Electrical Events Correlated with Contractile Vacuole Activity in the Suctorian, Heliophrya

SYNOPSIS. The surface membrane potentials of suctorian genus Heliophrya were studied with intracellular electrodes. Resting membrane potentials averaged -32 mV, and spontaneous depolarizing potentials occurring at apparently random intervals and having a variety of waveforms were routinely observed. Such spontaneous potentials were correlated in time with visually monitored contractile vacuole activity. Individual contractile vacuoles had unique, although somewhat variable, electrical signatures. In the presence of an intracellular electrode all vacuoles contracted independently, but at approximately the same frequency. The amplitude of the electrical potentials increased when the membrane was hyperpolarized and decreased when it was depolarized. The sign of such potentials reversed at between -10 mV and the zero membrane potential. A 20% decrease in the membrane resistance was measured at the peak of the spontaneous depolarizing potentials.     

Ultrastructure and Vacuolar Movements in the Free-Living Diplomonad Trepomonas agilis Klebs*

SYNOPSIS. The structure of Trepomonas agilis communis Klebs is described from light and electron microscope observations on 2 clone isolates of the organism. The surface membrane shows marked differentiation into an extremely thick (16 nm) symmetric membrane which covers the greater part of the body, and a thinner (∼ 10–12 nm) asymmetric membrane which lines the 2 lateral oral grooves and the posterior channel connecting them; a similar asymmetric membrane covers the flagella. Thorium dioxide staining suggests a denser distribution of acidic carbohydrate groups on the asymmetric membrane. The pathways of cytoplasmic streaming observed in the living flagellate coincide with those of microtubule bands arising close to the flagellar basal bodies and it is suggested that the bands play an orienting role in the streaming of food vacuoles. The contractile vacuole undergoes diastole in the anterior (postnuclear) cytoplasm, and is formed by coalescence of smaller vesicles. At systole the entire vacuole migrates to the posterior extremity to discharge into the posterior channel; the route of exit lacks guiding structural elements. Features of the flagellate's physiology and organization are discussed in relation to the observed lack of mitochondria, microbodies and Golgi apparatus in diplomonads.     

Neural Coding with Graded Membrane Potential Changes and Spikes

The neural encoding of sensory stimuli is usually investigated for spike responses, although many neurons are known to convey information by graded membrane potential changes. We compare by model simulations how well different dynamical stimuli can be discriminated on the basis of spiking or graded responses. Although a continuously varying membrane potential contains more information than binary spike trains, we find situations where different stimuli can be better discriminated on the basis of spike responses than on the basis of graded responses. Spikes can be superior to graded membrane potential fluctuations if spikes sharpen the temporal structure of neuronal responses by amplifying fast transients of the membrane potential. Such fast membrane potential changes can be induced deterministically by the stimulus or can be due to membrane potential noise that is influenced in its statistical properties by the stimulus. The graded response mode is superior for discrimination between stimuli on a fine time scale.     

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.     

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 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.     

Modulation of the Cell Membrane Potential and Intracellular Protein Transport by High Magnetic Fields

To explore cellular responses to high magnetic fields (HMF), we present a model of the interactions of cells with a homogeneous HMF that accounts for the magnetic force exerted on paramagnetic/diamagnetic species. There are various chemical species inside a living cell, many of which may have large concentration gradients. Thus, when an HMF is applied to a cell, the concentration‐gradient magnetic forces act on paramagnetic or diamagnetic species and can either assist or oppose large particle movement through the cytoplasm. We demonstrate possibilities for changing the machinery in living cells with HMFs and predict two new mechanisms for modulating cellular functions with HMFs via (i) changes in the membrane potential and (ii) magnetically assisted intracellular diffusiophoresis of large proteins. By deriving a generalized form for the Nernst equation, we find that an HMF can change the membrane potential of the cell and thus have a significant impact on the properties and biological functionality of cells. The elaborated model provides a universal framework encompassing current studies on controlling cell functions by high static magnetic fields. Bioelectromagnetics. 2021;42:27–36. © 2020 Bioelectromagnetics Society.     

利用线粒体膜电位测定筛选参与细胞凋亡的人类新基因

细胞凋亡(apoptosis)属于细胞程序化死亡(programmed cell death),是细胞内涉及到许多生化反应的复杂过程.建立了基于细胞水平的凋亡筛选模型,用于筛选人类基因组中功能未知的序列,以发现与细胞凋亡相关的新基因.通过构建人类未知基因的表达文库,并将未知基因表达载体瞬时转染HeLa细胞,用阳离子染料JC-1标记HeLa细胞线粒体内膜并检测线粒体跨膜电位,用流式细胞术进行阳性结果的验证.经过对未知基因表达文库内600个新基因的筛选,得到7个线粒体跨膜电位下降相关新基因(CHMP6、CGI-38、hCAP-H2、NUDT16L1、ARMC1、PHF17和FLJ21103),经实验验证,其中3个基因(CHMP6、CGI-38和hCAP-H2)与细胞凋亡相关.结果表明,所建立的基于细胞的凋亡筛选模型稳定高效,3个细胞凋亡相关基因将被进行深入研究.     

桑葚花色苷提取物对乳腺癌细胞凋亡及线粒体膜电位的影响

目的:观察桑葚花色苷提取物对人乳腺癌细胞株MDA-MB-453、MDA-MB-231和MCF-7细胞凋亡及线粒体膜电位的影响.方法:利用超声辅助乙醇萃取法提取桑葚花色苷,pH示差法测定提取物花色苷总含量,以50、100和150 mg/mL桑葚花色苷提取物作用三种乳腺癌细胞MDA-MB-231、MDA-MB-453和MCF-7 24h,采用Annexin V/PI双染流式细胞分析法检测细胞凋亡水平变化,JC-1探针染色激光共聚焦扫描显微镜观察MDA-MB-453细胞线粒体膜电位水平变化.结果:凋亡分析结果表明,桑葚花色苷提取物作用后三种乳腺癌细胞凋亡率均升高,显示出促凋亡效应,且具有剂量-效应关系,100和150 mg/mL组凋亡率显著升高(P＜0.05).激光共聚焦扫描显微镜检测结果显示,桑葚花色苷提取物作用24h,可使MDA-MB-453细胞线粒体膜电位显著下降,表现为红色/绿色荧光的比值显著降低(P＜0.05).结论:桑葚花色苷提取物可显著降低乳腺癌细胞线粒体膜电位,并促发细胞凋亡.     

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (ΔΨm) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of ΔΨm renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine ΔΨm and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine Δψm in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H₂DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess ΔΨm or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of ΔΨm or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of ΔΨm or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H₂DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H₂O₂. This protocol (with minor modifications) can be also used to determine changes in ∆Ψm and ROS in different cell types and in neurons isolated from other brain regions.     

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.     

Respiration and Mitochondrial Membrane Potential Are not Required for Apoptosis and Anti-apoptotic Action of Bcl-2 in HeLa Cells

The release of cytochrome c from intermembrane space of mitochondria into cytosol is one of the critical events in apoptotic cell death. The important anti-apoptotic oncoprotein Bcl-2 inhibits this process. In the present study it was shown that apoptosis and release of cytochrome c induced by staurosporine or by tumor necrosis factor- in HeLa cells were not affected by inhibitors of respiration (rotenone, myxothiazol, antimycin A) or by uncouplers (CCCP, DNP) that decrease the membrane potential at the inner mitochondrial membrane. The inhibitors of respiration and the uncouplers did not affect also the anti-apoptotic activity of Bcl-2.     

Potential Changes in the Distributions of Western North America Tree and Shrub Taxa under Future Climate Scenarios

Increases in atmospheric greenhouse gases are driving significant changes in global climate. To project potential vegetation response to future climate change, this study uses response surfaces to describe the relationship between bioclimatic variables and the distribution of tree and shrub taxa in western North America. The response surfaces illustrate the probability of the occurrence of a taxon at particular points in climate space. Climate space was defined using three bioclimatic variables: mean temperature of the coldest month, growing degree days, and a moisture index. Species distributions were simulated under present climate using observed data (1951–80, 30-year mean) and under future climate (2090–99, 10-year mean) using scenarios generated by three general circulation models—HADCM2, CGCM1, and CSIRO. The scenarios assume a 1% per year compound increase in greenhouse gases and changes in sulfate (SO₄) aerosols based on the Intergovernmental Panel on Climate Change (IPCC) IS92a scenario. The results indicate that under future climate conditions, potential range changes could be large for many tree and shrub taxa. Shifts in the potential ranges of species are simulated to occur not only northward but in all directions, including southward of the existing ranges of certain species. The simulated potential distributions of some species become increasingly fragmented under the future climate scenarios, while the simulated potential distributions of other species expand. The magnitudes of the simulated range changes imply significant impacts to ecosystems and shifts in patterns of species diversity in western North America. Received 12 May 2000; accepted 20 December 2000.     

Germination and <Emphasis Type="Italic">In Vitro</Emphasis> Growth of Petunia Male Gametophyte Are Affected by Exogenous Hormones and Involve the Changes in the Endogenous Hormone Level

The data obtained characterize the changes in the contents of endogenous phytohormones (IAA, cytokinins, GA, and ABA) in germinating pollen grains and growing pollen tubes of a self-compatible clone of petunia (sPetunia hybrida L.) within an 8-h period under in vitro conditions. The hydration and initiation of germination of pollen grains brought the ABA content down to a zero level, while the levels of GA, IAA, and cytokinins increased 1.5–2-fold. Later, in the growing pollen tubes, the GA content increased twofold, while the levels of IAA and cytokinins decreased. The exogenous ABA and GA₃ considerably promoted pollen germination and pollen tube growth; however, only the treatment with GA₃ produced the maximum length of pollen tubes. The exogenous IAA promoted and the exogenous cytokinins hindered the growth of pollen tubes. The membrane potential, as assessed with a potential-sensitive dye diS-C₃-(5), considerably increased in the pollen grains treated with ABA and benzyladenine, whereas IAA and GA₃ did not practically affect it. The authors conclude that the mature pollen grains contain the complete set of hormones essential for pollen germination and pollen tube growth. ABA, GA, and IAA together with cytokinins control the processes of pollen grain hydration, germination, and pollen tube growth, respectively.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 584–590.Original Russian Text Copyright © 2005 by Kovaleva, Zakharova, Minkina, Timofeeva, Andreev.     

Changes in phenology and abundance of suction-trapped Diptera from a farmland site in the UK over four decades

1. Recently documented insect declines have caused major concerns and an increased interest in studies using long-term population-monitoring data. 2. Samples from a 12.2-m suction trap were used to examine trends in phenology and abundance of Diptera over four decades. 3. The timing of peak flight has advanced by an average of 17 days, from 23 July in 1974 to 6 July in 2014. 4. The abundance of flies has decreased by 37% over the studied period (from April to September), and peak abundance has decreased by 48%. The flight period has started earlier in recent years, and in 2014, the number of flies was higher in spring until the 31st of May than in 1974. Possible causes and impacts of these changes are discussed.     

Changes in the Light Scattering by Symbiosomes from Vicia faba Root Nodules as Indicator of Ion and Metabolite Transport across the Peribacteroid Membrane

Passive transport of ions and metabolites across the peribacteroid membrane (PBM) was investigated on symbiosome preparations isolated from the broad bean (Vicia faba L.) root nodules and suspended in a potassium-free medium. Optical density of the symbiosome suspension at 546 nm was monitored as an indicator of light-scattering changes. Depolarization of the PBM with tetraphenylphosphonium cation (TPP⁺) caused an increase in light scattering of symbiosome suspension. This effect was enhanced after adding a K⁺ ionophore valinomycin to the incubation medium. A similar effect was observed after supplementing the symbiosome suspension with nigericin, a K⁺/H⁺ antiporter. Similar experiments on bacteroid suspensions prepared from isolated symbiosomes did not reveal any appreciable changes in light scattering in the presence of the same membrane-active substances. The light scattering by symbiosome suspensions decreased after adding malate or succinate, while the subsequent addition of centimolar concentrations of K⁺ substantially accelerated this process. Light scattering by the symbiosome suspension was insensitive to the addition of glutamate, a substance normally impermeant through the PBM of legume root nodules. These results suggest that the changes in light scattering by symbiosomes reflect the osmotically induced changes of symbiosome volume. These volume changes were assigned to alteration of the peribacteroid space (PBS). The incubation of symbiosomes in a potassium-free medium acidified their the PBS; this acidification was accelerated by valinomycin, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), and nigericin, and it was abolished in the presence of comparatively high concentrations of K⁺ in the incubation medium. The results indicate a relatively high permeability of the PBM to K⁺ ions.     

Factors and Processes Shaping Land Cover and Land Cover Changes Along the Wisconsin River

Land use can exert a powerful influence on ecological systems, yet our understanding of the natural and social factors that influence land use and land-cover change is incomplete. We studied land-cover change in an area of about 8800 km² along the lower part of the Wisconsin River, a landscape largely dominated by agriculture. Our goals were (a) to quantify changes in land cover between 1938 and 1992, (b) to evaluate the influence of abiotic and socioeconomic variables on land cover in 1938 and 1992, and (c) to characterize the major processes of land-cover change between these two points in time. The results showed a general shift from agricultural land to forest. Cropland declined from covering 44% to 32% of the study area, while forests and grassland both increased (from 32% to 38% and from 10% to 14% respectively). Multiple linear regressions using three abiotic and two socioeconomic variables captured 6% to 36% of the variation in land-cover categories in 1938 and 9% to 46% of the variation in 1992. Including socioeconomic variables always increased model performance. Agricultural abandonment and a general decline in farming intensity were the most important processes of land-cover change among the processes considered. Areas characterized by the different processes of land-cover change differed in the abiotic and socioeconomic variables that had explanatory power and can be distinguished spatially. Understanding the dynamics of landscapes dominated by human impacts requires methods to incorporate socioeconomic variables and anthropogenic processes in the analyses. Our method of hypothesizing and testing major anthropogenic processes may be a useful tool for studying the dynamics of cultural landscapes. Received 7 December 2000; accepted 2 October 2001.     

A Fast Axonally Transported Protein of the Frog Sciatic Sensory Axons Undergoes Similar Qualitative Changes During Regeneration In Vitro and In Vivo

The adult frog sciatic sensory neurons have been shown to regenerate in vitro. If a crush injury is made at the beginning of culture, regeneration starts after 3.4 days and proceeds at a rate of approximately 0.8 mm/day for several days. Two-dimensional gel electrophoresis was used to study the patterns of radiolabeled, fast axonally transported proteins during the first 7 days of regeneration. Interest was focused on one protein, referred to as rrp31 (regeneration-related protein 31), which changed in apparent pI from 4.9 to 5.3 when the outgrowth of new fibers started. The change was noticeable 3 days after injury and became prominent during day 5 of culturing. By day 7 the pI changed again, this time toward the original value. The in vitro results were supported by experiments in vivo. In this case the change occurred earlier, with a peak only 3 days after injury, after which the pI decreased. If adenosine at 1 mM was included in the culturing medium, the outgrowth of sensory axons was inhibited in a nontoxic way, and the pI changes of rrp31 were prevented. The temporal nature of the pI changes suggests a role for rrp31 in the initiation of the regeneration process.