

Variable histone modifications at the Avy metastable epiallele

The ability of environmental factors to shape health and disease involves epigenetic mechanisms that mediate gene-environment interactions. Metastable epiallele genes are variably expressed in genetically identical individuals due to epigenetic modifications established during early development. DNA methylation within metastable epialleles is stochastic due to probabilistic reprogramming of epigenetic marks during embryogenesis. Maternal nutrition and environment have been shown to affect metastable epiallele methylation patterns and subsequent adult phenotype. Little is known, however, about the role of histone modifications in influencing metastable epiallele expression and phenotypic variation. Utilizing chromatin immunoprecipitation followed by qPCR, we observe variable histone patterns in the 5′ long terminal repeat (LTR) of the murine viable yellow agouti (A^vy) metastable epiallele. This region contains 6 CpG sites, which are variably methylated in isogenic A^vy/a offspring. Yellow mice, which are hypomethylated at the A^vy LTR and exhibit constitutive ectopic expression of Agouti (a), also display enrichment of H3 and H4 di-acetylation (p = 0.08 and 0.09, respectively). Pseudoagouti mice, in which A^vy hypermethylation is thought to silence ectopic expression, exhibit enrichment of H4K20 tri-methylation (p = 0.01). No differences are observed for H3K4 tri-methylation (p = 0.7), a modification often enriched in the promoter of active genes. These results show for the first time the presence of variable histone modifications at a metastable epiallele, indicating that DNA methylation acts in concert with histone modifications to affect inter-individual variation of metastable epiallele expression. Therefore, the potential for environmental factors to influence histone modifications, in addition to DNA methylation, should be addressed in environmental epigenomic studies.Key words: epigenetics, metastable epiallele, viable yellow agouti, histone, environmental epigenomics     

The conformational dynamics of a metastable serpin studied by hydrogen exchange and mass spectrometry

Serpins are a class of protease inhibitors that initially fold to a metastable structure and subsequently undergo a large conformational change to a stable structure when they inhibit their target proteases. How serpins are able to achieve this remarkable conformational rearrangement is still not understood. To address the question of how the dynamic properties of the metastable form may facilitate the conformational change, hydrogen/deuterium exchange and mass spectrometry were employed to probe the conformational dynamics of the serpin human alpha(1)-antitrypsin (alpha(1)AT). It was found that the F helix, which in the crystal structure appears to physically block the conformational change, is highly dynamic in the metastable form. In particular, the C-terminal half of the F helix appears to spend a substantial fraction of time in a partially unfolded state. In contrast, beta-strands 3A and 5A, which must separate to accommodate insertion of the reactive center loop (RCL), are not conformationally flexible in the metastable state but are rigid and stable. The conformational lability required for loop insertion must therefore be triggered during the inhibition reaction. Beta-strand 1C, which anchors the distal end of the RCL and thus prevents transition to the so-called latent form, is also stable, consistent with the observation that alpha(1)AT does not spontaneously adopt the latent form. A surprising degree of flexibility is seen in beta-strand 6A, and it is speculated that this flexibility may deter the formation of edge-edge polymers.     

Role of secondary electrons and metastable atoms in the electron-beam activation of argon-silane mixtures

The energy and spatial degradation of the primary beam electrons and the production of high-energy secondary electrons in ionizing collisions are analyzed by solving the Boltzmann integral equation for the electron distribution function. The effect of the primary and secondary electrons on the direct ionization of an Ar-SiH₄ mixture, the production of metastable argon atoms, and the dissociation of monosilane molecules is investigated over a wide range of the beam electron energies, argon pressures, and monosilane concentrations. The influence of metastable Ar* atoms on the dissociation of SiH₄ is studied by using the balance equation for metastable argon atoms and the equation for the ambipolar diffusion of ions and low-energy secondary (plasma) electrons in the beam plasma. It is shown that the main contribution to the activation of an Ar-SiH₄ mixture in an electron-beam plasma is provided by secondary electrons with energies higher than the excitation threshold for argon and the dissociation threshold for monosilane, whereas the contribution from metastable argon atoms, though potentially being comparable with that from secondary electrons, is less than in gas-discharge plasmas.     

Effect of oscillating fluid flow stimulation on osteocyte mRNA expression

Structural adaptation of the bone tissue is mediated by loading-induced interstitial fluid flow within the bone microstructure. Within this framework, osteocytes fulfill the central mechanotransductive role in the bone remodeling process. While osteocytes have been demonstrated to be exquisitely sensitive to various forms of fluid flow stimulus in vitro, the effect of different oscillating fluid flow (OFF) parameters on osteocyte activity has yet to be systematically characterized. In this study, we investigate the effect of three OFF parameters on osteocyte activity in vitro and hypothesize that COX-2, RANKL, and OPG mRNA expression in osteocytes are sensitive to the OFF parameters: peak shear stress amplitude (0.5 Pa, 1 Pa, 2 Pa, and 5 Pa), oscillating frequency (0.5 Hz, 1 Hz, and 2 Hz), and total flow duration (1 h, 2 h, and 4 h). Our findings demonstrate that COX-2 mRNA levels are elevated in osteocytes subjected to higher peak shear stress amplitudes and longer flow durations, while RANKL/OPG mRNA levels decreased to a minimum threshold in response to higher peak shear stress amplitudes, faster oscillating frequencies, and longer flow durations. These findings suggest that dynamic fluid flow with higher peak shear stress amplitudes, faster oscillating frequencies, and longer loading durations provide the best conditions for promoting bone formation.     

Variable histone modifications at the Avy metastable epiallele

The ability of environmental factors to shape health and disease involves epigenetic mechanisms that mediate gene-environment interactions. Metastable epiallele genes are variably expressed in genetically identical individuals due to epigenetic modifications established during early development. DNA methylation within metastable epialleles is stochastic due to probabilistic reprogramming of epigenetic marks during embryogenesis. Maternal nutrition and environment have been shown to affect metastable epiallele methylation patterns and subsequent adult phenotype. Little is known, however, about the role of histone modifications in influencing metastable epiallele expression and phenotypic variation. Utilizing chromatin immunoprecipitation followed by qPCR, we observe variable histone patterns in the 5’ long terminal repeat (LTR) of the murine viable yellow agouti (A^vy) metastable epiallele. This region contains 6 CpG sites, which are variably methylated in isogenic A^vy/a offspring. Yellow mice, which are hypomethylated at the Avy LTR and exhibit constitutive ectopic expression of agouti (a), also display enrichment of H3 and H4 di-acetylation (p=0.08 and 0.09, respectively). Pseudoagouti mice, in which A^vy hypermethylation is thought to silence ectopic expression, exhibit enrichment of H4K20 tri-methylation (p=0.01). No differences are observed for H3K4 tri-methylation (p=0.7), a modification often enriched in the promoter of active genes. These results show for the first time the presence of variable histone modifications at a metastable epiallele, indicating that DNA methylation acts in concert with histone modifications to affect inter-individual variation of metastable epiallele expression. Therefore, the potential for environmental factors to influence histone modifications, in addition to DNA methylation, should be addressed in environmental epigenomic studies.     

Structure and metastability of N-lignocerylgalactosylsphingosine (cerebroside) bilayers

Differential scanning calorimetry (DSC) and X-ray diffraction have been used to study hydrated N-lignocerylgalactosylsphingosine (NLGS) bilayers. DSC of fully hydrated NLGS shows an endothermic transition at 69-70 degrees C, immediately followed by an exothermic transition at 72-73 degrees C; further heating shows a high-temperature (Tc = 82 degrees C), high-enthalpy (delta H = 15.3 kcal/mol NLGS) transition. Heating to 75 degrees C, cooling to 20 degrees C and subsequent reheating shows no transitions at 69-73 degrees C; only the high-temperature (82 degrees C), high-enthalpy (15.3 kcal/mol) transition. Two exothermic transitions are observed on cooling; for the upper transition its temperature (about 65 degrees C) and enthalpy (about 6 kcal/mol NLGS) are essentially independent of cooling rate, whereas the lower transition exhibits marked changes in both temperature (30----60 degrees C) and enthalpy (2.2----9.5 kcal/mol NLGS) as the cooling rate decreases from 40 to 0.625 Cdeg/min. On reheating, the enthalpy of the 69-70 degrees C transition is dependent on the previous cooling rate. The DSC data provide clear evidence of conversions between metastable and stable forms. X-ray diffraction data recorded at 26, 75 and 93 degrees C show clearly that NLGS bilayer phases are present at all temperatures. The X-ray diffraction pattern at 75 degrees C shows a bilayer periodicity d = 65.4 A, and a number of sharp reflections in the wide-angle region indicative of a crystalline chain packing mode. This stable bilayer form converts to a liquid-crystal bilayer phase; at 93 degrees C, the bilayer periodicity d = 59.1 A, and a diffuse reflection at 1/4.6 A-1 is observed. The diffraction pattern at 22 degrees C represents a combination of the stable and metastable low-temperature bilayer forms. NLGS exhibits a complex pattern of thermotropic changes related to conversions between metastable (gel), stable (crystalline) and liquid-crystalline bilayer phases. The structure and thermotropic properties of NLGS are compared with those of hydrated N-palmitoylgalactosylsphingosine reported previously (Ruocco, M.J., Atkinson, D., Small, D.M., Skarjune, R.P., Oldfield, E. and Shipley, G.G. (1981) Biochemistry 20, 5957-5966).     

Aeropin from the extremophile Pyrobaculum aerophilum bypasses the serpin misfolding trap

Serpins are metastable proteinase inhibitors. Serpin metastability drives both a large conformational change that is utilized during proteinase inhibition and confers an inherent structural flexibility that renders serpins susceptible to aggregation under certain conditions. These include point mutations (the basis of a number of important human genetic diseases), small changes in pH, and an increase in temperature. Many studies of serpins from mesophilic organisms have highlighted an inverse relationship: mutations that confer a marked increase in serpin stability compromise inhibitory activity. Here we present the first biophysical characterization of a metastable serpin from a hyperthermophilic organism. Aeropin, from the archaeon Pyrobaculum aerophilum, is both highly stable and an efficient proteinase inhibitor. We also demonstrate that because of high kinetic barriers, aeropin does not readily form the partially unfolded precursor to serpin aggregation. We conclude that stability and activity are not mutually exclusive properties in the context of the serpin fold, and propose that the increased stability of aeropin is caused by an unfolding pathway that minimizes the formation of an aggregation-prone intermediate ensemble, thereby enabling aeropin to bypass the misfolding fate observed with other serpins.     

1,10‐Phenanthroline–H2O2–KSCN–CuSO4–NaOH oscillating chemiluminescence system

In this paper, oscillating chemiluminescence (CL), 1,10‐phenanthroline H₂O₂–KSCN–CuSO₄–NaOH system, was studied in a batch reactor. The system described is a novel, slowly damped oscillating CL system, generated by coupling the well‐known Epstein–Orban, H₂O₂–KSCN–CuSO₄–NaOH chemical oscillator reaction with the CL reaction involving the oxidation of 1,10‐phenanthroline by hydrogen peroxide, catalyzed by copper(II) in alkaline medium. In this system, the CL reaction acts as a detector or indicator system of the far‐from‐equilibrium dynamic system. Narrow and slightly asymmetric light pulses of 1.2 s half‐width are emitted at 440 nm with an emitted light time of 200–1000 s, induction period of 3.5–357 s and oscillation period of 28–304 s depending on the reagent concentrations. In this report the effect of the concentration variation of components involved in the oscillating CL system on the induction period, the oscillation period and amplitude was investigated and the parameters were plotted with respect to reagent concentrations. Copper concentration showed a significant effect on the oscillation period. The possible mechanism for the oscillating CL reaction was also discussed. Copyright © 2008 John Wiley & Sons, Ltd.