Our research on proton pumping ATPases over three decades: their biochemistry,molecular biology and cell biology

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.     

A Spin Label Study of the Effects of NO2, SO2 and pH Upon the Pulmonary Alveolar Macrophage

Much of the in vivo research that has been conducted on the health effects of pollutants such as SO₂ and NO₂ has been concerned with their effects upon lung defense systems, with particular reference to their effect on functional properties of the alveolar macrophage (AM). The lungs are constantly exposed to the external environment with its variable content of irritants and infectious agents. Agents deposited in or below the region of the respiratory bronchioles are phagocytized by the AM. The specific capacity of the AM to perform its task is subject to many factors. Gaseous air pollutants have been shown to affect the functional state of the cells^(1). Phagocytosis is an energy dependent process and plasma membrane ATPase has been suggested to act as a mechanoenzyme making phagocytosis possible through the conversion of chemical energy in the form of ATPase to the mechanical energky required for attachement and ingestion ⁽²⁾. Since the bulk of the cellular ATPase activity is located in the plasma membrane of the AM⁽³⁾ the enzyme is easily accessible to inhaled pollutants. Furthermome, the activity of ATPase and other membrane bound enzymes is well know to be dependent upon the fluidity of membrane lipids.     

NMR studies of water compartmentation in carrot parenchyma tissue during drying and freezing

An analysis is presented of the effect of drying and freezing on the distribution and pulse spacing dependence of water proton transverse relaxation times in carrot parenchyma tissue. The relaxation behaviour can be interpreted by treating the effects of changing subcellular morphology with a numerical cell model and combining this with a two-site proton exchange model to take account of solutes dissolved in the vacuolar fluid. In this way it is shown that drying removes water primarily from the vacuolar compartment, causing cell shrinkage and concentration of dissolved solutes, with little change in the volume of intercellular air spaces. Freezing causes initial ice crystal formation in the vacuolar compartment which concentrates the dissolved solutes. However, even at 248 K substantial quantities of nonfreezing water are associated with the cell walls and dissolved biopolymers. Comparison is made with previous studies on apple tissue.     

Proton Beam Generated by Multi-Lasers Interaction with Rear-Holed Target

Multi-lasers are proposed to enhance the proton acceleration in laser plasma interaction. A rear-holed target is illuminated by three lasers from different directions. The scheme is demonstrated by two-dimensional particlein-cell simulations. The electron cloud shape is controlled well and the electron density is improved significantly. The electrons accelerated by the three lasers induce an enhanced target normal sheath acceleration(TNSA) which suppresses the proton beam divergence and improves the maximum proton energy. The maximum proton energy is 22.9 Me V, which increased significantly than that of a single-laser target interaction. Meanwhile, the average divergence angle(22.3?) is reduced. The dependence of the proton beam on the length of sidewall is investigated in detail and the optimal length is obtained.     

Adiabatic pumping mechanism for ion motive ATPases

An ion motive ATPase is a membrane protein that pumps ions across the membrane at the expense of the chemical energy of adenosine triphosphate (ATP) hydrolysis. Here we describe how an external electric field, by inducing transitions between several protein configurations, can also power this pump. The underlying mechanism may be very similar to that of a recently constructed adiabatic electron pump [Science 283, 1905 (1999)]].     

Quantitative analysis of proton imaging measurements of laser-induced plasmas

A method for obtaining quantitative information about electric field and charge distributions from proton imaging measurements of laser-induced plasmas is presented. A parameterised charge distribution is used as target plasma. The deflection of a proton beam by the electric field of such a plasma is simulated numerically as well as the resulting proton density, which will be obtained on a screen behind the plasma according to the proton imaging technique. The parameters of the specific charge distributions are delivered by a combination of linear regression and nonlinear fitting of the calculated proton density distribution to the measured optical density of a radiochromic film screen changed by proton exposure. It is shown that superpositions of spherical Gaussian charge distributions as target plasma are sufficient to simulate various structures in proton imaging measurements, which makes this method very flexible.     

Investigation of the effect of plasma waves excitation on target normal sheath ion acceleration using fs laser‐irradiating hydrogenated structures

Measurements of ion acceleration in polymethylmethacrylate foils covered by a thin copper film irradiated by fs laser in target normal sheath acceleration regime are presented. The ion acceleration depends on the laser parameters, such as the pulse energy; depends on the irradiation conditions, such as the focal point position of the laser with respect to the target surface; and depends on the target properties, such as the metallic film thickness. The proton acceleration increases in the presence of the metallic film enhancing the plasma electron density, reaching about 1.6 MeV energy for a focal position on the target surface. The plasma diagnostics uses SiC detectors, absorber foils, Faraday cups, and gafchromic films. Employing p‐polarized laser light and a suitable oblique incidence, it is possible to increase the proton acceleration up to about 2.0 MeV thanks to the effects of laser absorption resonance due to plasma waves excitation.     

Spectral dips in ion emission emerging from ultrashort laser-driven plasmas

Deep dips in MeV ion spectra are obtained from water droplet targets irradiated by intense [(0.5-1.2) x 10(19) W/cm(2)] and ultrashort (35 fs) laser pulses. The existence of these dips is ascribed to the generation of a multielectron-temperature plasma, which is confirmed by our experiments. An existing fluid model based on hot-electron components with significantly different temperatures is consistent with the behavior we observe in the ion spectra of the femtosecond laser-driven interaction. The model provides a good simulation of the observed spectral dips and allows us to establish important parameters such as hot- and cold-electron temperatures and the respective electron density ratios. The result may be of interest for spectral tailoring of proton spectra in future applications of laser-generated proton beams.     

Pore-filling electrolyte membranes based on plasma-activated microporous PE matrices and sulfonated hydrogenated styrene butadiene block copolymer (SHSBS): Single cell test and impedance spectroscopy in symmetrical mode

In this research the performance of proton exchange membrane fuel cells (PEMFC's) was studied, using pore-filling electrolyte membranes based on plasma-activated microporous polyethylene (PE) matrices coated with sulfonated hydrogenated butadiene–styrene block copolymer (SHSBS). The voltage–current and power density curves were recorded under different experimental temperature and pressure conditions. In addition, an electrochemical study was completed by means of electrochemical impedance spectroscopy (EIS) in the symmetrical mode, adjusting the electrical response obtained to an equivalent circuit, which allows for isolation of the different processes occurring within the system. Two parameters were taken into account in the study: the membrane's proton transport or ion resistance (R₁) and its charge transfer resistance (R₂).The results obtained indicate that SHSBS shows a single cell behaviour which is comparable to that of the commercial membrane Nafion®. In contrast, the performance of the PE–SHSBS pore-filling electrolyte membranes was lower than that of Nafion®. Likewise it was found that the different plasma treatments applied to the microporous PE matrix have an effect on the proton exchange capacity of the pore-filling electrolyte membrane. EIS allowed to determine the ion resistance of the proton exchange membrane, and it was demonstrated that the kinetics of the cathodic reaction and the cathode itself are decisive elements in membrane performance and hence prime objectives to be optimized, when the reduction of the overvoltages is at stake, which are currently observed in the polarization curve at low and high power densities.     

质子交换膜中的传质分析

质子交换膜燃料电池是最有应用前景的汽车动力替代系统。质子交换膜中的传质是质子交换膜燃料电池性能的控制因素之一。论文从宏观和微观角度分析了质子交换膜中的质子和水分的传递机理,分析了操作参数对质子在质子交换膜中传递的影响。研究发现:外载荷对质子和水分在质子交换膜中的传递有很大影响;（H₅O₂）⁺是水合质子的主要结构形式;通过（H₅O₂）⁺中氢氧键不断形成与断裂,电荷在质子交换膜中得以传递。研究结果对理解质子交换膜中的传质机理及其推广应用具有积极意义。     

On the philosophy of optimizing contrast agents. An analysis of 1H NMRD profiles and ESR lineshapes of the Gd(III)complex MS-325+HSA

A generalization of the modified SBM theory is developed in closed analytical form. The theory is applied to describe the paramagnetically enhanced water proton spin-lattice relaxation rates of the aqueous-systems containing a gadolinium(S=7/2) complex(MS-325) in the presence or absence of human serum albumin (HSA). MS-325 binds to HSA: in the absence of the protein the reorientational time, tauR, is short, but when HSA is added tauR becomes much longer. In this way, the effect of reorientational motion, static (Delta s), and transient (Delta t) zero-field splitting (ZFS) interactions on both the water proton relaxivity and the Gd ESR lineshapes are investigated. Two dynamic models of electron spin relaxation are presented, characterized by transient and static ZFS-interactions. X-, Q-, and W-bands ESR spectra of MS-325+HSA are analyzed in order to describe the effect on the electron spin system upon binding to a macromolecule. A computer program based on this theory is developed which calculates solvent water proton T1 NMRD profiles and the corresponding X-, Q-, U-, and W-bands ESR lineshapes.     

Nonequilibrium fluctuations, traveling waves, and instabilities in active membranes

The stability of a flexible fluid membrane containing a distribution of mobile, active proteins (e.g., proton pumps) is shown to depend on the structure and functional asymmetry of the proteins. A stable active membrane is in a nonequilibrium steady state with height fluctuations whose statistical properties are governed by the protein activity. Disturbances are predicted to travel as waves at sufficiently long wavelength, with speed set by the normal velocity of the pumps. The unstable case involves a spontaneous, pump-driven undulation of the membrane, with clumping of the proteins in regions of high activity.     

Caveolae and caveolin isoforms in rat peritoneal macrophages

Caveolea are special (highly hydrophobic) plasma membrane invaginations with a diameter of 50-100 nm. Their characteristic features are the flask- or omega-shape and the lack of basket-like coat composed of clathrin. Caveolin-an integral membrane protein-is the principal component of caveolae membranes in vivo. Multiple forms of caveolin have been identified: caveolin-1alpha, caveolin-1beta, caveolin-2 and caveolin-3. They differ in their specific properties and tissue distribution. In this paper we summarize the morphological and biochemical data providing strong evidence about the existence and function of caveolae in rat peritoneal macrophages. When studied electron microscopically, the surface of both resident and elicited macrophages exhibited omega- or flask-shaped plasma membrane invaginations. There was a significant difference, however, in the number of these profiles: whereas in resident cells only a small amount of them was found on the cell surface, in elicited cells they were abundantly present on the plasma membrane. Using an antibody against the VIP21/caveolin-1 isoform we showed that these plasma membrane pits were indeed caveolae. The number and the appearance of caveolae were found to be in close correlation with the functional activity of these phagocytotic cells, indicating that the formation of caveolae is a highly regulated process.Using Western blot analysis two different proteins ( approximately 29 and approximately 20 kDa)-both labelled with anti-caveolin antibodies-were identified in resident and elicited macrophages that have been isolated from rat peritoneal cavity. The approximately 20 kDa protein was labelled specifically only by anti-VIP21/caveolin-1, while the approximately 29 kDa protein was labelled by both anti-VIP21/caveolin-1 and anti-caveolin-2 antibodies. The presence of the approximately 29 kDa protein was highly characteristic of resident cells, and only a small amount of approximately 20 kDa protein was detected in these cells. Elicitation has resulted in a significant increase in the amount of approximately 20 kDa protein labeled only with anit-VIP21/caveolin-1. Our morphological (confocal and electron microscopical) studies have shown that in resident cells caveolin was present in the cytoplasm, in smaller vesicles and multivesicular bodies around the Golgi area. Only a very small amount of caveolae was found on the cell surface of these cells. In elicited macrophages, caveolae (labelled with anti-VIP21/caveolin-1 antibody) appeared in large numbers on the cell surface, but caveolin detected by anti-caveolin-2 was also found in small vesicles and multivesicular bodies. These data support the idea that the expression of the approximately 29 kDa (caveolin-related) protein is insufficient for caveolae formation in resident cells, it can function as a modified, macrophage-specific caveolin-2 isoform.Our results strongly suggest that caveolin-1 plays a crucial role in the formation of caveolae: it is the amount of caveolin-1 that regulates the appearance of caveolae on the plasma membrane.Studying the endocytotic processes of resident and elicited macrophages we have found that elicited macrophages bound and internalized significantly larger amounts of fluid phase marker (HRP) and immune complex (peroxidase-antiperoxidase-PAP) than resident cells. Serial section analysis, double labelled immunocytochemistry, and filipin treatment were used to demonstrate that caveolae can pinch off from the plasma membrane and can take part in endocytotic processes as alternative carriers in elicited macrophages.     

Polyelectrolyte Nanocomposite Membranes Using Imidazole-Functionalized Nanosilica for Fuel Cell Applications

The preparation and characterization of a new type of nanocomposite polyelectrolyte membrane, based on DuPont Nafion/imidazole-modified nanosilica (Im-Si), for direct methanol fuel cell applications is described. Related to the interactions between the protonated imidazole groups, grafted on the surface of nanosilica, and negatively charged sulfonic acid groups of Nafion, new electrostatic interactions can be formed in the interface of Nafion and Im-Si which result in both lower methanol permeability and also higher proton conductivity. Physical characteristics of these manufactured nanocomposite membranes were investigated by scanning electron microscopy, thermogravimetry analysis, differential scanning calorimetry, Fourier transform infrared spectroscopy, water uptake, methanol permeability, and ion-exchange capacity, as well as proton conductivity. The Nafion/Im-Si membranes showed higher proton conductivity, lower methanol permeability and, as a consequence, higher selectivity parameter in comparison to the neat Nafion or Nafion/silica membranes. The obtained results indicated that the Nafion/Im-Si membranes could be utilized as promising polyelectrolyte membranes for direct methanol fuel cell applications.     

Interaction between explosive and analyte layers in explosive matrix-assisted plasma desorption mass spectrometry

An HMX/insulin two-layer system was chosen as a model for further investigation of the matrix properties of explosive materials for protein analytes in plasma desorption mass spectrometry. The dependencies of the molecular ion yield and average charge state as a function of the analyte thickness were studied. An increase in the charge state of multiply protonated molecular species was confirmed as the major matrix effect, with the average charge state z at the smallest thickness studied being higher than in matrix-assisted laser desorption/ionization and closer to the value obtained in electrospray ionization under standard acidic conditions. Observed charge state distributions are significantly narrower than the corresponding Poisson distributions, which suggests that the protonation of insulin is limited in plasma desorption by the number of basic sites in the molecule, similar to electrospray ionization. Both the curve displaying total molecular ion yield and the one showing the total charge (proton) yield as a function of the insulin thickness have maxima at a thickness different from an insulin monolayer. These observations diminish the significance of a matrix/analyte interface mechanism for the explosive matrix assistance. Instead, a mechanism related to the chemical energy release during conversion of the explosive after the ion impact is proposed. As additional mechanisms, enhanced protonation of the analyte through collisions with products of the explosive decay is considered, as well as electron scavenging by other products, which leads to a higher survival probability of positively charged protein molecular ions. Copyright 1999 John Wiley & Sons, Ltd.     

Triazole and triazole derivatives as proton transport facilitators in polymer electrolyte membrane fuel cells

Some basic aspects pertaining to the application of triazole and its derivatives as proton transport facilitators for membranes for high temperature fuel cell operations are investigated. Performance as proton transport facilitators is studied for compounds in their native solid state and as a dopant in a polymer membrane. Some key parameters which influence the proton transport in the system are the proton affinity, pKa or acidity, activation energy and the ease of formation of hydrogen bonding network. Theoretical calculations of the proton affinity of the compounds are presented. The effect of proton affinity of the compound on the activation energies for proton transport is investigated. Proton conductivity is measured for acid doped triazoles in both pellet form (powder triazole mixed with acid) and in composite forms wherein the acid group is contained in a polymer matrix. The effect of formation of a hydrogen bonding network by the triazoles and its impact on the proton conductivity are studied. Also, the effect of ion exchange capacity (IEC) of the host polymeric electrolytes and loading of triazoles in the composites were investigated.     

Influences of finite Larmor radius on wake effects and stopping power for proton moving in magnetized two-component plasma

Considering finite Larmor radius (FLR) effects, wake effects and stopping power induced by proton projectile in two-component magnetized plasma are investigated within a linear response framework. Numerical results show that, FLR lessens wake effects and stopping power, essentially through excitation of collective plasma electron modes.     

鞘场加速机理中质子束的特性与其初始尺寸的关系

为了研究激光鞘场中质子层的尺寸对质子束特性的影响,本文应用中国工程物理研究院 激光聚变研究中心的二维Particle-In-Cell (2D-PIC)数值模拟程序Flips2D进行了相关数值模拟研究. 研究了质子束总能量随时间的变化,得出了加速持续过程与激光脉冲持续时间的关系; 研究了质子层的宽度对加速后质子束发散角和能谱的影响;研究了质子层的厚与加速后质子束 发散角和能谱的关系;得出了质子层的初始尺寸对加速后质子特性的影响规律.