The Relation between Membrane Potential and the Transport Activity of Systems a and L in Plasma Membrane Vesicles of the Ehrlich Cell

Plasma membrane vesicles isolated from Ehrlich ascites tumor cells have been used to investigate the role of the transmembrane potential in the energetics of Systems A and L. As expected, Na⁺-dependent System A was responsive to changes in membrane potential. System L activity, as measured by transport of 2-aminonorbornane-2-carboxylic acid (BCH), was shown to be Na⁺-independent and was not altered by changes in the membrane potential. The combination of valinomycin and nigericin decreased accumulation of MeAIB but not that of BCH. The presence of nigericin alone caused a significant decrease in uptake by System A and a decrease in uptake by System L to a lesser degree. The inhibitory action of nigericin might reflect its ability to dissipate the Na⁺ gradient rather than an effect on K⁺ or H⁺ flows. The results indicate that modes of energization not produced through the transmembrane potential must account for any uphill operation of System L.     

In Vitro Evaluation of the Effect of Stimulation with Magnetic Fields on Chondrocytes

Magnetic fields (MFs) have been used as an external stimulus to increase cell proliferation in chondrocytes and extracellular matrix (ECM) synthesis of articular cartilage. However, previously published studies have not shown that MFs are homogeneous through cell culture systems. In addition, variables such as stimulation times and MF intensities have not been standardized to obtain the best cellular proliferative rate or an increase in molecular synthesis of ECM. In this work, a stimulation device, which produces homogeneous MFs to stimulate cell culture surfaces was designed and manufactured using a computational model. Furthermore, an in vitro culture of primary rat chondrocytes was established and stimulated with two MF schemes to measure both proliferation and ECM synthesis. The best proliferation rate was obtained with an MF of 2 mT applied for 3 h, every 6 h for 8 days. In addition, the increase in the synthesis of glycosaminoglycans was statistically significant when cells were stimulated with an MF of 2 mT applied for 5 h, every 6 h for 8 days. These findings suggest that a stimulation with MFs is a promising tool that could be used to improve in vitro treatments such as autologous chondrocyte implantation, either to increase cell proliferation or stimulate molecular synthesis. Bioelectromagnetics. 2020;41:41–51 © 2019 Bioelectromagnetics Society     

Spatial Distribution of the Potential on a Cylindrical Surface Simulating the Somatic Membrane: Model Studies

In our study, we represent the theoretical and numerical analysis of a stochastic version of the Hodgkin–Huxley model applied to a two-dimensional spatial cylindrical area simulating the neuronal somatic membrane. We characterized the spatiotemporal dynamics of the membrane potential by its local value V _m (x, y, t) and the integral of this value with respect to time F(x, y, T) within an interval [0, T]. Analysis of the model showed that (i) there are nonzero gradients of F(x, y, T) at any distribution of ion channels; (ii) the maximum gradient F(x, y, T) decreases down to zero with the time T, if the channels are distributed homogeneously, and acquire some positive constant value, if the channels are distributed inhomogeneously; the gradient F(x, y, T) depends on the gradient of spatial distribution of the channels; and (iii) under conditions of spatial redistribution of the channels with preservation of their number, the dynamics of V _m (x, y, t) does not change.     

Possible Mechanism for Synchronized Detection of Weak Magnetic Fields by Nerve Cells

We propose that biological systems may detect static and slowly varying magnetic fields by the modification of the timing of firing of adjacent nerve cells through the local influence of the magnetic field generated by current from one cell's firing on its nearest neighbors. The time delay of an adjacent nerve cell pulse with respect to the initial clock nerve cell pulse could serve as a signal for sensing the magnitude and direction of the magnetic field in a direction perpendicular to the current flows in the cells. It has been shown that changes in static magnetic fields modify concentrations of reactive oxygen species, calcium, pH, the growth rates of fibrosarcoma cells, and membrane potentials. These are linked to changes in membrane potentials that can either inhibit or accelerate the firing rate of pacemaker or clock cells. This mechanism may have applications to animals' use of magnetic fields for navigation or other purposes, possibly in conjunction with other mechanisms. Bioelectromagnetics. © 2020 Bioelectromagnetics Society.     

内植静磁柱联合脉冲电磁场对骨组织修复作用的Micro-CT研究

目的:以永磁体构建静磁柱体内植入物,应用Micro-CT技术,探索研究内植静磁柱联合脉冲电磁场对骨组织的修复作用。方法:选取新西兰兔24只建立右侧股骨髁骨缺损模型,并随机分为联合磁场组,脉冲电磁场组和对照组。联合磁场组自兔右侧股骨干远端向髓内植入静磁柱,并联合施加体外脉冲电磁场(1 h/d);脉冲电磁场组及对照组均将无磁性钛合金棒植入右侧股骨干髓腔,同时,脉冲电磁场组于体外加载脉冲电磁场(1 h/d)。术后5周处死取材,应用Micro-CT对股骨远端样本进行扫描检测,并对骨缺损区域显微结构进行三维重建分析。结果:与非磁场组相比,联合磁场组骨缺损区域骨小梁相对体积及数目明显增加(P均0.05),骨小梁分离度下降(P0.01)。与传统脉冲电磁场组相比,联合磁场组骨小梁数目增加(P0.05),骨小梁分离度下降(P0.01)。扫描及重建图像可见,联合磁场组骨缺损区域骨量增多,外侧皮质区愈合完整,骨小梁结构连续致密,具有更好的骨显微结构。结论:内植静磁柱与脉冲电磁场的联合磁场干预方式能够在早期获得更好的成骨修复能力,为磁场治疗相关骨病的临床及基础研究提供了新的方向。     

Diffusion and Active Transport of NCAM within the Neuronal Plasma Membrane

In our study, we showed that distribution of NCAM along the surface of actively growing in vitro neurites of murine hippocampal neurons comprises at least two components. The first component is reflected in exponential decline of the NCAM immunoreactivity from the soma and growth cone to a central part of the neurite. This component can be described by a model assuming diffusive redistribution of NCAM from the sites of its preferential insertion (from the neuron's soma and growth cones). The second component manifests itself as clusters of NCAM immunoreactivity irregularly distributed along the neurites. Some of these NCAM clusters, which were immunolabeled in a living neuron, can intermittently move back and forth along the neurites with a velocity up to 0.5 m/sec. Our data demonstrate that, besides passive NCAM diffusion, an active mechanism of NCAM redistribution exists in the central neurite part.     

The Effect of Concanavalin A on Membrane Potential and Intracellular pH during Activation In Vitro of Tobacco Pollen Grains

We studied the effects of short-term (5–10 min) treatment of Nicotiana tabacum L. pollen grains with concanavalin A (ConA) on their activation (changes in the membrane potential and intracellular pH) and germination in vitro. ConA (10–1000 g/ml) induced plasma membrane hyperpolarization in the vegetative cell and enhanced pollen grain germination. These effects depended on ConA concentration and were interrelated: the value of the membrane potential was negatively correlated with the number of pollen grains germinated for 1 h of their incubation (r = –0.96). In addition, ConA (100 g/ml) increased the intracellular pH value by 0.3 unit. All these effects of ConA are determined by its specific interaction with carbohydrate determinants because a competitive sugar methyl--mannopyranoside (0.1 M) completely blocked ConA effects. The data obtained presume that the specific receptors are present on the surface of pollen grains, evidently on their plasma membrane, and their interaction with lectins has a functional significance for pollen grain activation and germination.     

The Sensitivity of Cognitive Processes to the Inhomogeneity of Natural Magnetic Fields

Three experiments were carried out to study the influence of inhomogeneity of natural magnetic field (MF) on animal cognition. Wistar rats (n = 90) were placed in a complicated problem environment, in which they were to form food-operant behavior under conditions of natural MF (Cond. 1) and MFs produced by iron objects (Cond. 2) or by magnets (Cond. 3). Unlike the control group (Cond. 1), all rats in Cond. 2 and 3 were unable to form operant behavior. Weak MF caused both locomotor and emotional depression, and there was no exploratory activity shown during 6 sessions. Brief external stimulation removed locomotor depression, and animals formed operant behavior similar to latent learning (Cond. 2) or “insight” (Cond. 3). Performance efficiency was lower while the level of stress manifestation was higher in Cond. 2 and 3 than in the control at the stage of stabilization. It has been proposed that MF reduces the activity of brain motivation centers and prevents the development of complicated forms of cognitive activity.     

O_2~-·与红细胞膜阴离子转运蛋白相互关系的研究

利用４,４′二硫氰２,２′二磺基芪（ＤＩＤＳ）对带３（ｂａｎｄ３）蛋白阴离子转运功能的专一抑制作用,研究了超氧阴离子（Ｏ－２·）进入红细胞对带３蛋白的依赖关系;同时根据红细胞在等渗氯化铵溶液中溶血的ｔ１／２,探讨了细胞外大量产生Ｏ－２·对带３蛋白转运阴离子功能的影响。结果表明：４０μｍｏｌ／ＬＤＩＤＳ完全抑制带３蛋白的阴离子转运功能,但并不影响Ｏ－２·进入细胞,证明Ｏ－２·进入细胞对ｂａｎｄ３蛋白无依赖关系;Ｏ－２·通过氧化修饰红细胞膜蛋白的－ＳＨ,使带３蛋白的阴离子转运能力下降,并可用－ＳＨ还原剂ＤＴＴ与巯基乙醇修复。     

Effect of Colchicine on Cell Membrane and on Biopterin Transport in Crithidia fasciculata*

SYNOPSIS. Incorporation of ¹⁴C-labeled biopterin into Crithidia fasciculata was inhibited by 1 mM colchicine or lumicolchicine. These substances do not penetrate the cell membrane, hence they cannot interact with the subpellicular microtubules. In view of this, interference of colchicine with biopterin transport must occur on the outer surface of the cell membrane. Binding of colchicine to Crithidia was not temperature-dependent and did not exhibit saturation kinetics. These facts exclude a binding as in the case of tubulin, or similar proteins which may be present in the membrane. The results suggest an inhibition reflecting steric hindrance of the biopterin carrier system.     

植物细胞程序性死亡的分类和膜通透性调控蛋白研究进展

程序性细胞死亡是由基因调控的贯穿于真核细胞生理和发育过程的细胞自杀行为。动物细胞的程序性死亡分成3类凋亡、自噬和坏死;线粒体和溶酶体分别在前两个过程中起关键作用。关于植物细胞程序性死亡的分类还存在很多争议,焦点是植物是否有细胞凋亡这种形式,核心问题是植物细胞的线粒体外膜上没有Bcl-2家族的膜通透性调控蛋白。近年,程序性细胞死亡也在细菌中发现,LrgAB家族的膜通透性调控蛋白起着重要作用。最近的研究表明,植物叶绿体外被膜上也有LrgAB家族的同源蛋白,它们在控制叶绿体发育和程序性细胞死亡方面起重要作用。因此,叶绿体在植物细胞死亡调控中的作用应该更加受到关注。     

Quantitative Analysis of Membrane Protein Transport Across the Nuclear Pore Complex

Nuclear transport of the Saccharomyces cerevisiae membrane proteins Src1/Heh1 and Heh2 across the NPC is facilitated by a long intrinsically disordered linker between the nuclear localization signal (NLS) and the transmembrane domain. The import of reporter proteins derived from Heh2 is dependent on the FG‐Nups in the central channel, and the linker can position the transport factor‐bound NLS in the vicinity of the FG‐Nups in the central channel, while the transmembrane segment resides in the pore membrane. Here, we present a quantitative analysis of karyopherin‐mediated import and passive efflux of reporter proteins derived from Heh2, including data on the mobility of the reporter proteins in different membrane compartments. We show that membrane proteins with extralumenal domains up to 174 kDa, terminal to the linker and NLS, passively leak out of the nucleus via the NPC, albeit at a slow rate. We propose that also during passive efflux, the unfolded linker facilitates the passage of extralumenal domains through the central channel of the NPC .     

Lidocaine Hydrochloride and Acetylsalicylate Kill Bacteria by Disrupting the Bacterial Membrane Potential in Different Ways

Lidocaine hydrochloride (LH), a local anesthetic, and acetylsalicylate (AcSAL), show antibacterial activity for both gram-negative and gram-positive bacteria. Kinetic studies indicated that antibacterial activity of LH was different from that of AcSAL. A subinhibitory concentration of LH and AcSAL enhanced the sensitivity of Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa to novobiocin and nalidixic acid. The synergistic effect of AcSAL with novobiocin and nalidixic acid was higher than that of LH. The effect of both drugs on the membrane potential of inner membrane was also studied using inverted membrane vesicles of bacteria. Both LH and AcSAL depolarized the membrane potential after the vesicles were energized with nicotinamide adenine dinucleotide. However, unlike AcSAL, pre-treatment of vesicles with LH had no effect on the generation of membrane potential. These results suggest that depolarization of the cytoplasmic membrane, preceded by the permeabilization of the outer membrane for gram-negative bacteria, is associated with antibacterial activity of LH and AcSAL. The difference in actions of LH and AcSAL was discussed.     

The Non-structural Protein of Crimean-Congo Hemorrhagic Fever Virus Disrupts the Mitochondrial Membrane Potential and Induces Apoptosis

Viruses have developed distinct strategies to overcome the host defense system. Regulation of apoptosis in response to viral infection is important for virus survival and dissemination. Like other viruses, Crimean-Congo hemorrhagic fever virus (CCHFV) is known to regulate apoptosis. This study, for the first time, suggests that the non-structural protein NSs of CCHFV, a member of the genus Nairovirus, induces apoptosis. In this report, we demonstrated the expression of CCHFV NSs, which contains 150 amino acid residues, in CCHFV-infected cells. CCHFV NSs undergoes active degradation during infection. We further demonstrated that ectopic expression of CCHFV NSs induces apoptosis, as reflected by caspase-3/7 activity and cleaved poly(ADP-ribose) polymerase, in different cell lines that support CCHFV replication. Using specific inhibitors, we showed that CCHFV NSs induces apoptosis via both intrinsic and extrinsic pathways. The minimal active region of the CCHFV NSs protein was determined to be 93–140 amino acid residues. Using alanine scanning, we demonstrated that Leu-127 and Leu-135 are the key residues for NSs-induced apoptosis. Interestingly, CCHFV NSs co-localizes in mitochondria and also disrupts the mitochondrial membrane potential. We also demonstrated that Leu-127 and Leu-135 are important residues for disruption of the mitochondrial membrane potential by NSs. Therefore, these results indicate that the C terminus of CCHFV NSs triggers mitochondrial membrane permeabilization, leading to activation of caspases, which, ultimately, leads to apoptosis. Given that multiple factors contribute to apoptosis during CCHFV infection, further studies are needed to define the involvement of CCHFV NSs in regulating apoptosis in infected cells.     

高效亲和膜色谱快速分析及小量制备蛋白质

以甲基丙烯酸缩水甘油酯纤维素复合膜为基质,分别以蛋白A(Protein A)、人免疫球蛋白G(HIgG)、三嗪染料(Cibacron blue F3GA)、亚胺二乙酸铜离子为配基,用不同方法制备了适合于分析及小量制备的高效亲和膜色谱介质,并对高效亲和柱的基本性能及其应用于各种相应蛋白的定量测定情况进行了考察。利用这种方法可以针对不同的目标蛋白及所存在的环境采用不同的配基,对各种蛋白的定量测定及小量制备可达到较为满意的结果。     

Low-Frequency Magnetic Fields in Cars and Office Premises and the Geomagnetic Field Variations

In this study, we measured and analyzed the spectral characteristics of a low-frequency magnetic field (MF) inside several gasoline-powered cars while driving on busy city roads. The spectra obtained upon measurements in the interior of the cars are compared with those measured in office locations at different times of the day and with different disturbances of the geomagnetic field (k-index of disturbance 2–8). The power spectral density of the electromagnetic field in cars moving on busy roads in the frequency range of 10⁻³–10² Hz is one to three orders of magnitude higher than that in urban offices. This raises a question regarding the possible influence of these MFs on the psychophysiological state of drivers. In turn, in the daytime, the MF power in the range from 10⁻³ to 1 Hz inside the locations is three times higher compared with the power of a strong geomagnetic storm. Despite such an overwhelming magnetic background, geomagnetic storms affect various organisms. The nonspecific effect of magnetic storms is supposed to be associated with relatively long (lasting several hours or more [frequency range of 10⁻⁴−10⁻⁵ Hz]) periods of enhancement or weakening of the local geomagnetic field. In this range, especially at night, the power spectral density of geomagnetic disturbances is comparable to and can even exceed the power density of urban MFs. © 2020 Bioelectromagnetics Society.     

Potential Roles of Electrogenic Ion Transport and Plasma Membrane Depolarization in Apoptosis

Apoptosis is characterized by the programmed activation of specific biochemical pathways leading to the organized demise of cells. To date, aspects of the intracellular signaling machinery involved in this phenomenon have been extensively dissected and characterized. However, recent studies have elucidated a novel role for changes in the intracellular milieu of the cells as important modulators of the cell death program. Specially, intracellular ionic homeostasis has been reported to be a determinant in both the activation and progression of the apoptotic cascade. Several apoptotic insults trigger specific changes in ionic gradients across the plasma membrane leading to depolarization of the plasma membrane potential (PMP). These changes lead to ionic imbalance early during apoptosis. Several studies have also suggested the activation and/or modulation of specific ionic transport mechanisms including ion channels, transporters and ATPases, as mediators of altered intracellular ionic homeostasis leading to PMP depolarization during apoptosis. However, the role of PMP depolarization and of the changes in ionic homeostasis during the progression of apoptosis are still unclear. This review summarizes the current knowledge regarding the causes and consequences of PMP depolarization during apoptosis. We also review the potential electrogenic ion transport mechanisms associated with this event, including the net influx/efflux of cations and anions. An understanding of these mechamisms could lead to the generation of new therapeutic approaches for a variety of diseases involving apoptosis.     

膜转运蛋白的功能性超表达和纯化

膜转运蛋白结构和功能的研究是功能膜蛋白质组研究中的一个重要内容,而大量蛋白质的分离纯化是进行蛋白质的结构和功能研究的基础.目前,结构和功能膜蛋白质组学相关研究的瓶颈,在于不能有效地超量表达和纯化具有生物活性的膜转运蛋白.影响膜转运蛋白超量表达和纯化的关键因素,包括目标蛋白的拓扑学结构分析和去垢剂的选择.进行膜转运蛋白拓扑学结构的分析,对于构建用于活体表达的重组膜转运蛋白具有指导意义.去垢剂能够稳定去膜状态的膜蛋白,在膜转运蛋白的离体表达和亲和纯化以及包涵体的处理过程中具有重要的作用.本文就目前功能膜蛋白质组学研究中所涉及的有关膜转运蛋白功能性超表达和分离纯化策略及关键技术作一简述.