Manipulation of herpes simplex virus type 1 by dielectrophoresis

The frequency-dependent dielectrophoretic behaviour of an enveloped mammalian virus, herpes simplex virus type 1 is described. It is demonstrated that over the range 10 kHz–20 MHz, these viral particles, when suspended in an aqueous medium of conductivity 5 mS m^?1, can be manipulated by both positive and negative dielectrophoresis using microfabricated electrode arrays. The observed transition from positive to negative dielectrophoresis at frequencies around 4.5 MHz is in qualitative agreement with a simple model of the virus as a conducting particle surrounded by an insulating membrane.     

A Nomograph for Calculating the Optimal Frequency for Dielectrophoresis and the Characteristic Frequency of the β-Dispersion of Cell Membrane Vesicles

The first stage of the two-stage cell electrofusion technique involves the dielectrophoretic apposition, in an AC field, of protoplasts suspended in a medium of relatively low specific conductivity. A frequency at which the maximum dielectrophoretic force is exerted is given by the characteristic frequency for the dielectric relaxation by a Maxwell-Wagner type of mechanism. We provide a nomograph for the rapid calculation of this frequency.     

Dielectrophoretic study of human erythrocytes

This paper is concerned with the dielectrophoretic study of human erythrocytes under cylindrical field geometry. The influence of physical variables such as the frequency and voltage of the applied electric field, conductivity of the medium in which the cells are suspended, cell concentration and exposure time of the cell to the non-uniform electric field on dielectrophoretic collection rate (DCR) is determined in a systematic manner. It is interesting to note from the DCR spectrum of human erythrocytes that the DCR is minimum at one frequency, maximum at another and there is practically no yield over a certain frequency range. This may be attributed to the variation of complex dielectric constant of the particle and medium over that frequency range. From the DCR spectrum of different groups, it is clear that DCR behaviour is different in the frequency range from 0.3–1.5 MHz, under similar conditions of temperature, conductivity and concentration of erythrocyte suspension and strength of applied AC field. The response of DCR with voltage of the applied field, concentration of cell suspension and square root of elapsed time of the cells confirms the theory of dielectrophoresis.     

Modulation of ATP-dependent Ca2+ transport in rat parotid basolateral membrane vesicles by K+ + Cl- flux

In basolateral membrane vesicles (BLMV) isolated from rat parotid glands, the initial rate of ATP-dependent Ca2+ transport, in the presence of KCl, was approx. 2-fold higher than that obtained with mannitol, sucrose or N-methyl-D-glucamine (NMDG)-gluconate. Only NH4+, Rb+, or Br- could effectively substitute for K+ or Cl-, respectively. This KCl activation was concentration dependent, with maximal response by 50 mM KCl. An inwardly directed KCl gradient up to 50 mM KCl had no effect on Ca2+ transport, while equilibration of the vesicles with KCl (greater than 100 mM) increased transport 15-20%. In presence of Cl-, 86Rb+ uptake was 2.5-fold greater than in the presence of gluconate. 0.5 mM furosemide inhibited 86Rb+ flux by approx. 60% in a Cl- medium and by approx. 20% in a gluconate medium. Furosemide also inhibited KCl activation of Ca2+ transport with half maximal inhibition either at 0.4 mM or 0.05 mM, depending on whether 45Ca2+ transport was measured with KCl (150 mM) equilibrium or KCl (150 mM) gradient. In a mannitol containing assay medium, potassium gluconate loaded vesicles had a higher (approx. 25%) rate of Ca2+ transport than mannitol loaded vesicles. Addition of valinomycin (5 microM) to potassium gluconate loaded vesicles further stimulated (approx. 30%) the Ca2+ transport rate. These results suggest that during ATP dependent Ca2+ transport in parotid BLMV, K+ can be recycled by the concerted activities of a K+ and Cl- coupled flux and a K+ conductance.     

The growth response of cells in medium made hyperosmolal with electrolytes or mannitol.

A comparison of the growth rates of established human lymphoid and tumor cell lines was performed in nutrient medium made hyperosmolal with mannitol, NaCl, or mixtures of NaCl and KCl at a constant Na/K ratio. It was found that considerably higher osmolalities were attained with mannitol than electrolytes before a reduction in the growth rate of the culture was observed. This suggests that mannitol and electrolytes affected the growth rate through different mechanisms. Mannitol uptake was studied with two of the cell lines and both cell lines were found to be permeable to mannitol. This eventually would have eliminated the osmolality gradient between the interior of the cell and the medium, and could explain why higher osmolalities were obtained with mannitol before the growth rate was effected. In addition, initial experiments showed that these cell lines may also be able to metabolize mannitol.     

The intramitochondrial volume measured using sucrose as an extramitochondrial marker overestimates the true matrix volume determined with mannitol.

The matrix volume of isolated liver and heart mitochondria has been estimated at various osmolarities and in various osmotic supports using 36Cl- and [14C]sucrose, D-mannitol, D-3-methoxyglucose and choline as extramitochondrial markers. The use of 3-methoxyglucose was only possible at 0 degree C since it entered mitochondria at physiological temperatures. All extramitochondrial markers used gave linear plots of apparent matrix volume against the reciprocal of the osmolarity, but the slope of this plot was greater when sucrose was used than with the other extramitochondrial markers. When extrapolated to infinite osmolarity the mean matrix volume was zero when mannitol was used, but about 0.6 microliter/mg of protein for sucrose and Cl- and -0.4 microliter/mg of protein when choline was used. At physiological osmolarity (about 330 m-osmol) the mean matrix volume of de-energized liver mitochondria in KCl medium estimated using mannitol was 0.46 microliter/mg of protein, whereas that obtained using sucrose was 1.68 microliters/mg of protein. Values in mannitol, choline and sucrose media were similar when mannitol but not sucrose was used as extramitochondrial marker. It is argued that the 3H2O/[14C]mannitol space more accurately reflects the true mitochondrial matrix volume than does the 3H2O/[14C]sucrose space. The consequences of this for measurements of the protonmotive force and the intramitochondrial concentration of metabolites are discussed.     

Osmotic water permeability and solute reflection coefficients of rat kidney brush-border membrane vesicles

Solute reflection coefficients, sigma i, of rat kidney brush-border membrane vesicles were determined by the comparison of water flows induced by equiosmolal gradients of sucrose and NaCl, KCl or mannitol. The values of 0.53 for sigma NaCl and 0.56 for sigma KCl when compared with 0.92 for sigma mannitol suggested some interactions between salt and water pathways. Altering the membrane proteins with 0.4 mM HgCl2 decreased the osmotic water permeability of the vesicles by 70 to 80% and brought sigma NaCl and sigma KCl to a value not different from 1. This argued in favor of water protein pathways in the luminal membrane of kidney proximal cells which are partly accessible to NaCl and KCl.     

Factors influencing survival of mammalian cells exposed to hypothermia. II. Effects of various hypertonic media

Survival of Chinese hamster lung (V79) cells, exposed as a function of time to hypothermia in tissue culture, in isosmotic and various hypertonic media was measured using a colony assay. The mechanism of hypothermic cell killing is different above and below 7 degrees C in this cell line. Addition of NaCl or mannitol to increase the tonicity to 400 mOsm greatly decreased the survival at 10 degrees C while addition of KCl had no significant effect. When these experiments were repeated at 5 degrees C, addition of either NaCl, KCl, or mannitol was detrimental to long-term cell survival. Furthermore, addition of mannitol to the medium did not improve survival when cells were stored at 7 degrees C. Addition of KCl at 5 or 10 degrees C or NaCl at 5 degrees C only affected the cells' ability to accumulate sublethal damage, while addition of mannitol at 5 or 10 degrees C affected both of the above and the cold sensitivity of the cells. Addition of NaCl at 10 degrees C only affected the latter. These experiments suggest that prevention of cell swelling by these conditions, while possibly necessary during clinical hypothermic organ storage, is detrimental to single cell survival at these temperatures.     

Effects of medium osmolarity on the release of amino acids from isolated cotyledons of developing pea seeds

The release of endogenous amino acids from isolated, immature pea (Pisum sativum L. cv. Marzia) cotyledons was investigated in relation to their developmental stage and the osmolarity of the bathing medium. The water potential of the cotyledons was about-1.1 MPa from which it could be inferred that the osmolarity of their apoplastic fluids will be approximately 450 mosmol·l^?1. The time course of amino-acid release conformed to an exponential function. Rate constants of the release were in the range 0.3 to 0.9 · h ^?1. No indication was found for increased permeability of the plasmamembrane for amino acids at low medium osmolarity. Rate constants were even 1.5-fold lower in 0 mM mannitol than in medium with 400 mM mannitol. This effect could be ascribed to reduced protein synthesis in hypotonic media. In the presence of 400 mM mannitol the release was nearly proportional to the total amino-acid pool of the cotyledons and ranged from 12% to 8% for the various developmental stages. Amino-acid release was stimulated by incubation in a hypotonic medium (< 400 mM mannitol), up to fourfold in a medium without mannitol where as much as 45% of the cotyledonary amino-acid content could be released. The extra aminoacid release induced by the hypotonic condition declined during development and eventually vanished completely. Release of amino acids into a medium with 400 mM mannitol was more selective than into a medium without mannitol. For instance, arginine was one of the main constituents of the cotyledonary amino-acid pool (19%) as well as of the released amino-acid mixture when the medium contained no mannitol (10%), whereas it was virtually absent when the medium contained 400 mM mannitol. As an overall interpretation of these results, it is proposed that the hypotonic condition greatly enhances the permeability of the tonoplast (not that of the plasmalemma) for amino acids so that the otherwise well-sequestered amino acids in the vacuole become available for release into the bathing medium.     

阳离子及ATP对绿豆线粒体膨胀与收缩的影响

本文报道了一价阳离子 K~+、Na~+及两价阳离子 Mg~(++)、Ca~(++)以及 ATP 对绿豆线粒体膨胀和收缩的影响。K~+、Na~+在低渗条件下引起线粒体瞬时的迅速膨胀。在同样离子强度下K~+引起的膨胀大于 Na~+。ATP 和 Mg~(++)能诱发低渗条件下膨胀线粒体的收缩,但对等渗和高渗 KCl 或 Nacl 溶液中膨胀的线粒体无明显作用。生理浓度的 Mg~(++)、Ca~(++)在低渗条件下引起线粒体缓慢的但幅度较大的膨胀,5mmol/l ATP 引起这种膨胀线粒体的部分收缩。1mmol/lca~(++)在含0.125mmol/l KCl 或在含0.25mol/l甘露醇的等渗介质中几乎不引起膨胀,而ATP 促进大幅度膨胀,10mmol/l MgCl_2引起这种膨胀线粒体的部分收缩。2mmol/l MgCl_2在含有0.25mol/l 甘露醇的等渗介质中引起明显膨胀,ATP 促进这种膨胀。0.125mol/lKCl+2mmol/l MgCl_2为肌动蛋白从单体聚合成多聚体所必须的条件。在此条件下,线粒体几乎不膨胀,而加入 ATP 后则促进大幅度膨胀。在电子显微镜下观察了等渗及低渗条件下线粒体形态变化。     

Arabitol accumulation in Geotrichum candidum

Culture conditions which lead to the intracellular accumulation of arabitol and mannitol in Geotrichum candidum were investigated. The accumulation of arabitol was dependent on the concentrations of metabolizable hexoses, the non-metabolizable disaccharide sucrose, NaCl and KCl in the growth medium. In media containing 2% (w/v) glucose, fructose or l-sorbose cultures contained only mannitol after 48 h or 72 h growth. In media containing 10% (w/v) to 30% (w/v) glucose, or 25% (w/v) fructose or l-sorbose there was an increase in the total concentration of intracellular polyol due to the accumulation of arabitol. This pentitol was also found to accumulate intracellularly when the organism was grown in medium containing 34% (w/v) sucrose, 0.7 M NaCl or 0.7 M KCl in addition to 2% (w/v) glucose. Under the conditions tested no change in the accumulation of mannitol or ethanol-soluble carbohydrate, believed to be primarily composed of trehalose, was evident.Intracellular polyol was released during incubation of arthrospores obtained from media containing 25% or 10% glucose, in distilled water at 25° C, but no polyol was released under these conditions from arthrospores obtained from growth in 2% glucose medium.     

Survival of Saccharomyces cerevisiae Y5 during starvation in the presence of osmotic supports

The viability of starved or dying Saccharomyces cerevisiae Y5 cells was extended for up to 1,500 h by adding osmotic supports (0.6 M sorbitol or mannitol) to the starvation media. Replacement of these polyols with 0.3 M KCl, 0.5 M MgSO4, or 0.2 M MgCl2 postponed the death of cells incubated in N-free medium, but accelerated it in distilled water.     

Survival of Saccharomyces cerevisiae Y5 during starvation in the presence of osmotic supports.

The viability of starved or dying Saccharomyces cerevisiae Y5 cells was extended for up to 1,500 h by adding osmotic supports (0.6 M sorbitol or mannitol) to the starvation media. Replacement of these polyols with 0.3 M KCl, 0.5 M MgSO4, or 0.2 M MgCl2 postponed the death of cells incubated in N-free medium, but accelerated it in distilled water.     

Herpes simplex virus capsids are transported in neuronal axons without an envelope containing the viral glycoproteins

Electron micrographic studies of neuronal axons have produced contradictory conclusions on how alphaherpesviruses are transported from neuron cell bodies to axon termini. Some reports have described unenveloped capsids transported on axonal microtubules with separate transport of viral glycoproteins within membrane vesicles. Others have observed enveloped virions in proximal and distal axons. We characterized transport of herpes simplex virus (HSV) in human and rat neurons by staining permeabilized neurons with capsid- and glycoprotein-specific antibodies. Deconvolution microscopy was used to view 200-nm sections of axons. HSV glycoproteins were very rarely associated with capsids (3 to 5%) and vice versa. Instances of glycoprotein/capsid overlap frequently involved nonconcentric puncta and regions of axons with dense viral protein concentrations. Similarly, HSV capsids expressing a VP26-green fluorescent protein fusion protein (VP26/GFP) did not stain with antiglycoprotein antibodies. Live-cell imaging experiments with VP26/GFP-labeled capsids demonstrated that capsids moved in a saltatory fashion, and very few stalled for more than 1 to 2 min. To determine if capsids could be transported down axons without glycoproteins, neurons were treated with brefeldin A (BFA). However, BFA blocked both capsid and glycoprotein transport. Glycoproteins were transported into and down axons normally when neurons were infected with an HSV mutant that produces immature capsids that are retained in the nucleus. We concluded that HSV capsids are transported in axons without an envelope containing viral glycoproteins, with glycoproteins transported separately and assembling with capsids at axon termini.     

Effect of taurine on calcium binding to microsomes isolated from rat cerebral cortex

Effects of taurine on Ca⁺⁺ binding to microsomes isolated from rat cerebral cortex were investigated in a medium containing various concentrations of KCl and/or NaCl. Calcium binding to microsomes was inhibited in a dose-dependent fashion by taurine in the incubation medium containing 5 mM KCl and 115 mM NaCl, while there was no inhibition in the medium containing 115 mM KCl and 5 mM NaCl. Taurine also decreased Ca⁺⁺ binding in the medium containing 70 mM KCl without NaCl. A similar tendency toward inhibition of the Ca⁺⁺ binding was observed in the medium with 5 mM or 120 mM KCl without NaCl. Taurine did not influence the Ca⁺⁺ binding in the medium containing different concentrations of NaCl without KCl, or in the medium from which KCl and NaCl were omitted. Isethionate, glycine, γ-aminobutyric acid, β-alanine and L-leucine did not significantly alter the Ca⁺⁺ binding to microsomes in the medium containing 70 mM KCl without NaCl. Thus it would appear that taurine may modulate the binding of calcium to microsomes in conditions which resemble the state of depolarization, while it is inactive in the normal resting state. This effect is apparently specific to taurine amongst a series of putative “inhibitory” amino acids.     

Kinetic study of the self-assembly of brome mosaic virus capsid

The protein of brome mosaic virus can self assemble in-vitro to form empty capsids. In the absence of RNA at pH=7 and 0.5 M KCl there is a dynamic equilibrium between monomers and oligomers. At pH=5 the protein assembles into empty capsids. The kinetics of this assembly, triggered by pH jump from neutral to acidic pH, was investigated by X-ray and light scattering.Cryoelectron microscopy observations suggested that reconstitution is achieved by progressive incorporation of small building units in a spherical shell. This hypothesis has been tested by the analysis of the scattering data in terms of four classes of incomplete capsids represented as spherical shells with holes of different sizes. The time dependence of the population of each class was determined by a least squares analysis of the experimental data. Although the basic polymerizing unit has not been uniquely characterized, the results are compatible with a dimer for this species. The characteristic times for capsid assembly are found to vary as the inverse of the square of the concentration.     

Influence of Na+ on Synthesis of Macromolecules by a Marine Bacterium

Resting cells of Vibrio natriegens acquired the ability to take up (14)C-labeled mannitol in media containing Na(+) and K(+). But, the cells took up a significant quantity of the label as well in the presence of 0.3 m K(+) and no Na(+). The label was distributed throughout the cells in both systems. Cells incubated in mannitol minimal culture medium proliferated and synthesized approximately nine times as much protein in the presence of Na(+) and K(+) as those incubated in the presence of mannitol and 0.3 m K(+). The bacteria did not proliferate in the absence of Na(+). Cells incubated in medium containing mannitol and Na(+) and K(+) synthesized approximately twice the quantity of deoxyribonucleic acid and ribonucleic acid as those incubated in medium containing mannitol and 0.3 m K(+) but no Na(+). A significant amount of mannitolbinding protein was synthesized in the membranes of V. natriegens incubated in the presence of mannitol and Na(+) and K(+), but only a small quantity was produced in medium containing mannitol and 0.3 m K(+) but no Na(+). A binding fraction comprising at least two proteins (both with molecular weight near 34,000) was isolated by gel electrophoresis from other components of a K(2)CO(3)-extract of membrane protein from mannitol-grown cells. This binding fraction mediated phosphorylation of mannitol at the expense of either adenosine triphosphate or phosphoenolpyruvate. It was then found that mannitol-grown, but not broth-grown, cells contained nicotinamide adenine dinucleotide-linked mannitol-1-phosphate dehydrogenase. Neither contained mannitol dehydrogenase.     

Dielectrophoretic behavior of yeast cells: effect of growth sources and cell wall and a comparison with fungal spores.

Saccharomyces cerevisiae showed different dielectrophoretic behavior depending on the source of carbon for growth. Growth on fermentable carbon sources produced a dielectrophoretic response that decreased according to the amount of sugar present in the culture medium. Growth on nonfermentable carbon sources produced a constant dielectrophoretic yield, independent of the amount and source of carbon present in the medium. The dielectrophoretic yield, however, was independent of the nitrogen source. The yield spectrum for S. cerevisiae protoplasts was similar to that for the cells, although a decrease in the absolute value was observed. This decrease could be explained by the reduction in cell size and by assuming that the cell wall contributes a negative net charge to the yield. Fungal spores responded to the nonuniform electric field in the same range of frequencies as assayed for yeast cells.     

Delta-Pyrroline-5-carboxylic Acid Dehydrogenase in Barley, a Proline-accumulating Species

The characteristics of the enzyme Delta(1)-pyrroline-5-carboxylic acid dehydrogenase from etiolated barley (Hordeum distichum) shoots have been examined. The bulk of the enzyme activity was found in the 10,000g pellet fraction, this activity being displayed only after detergent treatment of the suspended pellet. The enzyme was most active at pH 8, and activity was NAD-dependent. Enzyme activity was unaffected by either mannitol or sucrose in the reaction mixture up to a concentration of 0.45 m but was strongly inhibited by Cl(-) and, to a lesser extent, SO(4) (2-). The inhibition attributable to KCl was reversed by increasing the concentration of Delta(1)-pyrroline-5-carboxylic acid in the reaction mixture.