Cycloheptaamylose-dansyl chloride complex as a fluorescent label of surface membranes in living ciliates

Labelling of surface membrane of living ciliates: Paramecium aurelia and Tetrahymena pyriformis with fluorescent compound--cycloheptaamylose-dansyl chloride complex (CDC) has been achieved. Fluorescence micrographs of the dried samples showed specific localization of CDC on the cell membrane without any intracellular penetration. On the contrary the ciliates which have been dead during labelling revealed a non-specific fluorescence of their whole bodies. Microspectrofluorimetric analysis of labelled Paramecium cells was performed with Leitz microspectrograph. Spectrum of fluorescence emission measured over the cell membrane level had maximum at 450 nm. Strikingly, the emission maximum of the cells dead at the moment of labelling was shifted 10 nm to a longer wavelength. The rate of photofading measured in this case was almost 3-fold higher than for the ciliates labelled as living ones. Fluorescence excitation spectra did not show any difference in the peak position. Thus CDC staining appears to be an useful method of supravital labelling of cell surface enabling also to distinguish--on the basis of spectral characteristics--the ciliates being alive from those dead at the moment of fluorochrome binding.     

Cycloheptaamylose-dansyl chloride complex as a fluorescent label of surface membranes in living ciliates

Summary Labelling of surface membrane of living ciliates: Paramecium aurelia and Tetrahymena pyriformis with fluorescent compound — cycloheptaamylose-dansyl chloride complex (CDC) has been achieved. Fluorescence micrographs of the dried samples showed specific localization of CDC on the cell membrane without any intracellular penetration. On the contrary the ciliates which have been dead during labelling revealed a non-specific fluorescence of their whole bodies. Microspectrofluorimetric analysis of labelled Paramecium cells was performed with Leitz microspectrograph. Spectrum of fluorescence emission measured over the cell membrane level had maximum at 450 nm. Strikingly, the emission maximum of the cells dead at the moment of labelling was shifted 10 nm to a longer wavelength. The rate of photofading measured in this case was almost 3-fold higher than for the ciliates labelled as living ones. Fluorescence excitation spectra did not show any difference in the peak position. Thus CDC staining appears to be an useful method of supravital labelling of cell surface enabling also to distinguish — on the basis of spectral characteristics — the ciliates being alive from those dead at the moment of fluorochrome binding.     

Design and validation of a pulsatile perfusion bioreactor for 3D high cell density cultures

This study presents the design and validation of a pulsatile flow perfusion bioreactor able to provide a suitable environment for 3D high cell density cultures for tissue engineering applications. Our bioreactor system is mobile, does not require the use of traditional cell culture incubators and is easy to sterilize. It provides real‐time monitoring and stable control of pH, dissolved oxygen concentration, temperature, pressure, pulsation frequency, and flow rate. In this bioreactor system, cells are cultured in a gel within a chamber perfused by a culture medium fed by hollow fibers. Human umbilical vein endothelial cells (HUVEC) suspended in fibrin were found to be living, making connections and proliferating up to five to six times their initial seeding number after a 48‐h culture period. Cells were uniformly dispersed within the 14.40 mm × 17.46 mm × 6.35 mm chamber. Cells suspended in 6.35‐mm thick gels and cultured in a traditional CO₂ incubator were found to be round and dead. In control experiments carried out in a traditional cell culture incubator, the scarcely found living cells were mostly on top of the gels, while cells cultured under perfusion bioreactor conditions were found to be alive and uniformly distributed across the gel. Biotechnol. Bioeng. 2009; 104: 1215–1223. © 2009 Wiley Periodicals, Inc.     

Estimation of lipid peroxidation of live cells using a fluorescent probe, diphenyl-1-pyrenylphosphine.

Diphenyl-1-pyrenylphosphine (DPPP), which reacts with lipid hydroperoxides stoichiometrically to yield fluorescent product DPPP oxide, was used as a fluorescent probe for lipid peroxidation in live cells. DPPP was successfully incorporated into U937 cells. Incorporation of DPPP into the cell membrane was confirmed by fluorescence microscopy. Reaction of DPPP with hydroperoxides was examined by monitoring increase in fluorescence intensity of the cell. It was found that lipid-soluble hydroperoxides such as methyl linoleate hydroperoxide preferably react with DPPP, whereas hydrogen peroxide did not react with DPPP located in the membrane. Linear correlation between increase in fluorescence intensity and the amount of methyl linoleate hydroperoxide applied to the cell was observed. DPPP gave little effect on cell proliferation, cell viability or cell morphology for at least 3 d. DPPP oxide, fluorescent product of DPPP, was quite stable in the membrane of living cells for at least 2 d. Fluorescence of DPPP-labeled cells was measured after treating with diethylmaleate (DEM), or 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH), or culturing with low serum content. These reagents and culture condition induced dose- and/or time-dependent increase in fluorescence. Addition of vitamin E effectively suppressed increase in fluorescence. When DPPP-labeled cells and DCFH-DA-labeled cells were treated with NO, H(2)O(2), AAPH, and DEM to compare the formation of hydoperoxides in the membrane and cytosol, distinct patterns of peroxide formation were observed. These results indicate that fluorescent probe DPPP is eligible for estimation of lipid peroxidation proceeding in the membrane of live cells, and use of this probe is especially advantageous in long-term peroxidation of the cell.     

Nano-probing of the membrane dynamics of rat pheochromocytoma by near-field optics

High-resolution analysis of activities of live cells is limited by the use of non-invasive methods. Apparatuses such as SEM, STM or AFM are not practicable because the necessary treatment or the harsh contact with system probe will disturb or destroy the cell. Optical methods are purely non-invasive, but they are usually diffraction limited and then their resolution is limited to approximately 1 microm. To overcome these restrictions, we introduce here the study of membrane activity of a live cell sample using a Scanning Near-field Optical Microscope (SNOM). A near field optical microscope is able to detect tiny vertical movement on the cell membrane in the range of only 1 nm or less, about 3 orders of magnitude better than conventional optical microscopes. It is a purely non-invasive, non-contact method, so the natural life activity of the sample is unperturbed. In this report, we demonstrated the nanometer-level resolving ability of our SNOM system analyzing cardiomyocytes samples of which membrane movement is known, and then we present new intriguing data of sharp 40 nm cell membrane sudden events on rat pheochromocytoma cell line PC12. All the measurements are carried out in culture medium with alive and unperturbed samples. We believe that this methodology will open a new approach to investigate live samples. The extreme sensitivity of SNOM allows measurements that are not possible with any other method on live biomaterial paving the way for a broad range of novel studies and applications.     

Cell death in lake phytoplankton communities

1. The fraction of living and dead phytoplankton cells in seven Florida lakes was assessed by using the cell digestion assay, a non‐staining membrane permeability test. The cell digestion assay is an effective method to analyse cell viability in complex natural phytoplankton communities. 2. The lakes examined ranged widely in phytoplankton abundance and community composition. The variability in the percentage of living cells (% LC) was high among the taxonomic groups forming the different phytoplankton communities, ranging from 19.7% to 98% LC. 3. All cells within single cyanobacteria filaments were determined to be either dead or alive, suggesting physiological integration of the cells within colonies. 4. Within each lake, the dominant taxa generally exhibited the highest proportion of living cells. A high proportion of living cells was found to be a characteristic of the different taxa forming the communities of eutrophic lakes. The average value for the % LC for all groups comprising the phytoplankton communities in each of the lakes ranged from 29.9 ± 7.2 to 80.4 ± 4.0 (mean ± SE) and varied strongly and positively with chlorophyll a concentration. 5. These results suggest phytoplankton cell death to be an important process structuring phytoplankton communities in lakes, particularly in oligotrophic ones.     

Detection of the absorption of glucose molecules by living cells using atomic force microscopy

A very small electrode (nanobiosensor) was constructed by immobilizing enzyme (glucose oxidase or hexokinase) on the surface of the cantilever of the atomic force microscope in order to detect the absorption of glucose molecules by living cells. If glucose is present, the nanobiosensor deflects, probably due to the reaction heat evolved in the process. Nanobiosensors built with inactivated enzyme or cantilevers without immobilized enzyme were not capable of producing this type of signal (deflection). This technique will be very useful in detecting the passage of specific molecules through a cell wall (or a cell membrane for other types of cells).     

The gene is not dead, merely orphaned and seeking a home

SUMMARY Despite announcements and obituaries, news of the death of the gene has been greatly exaggerated, or so says the gene as it struggles to survive and find a safe haven from which to steer its course through development and evolution. In this short piece, I consider recent claims that the gene is dead. I conclude that the gene is alive and well, living and functioning in the cell, which is both its natural home and a fundamental unit of evo-devo.     

Effect of proteases,phospholipases and polysaccharide-splitting enzymes on plasma membrane particles and on the synthesis of the fibrillar cell wall component in yeast protoplasts

Plasma membrane particles demonstrable by the freeze-etching technique, play, according to some authors, a role in the cell wall synthesis. On a model of yeast protoplast capable of regenerating the cell wall we studied the morphology of plasma membrane particles and the synthesis of the fibrillar cell wall component following a treatment with various enzymes and with lysolecithin. The enzymes used included proteases (trypsin, papain, pronase), polysaccharide-splitting enzymes (snail enzyme complex, mannosidase), phospholipases (A, C, D) and lipase. Upon treating living protoplasts with these substances in no case did we observe any morphologically demonstrable change in the particle structure or in their distribution in the plasma membrane. The fibrillar cell wall component was synthetized even in the presence of proteases and phospholipases. If the plasma membrane particles are assumed to represent enzyme systems synthesizing the cell wall component then in living protoplasts they are not located on the outer plasma membrane face or else are protected by some mechanism against the action of the corresponding enzymes.     

The Spatial Distribution of Mustelidae in France

We estimated the spatial distribution of 6 Mustelidae species in France using the data collected by the French national hunting and wildlife agency under the “small carnivorous species logbooks” program. The 1500 national wildlife protection officers working for this agency spend 80% of their working time traveling in the spatial area in which they have authority. During their travels, they occasionally detect dead or living small and medium size carnivorous animals. Between 2002 and 2005, each car operated by this agency was equipped with a logbook in which officers recorded information about the detected animals (species, location, dead or alive, date). Thus, more than 30000 dead or living animals were detected during the study period. Because a large number of detected animals in a region could have been the result of a high sampling pressure there, we modeled the number of detected animals as a function of the sampling effort to allow for unbiased estimation of the species density. For dead animals -- mostly roadkill -- we supposed that the effort in a given region was proportional to the distance traveled by the officers. For living animals, we had no way to measure the sampling effort. We demonstrated that it was possible to use the whole dataset (dead and living animals) to estimate the following: (i) the relative density -- i.e., the density multiplied by an unknown constant -- of each species of interest across the different French agricultural regions, (ii) the sampling effort for living animals for each region, and (iii) the relative detection probability for various species of interest.     

THE PHYSIOLOGY OF VIRUS DISEASES IN PLANTS

In this paper the movement in the plant of the causative agent of virus disease is discussed. The relevant data in the literature are summarised.
A method is described whereby a portion of the stem in the middle of a tomato plant was killed either by chloroform or by steam. In this way the living upper and lower portions of the plant were connected by a bridge of dead tissue. It is shown that the symptoms appeared in that part of the plant in which the inoculation was made. The virus agent did not travel across the dead region.
The xylem tracts were not materially affected by this treatment, and water travelled across the region. Evidence of this is the fact that the distal portion remained turgid and sometimes continued growth for a considerable time. If the stem were removed above the ground level and put into eosin solution, this travelled readily over the dead tissue. That the vessels were not occluded by protein plugs is shown by the fact that particulate substances were carried up the xylem tracts past the dead region.
No evidence of adsorption of the virus agent to the cell remains could be adduced, so it is assumed that it was not travelling in the xylem stream.     

Measurement of hydrodynamic shear by using a dissolved oxygen probe

When a dissolved oxygen (DO) probe is submerged in an air-saturated cell culture medium the thickness of the liquid film that exists outside the membrane of a DO probe changes with hydrodynamic shear. The response of the DO probe thus varies with the hydrodynamic shear environment near the DO probe in cell culture reactors. The thickness of the liquid film was estimated by using a three-layer model, which describes the flow of DO molecules through the liquid layer, the membrane, and the electrolyte, to the cathode of a DO probe. According to the three-layer model, the current output of the DO probe was a strong function of thickness of the liquid film outside the membrane of the DO probe. A correlation between shear rates on the surface of the probe and the DO saturation reading was obtained by using two concentric cylinders with a rotating inner cylinder. This correlation was then used to characterize the local hydrodynamic shear environment in a cell culture reactor. (c) 1993 John Wiley & Sons, Inc.     

Dead cell counts during serum cultivation are underestimated by the fluorescent live/dead assay

The live/dead fluorescent assay provides a quick method for assessing the proportion of live and dead cells in cell culture systems or tissues and is widely used. Dead cells are detected by the fluorescence produced when propidium iodide (PI) binds to DNA; PI and similar molecules are excluded from live cells but can penetrate dead cells because of their loss of membrane integrity. Here we investigated the effect of serum in the culture medium on the reliability of the method. We assessed viability of chondrocytes with/without serum using both a live/dead assay kit and also trypan blue staining. We found that after 2 days of culture, the DNA-binding dye PI could no longer detect dead cells if serum was present but they were readily detected in serum-free medium or if an inhibitor to DNase I was added to the serum-containing medium. Dead cells could be detected by trypan blue staining in all cultures. Hence dead cells are no longer detected as the DNase I present in serum degrades their DNA. DNA-binding dyes may thus not give a reliable estimate of the number of dead cells in systems that have been cultured in the presence of serum for several days.     

Temperature effects on soybean imbibition and leakage

As a part of an analysis of the nature of chilling injury to seeds, measurements were made of the initial linear rates of water entry into and solute leakage out of cotyledons of soybean at various temperatures. Arrhenius plots were approximately linear for water entry into both living and dead cotyledons, with the slope (and activation energy) for entry into living cells being insignificantly higher than for dead cells, suggesting little effect of membrane barriers on water entry. The plots for solute leakage showed 10-fold lower leakage rates from living than from dead tissues; a reversal of slope in the Arrhenius plot at temperatures below 15 C reflected increasing leakage rates, interpreted as a quantitative disruption of membrane reorganization at the temperatures associated with chilling injury.     

Distending stress of the cytoskeleton is a key determinant of cell rheological behavior

One fundamental question in cell biology is what determines rheological properties of living cells. If the cytoskeletal distending stress is a key determinant of cell rheology, then modulating this stress by cell stretching should have a major effect on cell rheological properties. If not, then other mechanisms must play a major role. We developed a stretchable cell culture device that could rapidly stretch cells and thus generate passive mechanical stress within the cytoskeleton. This device was placed inside a magnetic cytometry system to measure the effect of stretching on rheological properties of cultured human airway smooth muscle cells. A gradual increase in cell distension caused a systematic increase in cell dynamic stiffness in a manner which was consistent with earlier observations where the active component of the distending stress was modulated pharmacologically. These findings provide strong evidence that the cytoskeletal distending stress is a key determinant of cell rheological properties.     

Requirement for G protein activity at a specific time during embryonic chick myogenesis

Signaling between embryonic myoblasts involves prostaglandin metabolism, the activation of a membrane receptor and changes in polyphosphatidyl inositol metabolism. Many of these membrane-localized events occur between 33 to 35 h of differentiation, concomitant with a dramatic change in membrane organization, in myoblast aggregates in culture. Since many receptors affect inositol phosphate metabolism by activating a GTP-binding protein (G protein), we asked if there was evidence for such a protein in myogenic signaling. We show that during the period of differentiation in culture when prostaglandin is needed to bind to a transient receptor, a pertussis toxin-sensitive but cholera toxin-insensitive G protein must act. If this activation is blocked, the characteristic change in myoblast cell adhesion and subsequent membrane fusion do not occur. We suggest that a G protein couples the activated prostaglandin receptor and the change in polyphosphatidyl inositol metabolism and that this membrane transduction step is necessary for subsequent membrane differentiation events during myogenesis.     

Noncontact measurement of the local mechanical properties of living cells using pressure applied via a pipette

Mechanosensitivity in living biological tissue is a study area of increasing importance, but investigative tools are often inadequate. We have developed a noncontact nanoscale method to apply quantified positive and negative force at defined positions to the soft responsive surface of living cells. The method uses applied hydrostatic pressure (0.1-150 kPa) through a pipette, while the pipette-sample separation is kept constant above the cell surface using ion conductance based distance feedback. This prevents any surface contact, or contamination of the pipette, allowing repeated measurements. We show that we can probe the local mechanical properties of living cells using increasing pressure, and hence measure the nanomechanical properties of the cell membrane and the underlying cytoskeleton in a variety of cells (erythrocytes, epithelium, cardiomyocytes and neurons). Because the cell surface can first be imaged without pressure, it is possible to relate the mechanical properties to the local cell topography. This method is well suited to probe the nanomechanical properties and mechanosensitivity of living cells.     

Parenchyma cell respiration and survival in secondary xylem: does metabolic activity decline with cell age?

Sapwood respiration often declines towards the sapwood/heartwood boundary, but it is not known if parenchyma metabolic activity declines with cell age. We measured sapwood respiration in five temperate species (sapwood age range of 5-64 years) and expressed respiration on a live cell basis by quantifying living parenchyma. We found no effect of parenchyma age on respiration in two conifers (Pinus strobus, Tsuga canadensis), both of which had significant amounts of dead parenchyma in the sapwood. In angiosperms (Acer rubrum, Fraxinus americana, Quercus rubra), both bulk tissue and live cell respiration were reduced by about one-half in the oldest relative to the youngest sapwood, and all sapwood parenchyma remained alive. Conifers and angiosperms had similar bulk tissue respiration despite a smaller proportion of parenchyma in conifers (5% versus 15-25% in angiosperms), such that conifer parenchyma respired at rates about three times those of angiosperms. The fact that 5-year-old parenchyma cells respired at the same rate as 25-year-old cells in conifers suggests that there is no inherent or intrinsic decline in respiration as a result of cellular ageing. In contrast, it is not known whether differences observed in cellular respiration rates of angiosperms are a function of age per se, or whether active regulation of metabolic rate or positional effects (e.g. proximity to resources and/or hormones) could be the cause of reduced respiration in older sapwood.     

Mechanical response of a living human epidermal keratinocyte sheet as measured in a composite diaphragm inflation experiment

Sheets of normal human epidermal keratinocytes (NHEKs) were reconstituted in vitro on tensed but highly compliant, freestanding polydimethylsiloxane (PDMS) membranes, 5.0 mm in diameter and 10 mum thick. NHEK-PDMS composite diaphragm (CD) specimens were then subjected to cyclical axisymmetric inflation tests to investigate epithelial sheet rheology under conditions of physiologically severe deformations (~50% nominal polar biaxial strains). Because the compliance of the specially formulated PDMS membrane was greater than that of the attached cell layer, the finite load-deformation behavior (mechanical response) of the living NHEK sheet was inferred from differences between the mechanical behavior of the CD specimen and the response of the underlying PDMS membrane measured prior to cell culture. In these composite diaphragm inflation (CDI) experiments, interconnected NHEKs exhibited rheological behaviors that were suggestive of a viscoelastic-plastic stress response. Remarkably, specimens returned to quiescent culture following a sequence of inflation tests recovered at least 80% of their original ability to store elastic strain energy, evidence of biological adaptation and recovery or restitutio ad integrum. Unlike methodologies that assay the morphological or biochemical response of cultured cells to an applied mechanostimulus, CDI experiments can be used to probe the load-bearing functions of desmosomes and adherens junctions within a living epithelial sheet, as well as to assess the rheological behaviors of the intermediate filament and microfilament networks that these cell-cell junctions serve to interconnect.     

Flow cytometric studies of bicarbonate-mediated Ca2+ influx in boar sperm populations

Boar spermatozoa loaded with the Ca²⁺ probe fluo-3 were incubated in various Tyrode's-based media similar to those used for in vitro fertilization (IVF), and samples were then analysed by two-colour flow cytometry; propidium iodide was included in the media to detect membrane-damaged (“dead”) cells. If media contained bicarbonate/CO₂ (a component thought to promote capacitation), part of the live sperm population experienced a considerable influx of Ca²⁺ into both head and tail compartments. The percentage of responding cells reached a maximum after about 30 min, but both during and after this period there was also a steady increase in the number of dead cells. This bicarbonatemediated increase in cell death took place in the absence of external Ca²⁺. Evidence was obtained that the entry of propidium iodide was preceded by a change in permeability of the plasma membrane, detectable by leakage of carboxydichlorofluorescein, and it was therefore deduced that the Ca²⁺ influx detected by fluo-3 was due to destabilization of the plasma membrane. A similar response could be produced by both caffeine and papaverine (best known as phosphodiesterase inhibitors), but neither cyclic AMP nor activators of adenylate cyclase had any effect. There was no influence of substrate on the process, but, in comparison to poly(vinyl alcohol), serum albumin enhanced it. The precise relevance of this destabilization to capacitation is not yet clear, but it seems significant that the process is mediated or enhanced by components often specifically included in IVF media, and that different individual cells respond after different times. © 1993 Wiley-Liss, Inc.