Surface Charge Density Estimation of Vigna mungo Protoplasts Using a Fluorescent Dye, 9-Aminoacridine

We showed that the surface charge density of protoplasts canbe estimated by the 9-aminoacridine method. The estimated surfacecharge density of the protoplasts isolated from elongating regionsof Vigna mungo root was – 39 ? 8 mC/m². The negative surfacecharge density increased when protoplasts were treated withglutaraldehyde or when EDTA was added to the protoplast suspensionmedium. These results support the validity of our estimationof the surface charge density of protoplasts by the 9-aminoacridinemethod. The concentration of amino groups at the surface ofthe protoplasts was estimated to be 34 mC/m². (Received June 19, 1987; Accepted April 11, 1988)     

Electrophoretic Studies on Plant Protoplasts II. Relative Amounts of Various Charged Groups on the Surface of Barley Mesophyll Protoplasts

Electrophoretic mobilities of barley mesophyll cell protoplastsmodified by chemical and enzymatic treatments were measuredin media at various pH values to elucidate the contributionof phosphate, carboxylate and amino groups to the surface chargedensity. Existence of these charged groups was confirmed byresults of treatment of protoplasts with glutaraldehyde (foramino groups), acid phosphatase (for phosphate groups) and l-ethyl-3-(3-dimethylaminopropyl)carbodi-imidetogether with glycine methyl ester (for carboxylate groups).The relative amounts of these groups were estimated from thecurves of surface charge density () vs. surface pH (pH_s) ofthe treated protoplasts, in terms of simplified acid-base dissociationcurves. The estimated ratio of the amounts of phosphate, carboxylateand amino groups was approximate 0.5 :0.5 : 0.7 for the native(unmodified) barley mesophyll cell protoplasts, when the totalnegative charge on the native protoplasts was assumed to be1. (Received January 9, 1989; Accepted April 28, 1989)     

Electrophoretic Studies on Plant Protoplasts I. pH Dependence of Zeta Potentials of Protoplasts from Various Sources

Electrophoretic mobilities of plant protoplasts from varioussources were measured, as a function of the pH of the medium,by a micro-electrophoresis technique to characterize the protoplastsin terms of curves of zeta potential vs. protoplast surfacepH (pH_s). The shape of the curves of zeta potential vs. pH_scurves differed among preparations of protoplasts isolated fromvarious species and strains. The isoelectric points (pI) ofthe protoplasts measured in this study were between 3.0 and4.0. These differences among the protoplasts suggest that itmay be possible to develop an electrophoretic method for theselection of protoplasts. The shape of the curves of zeta potentialvs. pH_s also indicated that carboxyl groups, as well as phosphategroups, may contribute to the negative charges on the surfaceof protoplasts. (Received October 14, 1988; Accepted February 22, 1989)     

Correlation Between the Surface Charge Density of Whole Cells and Electrophoretic Movement of Isolated Somatic Antigen of Rhizobium trifolii

Lipopolysaccharide somatic antigens isolated from two strains of Rhizobium trifolii showed the same electrophoretic mobility relative to each other as did the whole bacteria of these strains.     

Surface Topography and Cytomatrix Elements in Protoplasts from Plant Cells

We studied the surface topography of protoplasts from callus of Daucus sativus(Hofm.) Roehl. and from mesophyll cells of Nicotiana tabacumL. We also followed the distribution of actin elements of the cytomatrix in the subcortical cytoplasm layer. Protoplasts were prepared for scanning electron microscopy by a modified method without drying in the critical point apparatus. After postfixation with OsO₄, carrot and tobacco protoplasts had a similar topography of the surface: it was rough and had few pores. When carrot protoplasts were not postfixed with OsO₄, their surface looked different: it was folded and had 1.5-m pores, which sometimes were bordered with globules 0.3 m in diameter, or it consisted of conical cells varying in depth and size of their bases. We believe that, when protoplasts were not fixed with OsO₄, they lost lipid-containing structures from their surface, and what remained was the protein carcass of the plasmalemma and the underlying layer of the cytoplasm. The inner surface of opened carrot protoplasts had elaborate topography, apparently produced by the elements of the cytomatrix, that is, a relatively thick layer of the cortical cytoplasm, where, using phalloidin–colloidal gold and transmission electron microscopy, we could observe a dense network of actin filaments.     

Staining Air Dried Protoplasts for Study of Plant Chromosomes

The air drying technique used in mammalian cytology was applied to isolated plant protoplasts for study of chromosomes. For cultured celery cells, this technique resulted in good spreads of metaphase chromosomes with high resolution. Mitotic chromosomes of Brassica species are relatively small, poorly stained by common stains, and difficult to spread by the squash technique. In this study, however, the chromosomes of B. carinata in callus culture were spread well and stained clearly with Giemsa staining solution. The chromosome preparations by the present techniques should also be amenable to chromosome banding studies in plants.     

Surface Structure of Yeast Protoplasts

The fine structure of the yeast cell wall during protoplast formation was studied by means of phase-contrast microscopy and the freeze-etching technique. The freeze-etching results indicated that at least in some cases the entire wall substance was not removed from the surface of the protoplasts. After a treatment of 30 min to 3 hr with 2% snail enzymes, an innermost thin wall layer as well as remnants of the fibrillar middle layer sometimes could be demonstrated.     

Enzymatic Activity of Cytochrome C-Oxidase Inserted into Magnetoliposomes Differing in Surface Charge Density

The role played by the surface charge density of the phospholipid coat of nanometer-sized Fe₃O₄ colloids (so-called “magnetoliposomes”) in the catalytic activity of beef heart cytochrome c oxidase was investigated. Screening of various binary mixtures of the anionic dimyristoylphosphatidylglycerol and the zwitterionic dimyristoylphosphatidylcholine demonstrated that the highest degree of reactivation was found in the lower negative charge range. Pre-incubation of the charged colloidal biocatalytic particles with cytochrome c induced aggregation and reduced overall enzymatic activity. The results are interpreted in terms of a different affinity of the substrate for the various membrane types and of a reorganisation of the enzyme within the membrane matrices.     

Molecular Mechanisms of Polymyxin B-Membrane Interactions: Direct Correlation Between Surface Charge Density and Self-Promoted Transport

We have studied the interaction of the polycationic peptide antibiotic polymyxin B (PMB) with asymmetric planar bilayer membranes via electrical measurements. The bilayers were of different compositions, including those of the lipid matrices of the outer membranes of various species of Gram-negative bacteria. One leaflet, representing the bacterial inner leaflet, consisted of a phospholipid mixture (PL; phosphatidylethanolamine, -glycerol, and diphosphatidylglycerol in a molar ratio of 81:17:2). The other (outer) leaflet consisted either of lipopolysaccharide (LPS) from deep rough mutants of PMB-sensitive (Escherichia coli F515) or -resistant strains (Proteus mirabilis R45), glycosphingolipid (GSL-1) from Sphingomonas paucimobilis IAM 12576, or phospholipids (phosphatidylglycerol, diphytanoylphosphatidylcholine). In all membrane systems, the addition of PMB to the outer leaflet led to the induction of current fluctuations due to transient membrane lesions. The minimal PMB concentration required for the induction of the lesions and their size correlated with the charge of the lipid molecules. In the membrane system resembling the lipid matrix of a PMB-sensitive strain (F515 LPS/PL), the diameters of the lesions were large enough (d= 2.4 nm ± 8%) to allow PMB molecules to permeate (self-promoted transport), but in all other systems they were too small. A comparison of these phenomena with membrane effects induced by detergents (dodecyltriphenylphosphonium bromide, dodecyltrimethylammonium bromide, sodiumdodecylsulfate) revealed a detergent-like mechanism of the PMB-membrane interaction. Received: 16 September 1997/Revised: 25 November 1997     

Surface Charge of Trypanosoma cruzi. Binding of Cationized Ferritin and Measurement of Cellular Electrophoretic Mobility*

SYNOPSIS The surface charge of epimastigote and trypomastigote forms of Trypanosoma cruzi was evaluated by means of binding of cationized ferritin to the cell surface as visualized by electron microscopy, and by direct measurements of the cellular microelectrophoretic mobility (EPM). Epimastigote forms had a mean EPM of -0.52 μm^.s^-1.V^-1.cm and were lightly labeled with cationized ferritin. In contrast, bloodstream trypomastigotes had a much higher EPM (-1.14), and the surface was heavily labeled with cationized ferritin. When trypomastigotes from stationary phase cultures were isolated on DEAE cellulose columns, the mean EPM was found to be significantly lower (-0.63), and labeling with cationized ferritin decreased. With a mixed population containing epimastigote, trypomastigote, and intermediate forms, EPM values ranging between -0.70 to -1.14 were found. From these observations we conclude that there is a definite increase in negative surface charge during development from epi- to trypomastigote forms of T. cruzi.     

Effects of Selected Herbicides on Plant Protoplasts

Plant protoplasts were released from immature tomato fruits by incubation with a 20% solution of polygalacturonase (Pectinol R-10, Rhom & Haas) dissolved in 0.1 M KCl + 0.1 M MgCl₂. In this salt solution the protoplasts remained stabilized for up to 8 h and were used as a source of exposed plasma membrane. Gross responses of protoplasts to selected chemicals and herbicides were recorded photomicroscopically. Paraquat (1,1′-dimethyl-4,4′-bipyridinium ion) treatments resulted in a characteristic response which was different from that of general denaturants (trichloroacetic acid, ethanol, and detergents) and of osmotic shock. Initial phases of the paraquat response were characterized by a segregation of the cytoplasm into isolated areas on the inner membrane surface. The final phase was a rupture of the plasma membrane and collapse of the cell. The herbicides, 2,4′-dinitro-4-trifluoromethyl-diphenylether (preforan); 1,1-dimethyl-3-(α,α,α-trifluoro-m-tolyl)urea (fluometuron); 3-(3-chloro-4-bromophenyl)-1-methoxy-1-methylurea (chlorbromuron); and α,α,α-trifluoro-2,6-dinitro-N-N-dipropyl-p-toluidine (trifluralin) produced no apparent structural effect on the protoplasts.     

High Yield Isolation of Nuclei from Plant Protoplasts

Nuclei were isolated from protoplasts obtained from Parthenocissus tricuspidata Crown Gall callus tissues. The effect of various isolation procedures, detergent or ultrasonication, on yield and quality of nuclei was studied. A standard procedure, based on the use of 5 × 10^?3% Triton × 100 — 6% PVP — 20% glycerol, may be carried out in 30 min and gives 80 to 90% yield of nuclei in which RNA polymerase activity is retained.     

Plant Regeneration from Mesophyll Protoplasts of Echinacea Purpurea

An efficient plant regeneration system was developed from isolated protoplasts of Echinacea purpurea L. using an alginate block/liquid culture system. Viable protoplasts could be routinely isolated from young leaves of Echinacea seedlings in an isolation mixture containing 1.0% cellulase Onozuka R-10, 0.5% pectinase and 0.3 mol l^–1 mannitol. Purified protoplasts were embedded in 0.6% Na-alginate block at a density of 1 × 10⁵/ml and cultured in a modified MS medium containing 0.3 mol l^–1 sucrose, 2.5 µmol l^–1 BA and 5.0 µmol l^–1 2,4-D. Cell colonies were observed after 4 weeks of culture, and the protoplast-derived colonies formed calluses when transferred onto 0.25% gellan gum-solidified MS medium supplemented with 1.0 µmol l^–1 BA and 2.0 µmol l^–1 IBA. Shoot organogenesis from protoplast-derived callus was induced on MS medium supplemented with 5.0 µmol l^–1 BA and 2.0 µmol l^–1 IBA. Complete plantlets were obtained from the regenerated shoots on MS basal medium. The protoplast to plant regeneration protocol developed in this study provides the prerequisite for creating novel genotypes of this valuable medicinal species through genetic manipulation.     

用TUNEL法检测植物原生质体的调亡

ＴＵＮＥＬ是近年来发展的一种凋亡细胞进行原位检测的方法,可以特异性地标记完整的凋亡细胞核或凋亡小体的染色体３’－ＯＨ断裂末端,但在植物细胞中的应用还不多。本文报道应用ＴＵＮＥＬ法检测胡萝卜原生质体的凋亡,并与ＤＮＡ电泳、彗星电泳等方法进行了比较,结果表明它是一种适用于植物原生质体凋亡检测的灵敏度较高的方法。     

The Functional Surface Charge Density of a Fast K Channel in the Myelinated Axon of Xenopus laevis

The action of Mg²⁺ on the putative xKv1.1 channel in the myelinated axon of Xenopus laevis was analyzed in voltage clamp experiments. The main effect was a shift in positive direction of the open probability curve (16 mV at 20 mm Mg²⁺), calculated from measurements of the instantaneous current at Na reversal potential after 50–100 msec steps to different potentials. The shift was measured at an open probability level of 25% to separate it from shifts of other K channel populations in the nodal region. The results could be explained in terms of screening effects on fixed charges located on the surface of the channel protein. Using the Grahame equation the functional charge density was estimated to −0.45 e nm⁻². Analyzing this value, together with previously estimated values from other K channels, with reference to the charge of different extracellular loops of the channel protein, we conclude that the loop between the transmembrane S5 segment and the pore forming P segment determines the functional charge density of voltage-gated K channels. Received: 11 December 1997/Revised: 24 April 1998     

Plant Regeneration from Mesophyll Protoplasts of Several Nicotiana Species

In a search for model systems in plant cell genetics studies mesophyll protoplasts from eleven species of Nicotiana with low chromosome number (N. acuminata, N. alata, N. glauca, N. glutinosa, N. langsdorffii, N. longiflora, N. otophora, N. paniculata, N. plumbaginifolia, N. suaveolens, N. sylvestris) were shown to divide in a liquid culture medium. Plants were recovered from calli originating from protoplasts of all these species except N. glutinosa.     

Introduction of Functional RNA into Plant Protoplasts by Electroporation

A simple apparatus was constructed for producing electric dischargeof varying intensities between two electrodes in a spectrophotometercuvette. This apparatus was used to study the conditions forefficient introduction of functional RNA into plant protoplastsusing RNAs of tobacco mosaic virus (TMV) and cucumber mosaicvirus (CMV). Electroporation under optimal conditions resultedin the production of TMV and CMV in 80% of the protoplasts fromtobacco cell line BY2. Infection by TMVRNA occurred in 60% ofVinca rosea suspension culture protoplasts and in 40% of tobaccomesophyll protoplasts. Electroporation in the presence of TMVand CMV particles also resulted in infection, suggesting thatpores larger than 30 nm are formed. The advantages and usesof the electrical method for introduction of functional RNAare discussed. (Received December 21, 1985; Accepted March 1, 1986)