Flow cytometric analysis of liposome-nuclei interaction: transfer and intranuclear release of carboxyfluorescein

Flow cytometric analysis of the transfer of liposome-encapsulated carboxyfluorescein to isolated rat liver nuclei indicated that the fluorophore is actively taken up in this form, while negligible amounts are transferred when the dye is free in the reaction medium. The kinetic analysis of the uptake indicated a time- and dose-dependent reduction of the slope in the absence of transport saturable sites on the nuclear surface and of quenching phenomena. The comparison between entire and membrane-deprived nuclei demonstrated that the initial rate of uptake was higher in the absence of the complete nuclear envelope. The intranuclear binding sites were considered on the basis of the fluorescence distribution and of quantitative estimates of the amount of linked dye. The possibility of employing flow cytometry to monitor the interactions between liposomes and isolated nuclei by means of a fluorescent probe is discussed.     

Low pH fusion of mouse liver nuclei with liposomes bearing covalently bound lysozyme

Lysozyme covalently bound to liposomes induces the fusion of liposomes with isolated mouse liver nuclei. The fusion behavior is very similar to the case of erythrocyte ghosts (Arvinte, T., Hildenbrand, K., Wahl, P. and Nicolau, C. (1986) Proc. Natl. Acad. Sci. USA 83, 962-966). Kinetic studies showed that membrane lipid mixing was completed within 15 min, as indicated from the resonance energy transfer (RET) measurements. For the resonance energy transfer kinetic measurements the liposomes contained L-alpha-dipalmitoylphosphatidylethanolamine (DPPE), labeled at the free amino group with the energy donor 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) or with the energy acceptor tetramethylrhodamine. The lipid mixing at equilibrium was studied by the fluorescence recovery after photobleaching technique (FRAP). Liposomes (with/without lysozyme) containing Rh-labeled DPPE in their membranes were incubated with nuclei at 37 degrees C, pH 5.2, for 30 min. After washing of nuclei by three centrifugations, 60-70% of the initial amount of labeled DPPE was associated with the nuclei in the case of liposomes bearing lysozyme and only 7-10% in the case of liposomes without lysozyme. For the nuclei incubated with liposomes having lysozyme, about 70% of the total Rh-labeled lipids present in the nuclei diffused in the nuclear membrane(s) (lateral diffusion constant of D = (1.4 +/- 0.5) X 10(-9) cm2/s). By encapsulating fluorescein isothiocyanate-labeled dextran of 150 kDa molecular mass into the liposomes and using a microfluorimetric method, it was shown that after the fusion a part of the liposome contents is found in the nuclei interior. In this lysozyme-induced fusion process between liposomes and nuclei or erythrocyte ghosts, the binding of lysozyme to the glycoconjugates contained in the biomembranes at acidic pH seems to be the determining step which explains the high fusogenic property of the liposomes bearing lysozyme.     

Interaction of negatively charged liposomes with nuclear membranes: adsorption, lipid mixing and lysis of the vesicles

Fluorescence energy transfer studies reveal that negatively charged lipid vesicles interact with nuclei from mouse liver cells. This interaction was observed with charged lipid vesicles composed of PA or PS but not with the uncharged PC or PE:PC vesicles. The vesicles were prepared by bath sonication and contained either a fluorescent marker in the lipid bilayer or in the vesicular interior. The negatively charged vesicles showed an adsorption to the nuclear membrane visible by fluorescence microscopy. The results obtained by resonance energy transfer experiments are interpreted in terms of a mixing of the lipids from the vesicles with the nuclear membrane. Encapsulation studies documented a staining of the nuclei only if the dye molecules of high or low molecular weight were encapsulated inside negatively charged vesicles. As consequence of the vesicle-nuclei interaction morphological changes on the nuclear surface became visible.     

Cell age-related differences in the interaction of a potential-sensitive fluorescent dye with nuclear envelopes of Acetabularia mediterranea

Abstract. To study whether an electrical potential difference exists across the nuclear envelope or inner nuclear membrane of plant cells, the authors have used an optical probe of membrane potential, the cationic fluorescent dye, DiOC₆(3) (MW = 572.5). This dye was microinjected into the nucleoplasm of isolated Acetabularia nuclei (which are still surrounded by a thin layer of cytoplasm) and its subnuclear localization visualized by fluorescence microscopy. Striking differences, which seemed to be correlated with the developmental stage of the isolated nucleus, were observed. In nuclei isolated from cells at the stage of early cap stage formation, the dye was restricted to the nuclear envelope. In nuclei isolated from cells with intermediate or fully developed caps, there was increased nucleoplasmic staining, and the staining of the envelope was frequently diminished or abolished. In all nuclei, the dye remained within the nucleus after injection. Cytoplasmic staining was only observed when nuclei isolated from cells at the stage of early cap formation were incubated in a hyper- or hypo-tonic medium. Various ionophores, injected before the dye into the nucleoplasm, had no effect on the subsequent nuclear localization of DiOC₆(3), although they did rapidly induce nucleolar condensation in nuclei isolated from cells at the stage of early cap formation. The results suggested that the electrical properties of Acetabularia nuclear envelopes or inner nuclear membranes change during cell maturation. Furthermore, the retention of the dye in the nucleoplasm under isotonic conditions indicated that the nuclear pores were not open channels for molecules of this size.     

Resonance energy-transfer and fluorescence intensity studies of the transport of liposome-encapsulated molecules into isolated mouse liver nuclei

We present evidence that liposomes (composed of egg yolk L-alpha-phosphatidylcholine/phosphatidylethanolamine/cholesterol, in a molar ratio of 4:5:1) fuse with isolated mouse liver nuclei at low pH. Using the resonance energy-transfer assay, we determined the rate and extent of liposome and nuclear membrane lipid mixing. Fusion was substantial when the pH was below 5. The half-time of lipid mixing decreased by acidification of the solvent, reaching about 2 min at pH 4.5. In order to study the transport of the liposome-aqueous contents to the interior of the nuclei during the process, we developed fluorescence assays in which fluorescein isothiocyanate labeled dextrans of 150 kDa molecular mass (FITC-D150) were encapsulated in liposomes. These liposomes also included in their bilayers the fluorescent lipid N-tetramethylrhodamine-L-alpha-dipalmitoylphosphatidylethanolamine (N-Rh-DPPE). After incubation of these liposomes with mouse liver nuclei (pH 4.5, 37 degrees C, 30 min), we measured the fluorescence spectra of a suspension of washed nuclei and of nuclei treated by the detergent Triton X-100 (membrane-denuded nuclei). These Triton X-100 treated nuclei had no N-Rh-DPPE fluorescence while they showed a FITC-D150 fluorescence which amounted to 20% of that of the intact nuclei. In another assay, a laser beam was focused on single nuclei by a microscope epiexcitation device. The variation of the N-Rh-DPPE and FITC-D150 fluorescence with the nuclear radius was determined with the microphotometric attachment of the microscope.(ABSTRACT TRUNCATED AT 250 WORDS)     

A large conductance ion channel in the nuclear envelope of a higher plant cell.

To detect and characterize ion channel activity in the nuclear envelope of a higher plant cell, we performed patch clamp experiments on nuclei isolated from coconut endosperm cells and on giant liposomes containing nuclear envelope fragments prepared from the same cells. An ion channel exhibiting a number of conductance substates, with a maximum of ca. 1,000 pS, was observed. Above an applied potential of +/- 100 mV, the behavior of the channel was similar in isolated nuclei and liposomes, indicating that both patch clamp modes were detecting the same channel. That such a channel has now been identified in members of both the animal and plant kingdoms reinforces the notion that the nuclear pores are not always open to ions.     

Interaction between oleic acid-containing pH-sensitive and plain liposomes. Fluorescent spectroscopy studies.

The energy transfer method has been applied to study the interaction between pH-sensitive liposomes (phosphatidyl ethanolamine/oleic acid/cholesterol, 4:2:4 molar ratio) and plain liposomes (phosphatidyl choline/phosphatidyl ethanolamine/cholesterol, 4:2:3 molar ratio). It was shown that a slow fusion process occurs between two types of liposomes. Also, the transfer of oleic acid from pH-sensitive liposomes to plain liposomes takes place. This transfer results in the increased permeability of both pH-sensitive and plain liposomes, facilitating the release of liposome-entrapped fluorescent dye. The data obtained were used for a possible explanation of the mechanism of intracytoplasmic drug delivery by pH-sensitive oleic acid-containing liposomes.     

Liposome-mediated DNA transfer in eukaryotic cells: Dependence of the transfer efficiency upon the type of liposomes used and the host cell cycle stage

The pBR322 plasmid containing the sequence encoding β-lactamase, the enzyme conferring resistance to ampicillin, was encapsulated in liposomes of different phospholipid composition and incubated with synchronized cells. In mitotic cells as compared to cells synchronized in G₁, twice as many exogeneous DNA molecules were found associated with the cell nuclear DNA, when fluid, neutral liposomes were used. These liposomes are taken up by the cells mainly via endocytosis. When fluid, negatively charged liposomes were used as carriers about the same number of exogeneous DNA molecules were found associated with the nuclear DNA both in mitotic and in G₁-synchronized cells. The efficiency for gene transfer of liposomes entering the cells by different mechanisms was further studied and expressed both by the fraction of the radioactive plasmid associated with the nuclear DNA and by the level of the β-lactamase activity detected in the transfected cells. It appears that liposomes entering the cells mainly via an energy-dependent mechanism are more efficient for this type of DNA transfer.     

Influence of Temperature on Stability of Multilamellar Liposomes in Wool Dyeing

Liposomes are lipid vesicles that are composed of amphiphile molecules and can carry hydrophobic and hydrophilic materials. In this research work liposomes used as carrier for transfer of dye molecules into wool fibers. The preparation and production of multilamellar liposomes (MLV) from Soya lecithin were carried out and the behavior of liposomes at different temperature was studied. The effect of different concentration of liposomes in the dye exhaustion profile of two dyes (Namely, Irgalan Blue FBL and Lanaset Blue 2R) at two different temperatures of 85°C and 95°C on the wool fabric was investigated. The results showed that presence of liposomes in the dye-bath helps to increase the dye absorption on the wool fabric before 80°C. Dyeing at higher temperature and longer time leads to a decrease in the final exhaustion along with increase in the liposomes concentration. Liposomes at high temperature converted to the disperse phospholipids unimers that may deposited on the fabric surface and may produce a hydrophobic barrier against absorption of dye. The presence of 1% o.w.f. (on weight of fabric) of liposomes at 85°C improved the dye exhaustion of Irgalan Blue FBL on the wool fabric. The wash fastness properties of samples which dyed in the dye-bath containing liposomes also improved.     

Influence of temperature on stability of multilamellar liposomes in wool dyeing

Liposomes are lipid vesicles that are composed of amphiphile molecules and can carry hydrophobic and hydrophilic materials. In this research work liposomes used as carrier for transfer of dye molecules into wool fibers. The preparation and production of multilamellar liposomes (MLV) from Soya lecithin were carried out and the behavior of liposomes at different temperature was studied. The effect of different concentration of liposomes in the dye exhaustion profile of two dyes (Namely, Irgalan Blue FBL and Lanaset Blue 2R) at two different temperatures of 85 degrees C and 95 degrees C on the wool fabric was investigated. The results showed that presence of liposomes in the dye-bath helps to increase the dye absorption on the wool fabric before 80 degrees C. Dyeing at higher temperature and longer time leads to a decrease in the final exhaustion along with increase in the liposomes concentration. Liposomes at high temperature converted to the disperse phospholipids unimers that may deposited on the fabric surface and may produce a hydrophobic barrier against absorption of dye. The presence of 1% o.w.f. (on weight of fabric) of liposomes at 85 degrees C improved the dye exhaustion of Irgalan Blue FBL on the wool fabric. The wash fastness properties of samples which dyed in the dye-bath containing liposomes also improved.     

Transfer of Isolated Nuclei into Protoplasts of Trichoderma harzianum

Protoplasts released from young hyphae of Trichoderma harzianum contained 0 to 10 nuclei per protoplast, and most (about 80%) contained from 4 to 6 nuclei. Most protoplasts were larger than 3 μm in diameter. Nuclei were isolated from protoplasts of an auxotrophic mutant of T. harzianum and transferred into protoplasts obtained from another auxotroph of the same strain. This intrastrain nuclear transfer gave rise to numerous progeny which were stable, prototrophic, and heterokaryotic. Interstrain transfers in which nuclei from a wild-type prototroph of one strain were transferred into protoplasts from a lysine-deficient auxotroph of a second strain were also done. Heterokaryotic progeny were recovered from these interstrain transfers when the regenerating protoplasts were provided with a low concentration of lysine 48 h after the initial plating. Heterokaryotic progeny contained 11 to 17% of donor-type nuclei. Progeny homokaryotic for donor-type nuclei were obtained as single-spore isolates. These homokaryotic isolates expressed the isozyme pattern and colony morphology phenotype of the nuclear donor. When regenerating protoplasts were provided with lysine 10 days after the initial plating, only a single progeny was obtained. However, single-spore subprogeny of this nuclear transfer were prototrophic and exhibited a wide range of unstable morphological phenotypes.     

Mechanism of fluorescence concentration quenching of carboxyfluorescein in liposomes: energy transfer to nonfluorescent dimers

When 5(6)-carboxyfluorescein (6CF) is encapsulated in liposomes at 0.2 M, 97-98% of the fluorescence is quenched. We have studied the mechanism of this effect. The dye-liposome system is a special case of concentration quenching of dyes, a phenomenon recognized for 100 years. Absorption spectra of encapsulated dye show that 6CF dimerizes, and the dimer is nonfluorescent. The dimerization constant was estimated, and it was concluded that dimerization can account for only part of the quenching. In 6CF solutions, the fluorescence lifetime decreased drastically as concentration was changed over the narrow range 0.02-0.05 M, a finding which was attributed to energy transfer to dimers. Inhibition of dimerization by propylene glycol also inhibited the shortening of lifetime. F?rster critical transfer distances were calculated to be 51 and 57 A for monomer-monomer and monomer-dimer transfer, respectively. Monomer-monomer transfer was demonstrated directly by steady-state or time-resolved anisotropy experiments, while transfer to dimer was modeled by using sulforhodamine B, which has a critical transfer distance like that for the dimer and also quenches 6CF emission. No direct evidence for collisional self-quenching of 6CF could be found, although a model compound, salicylate, did quench weakly. For xanthene dyes, the rate of energy transfer is much faster than that for quenching collisions, implying that collisional quenching in the usual 6CF-liposome system is insignificant. The reason why 6CF is not 100% quenched in liposomes is attributed to dye interaction with lipid as evidenced by (i) multiexponential decay of 6CF in liposomes with a long component of 3-4 ns, (ii) inhibition of dimerization in liposomes, (iii) partial protection of dye from quenching by KI, (iv) differing amounts of dimerization in liposomes made from different kinds of phospholipid, and (v) enhancement of fluorescence lifetime in the presence of Triton X-100.     

Changes in ribonucleoprotein particle and chromatin organization induced by liposomes in isolated nuclei

Nuclei isolated from rat liver, incubated in the presence of liposomes of different phospholipids, undergo typical modifications: chromatin dispersion and reduction of the interchromatin granules in nuclei incubated with negatively charged liposomes and increase of the chromatin density and of the number and size of the interchromatin granules in nuclei incubated with neutral liposomes. The possibility that the observed modifications are caused by an impairment of the transport and translocation of ribonucleoproteins belonging to the inner nuclear matrix, is suggested by the results obtained by radiotracer techniques on the release of RNA from liposome-incubated nuclei.     

Interaction of unilamellar liposomes with serum lipoproteins and apolipoproteins

The effect of rat whole blood plasma, serum, serum lipoproteins, and apolipoproteins on the stability of unilamellar liposomes prepared with French pressure cell was evaluated by measuring the release of entrapped carboxyfluorescein and by electron microscopy. In the absence of serum components, dye escaped very slowly (hours) from egg phosphatidylcholine and phosphatidylcholine-cholesterol (43 mol % cholesterol) vesicles without apparent change in liposomal structure. This slow release was both temperature- and size-dependent. serum and some of its constituents induced a far more rapid (seconds) loss of entrapped dye from phosphatidylcholine liposomes, associated with structural changes. For equal masses of protein the order of potency of this induced activity was: free apolipoproteins (apo A-I, apo E) > isolated lipoproteins (HDL and VLDL) > whole serum or whole plasma. Substantial activity was found in three preparations of bovine serum albumin. This activity could be attributed to small and variable amounts of contaminating lipoprotein-like particles and apolipoprotein A-I. Induced release of dye from liposomes by apolipoproteins was usually associated with rapid formation of discs although other structures were sometimes formed. Purified rat apolipoproteins A-I and E appeared to interact identically with liposomes to induce dye release. This effect was progressively impaired for both apoproteins by increasing amounts of cholesterol and was completely inhibited when liposomes contained 37 mol % cholesterol.     

Somatic cell nuclear transfer in the pig: control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy

To clone a pig from somatic cells, we first validated an electrical activation method for use on ovulated oocytes. We then evaluated delayed versus simultaneous activation (DA vs. SA) strategies, the use of 2 nuclear donor cells, and the use of cytoskeletal inhibitors during nuclear transfer. Using enucleated ovulated oocytes as cytoplasts for fetal fibroblast nuclei and transferring cloned embryos into a recipient within 2 h of activation, a 2-h delay between electrical fusion and activation yielded blastocysts more reliably and with a higher nuclear count than did SA. Comparable rates of development using DA were obtained following culture of embryos cloned from ovulated or in vitro-matured cytoplasts and fibroblast or cumulus nuclei. Treatment of cloned embryos with cytochalasin B (CB) postfusion and for 6 h after DA had no impact on blastocyst development as compared with CB treatment postfusion only. Inclusion of a microtubule inhibitor such as nocodozole with CB before and after DA improved nuclear retention and favored the formation of single pronuclei in experiments using a membrane dye to reliably monitor fusion. However, no improvement in blastocyst development was observed. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by cotransfer of parthenogenetic embryos.     

Some aspects of the value of Sudan Black B in lipid histochemistry.

The lipid dye Sudan Black B, as generally used to demonstrate lipids in the interior of the cell nucleus, was studied with regard to its staining properties for isolated nuclei in relation with its chromatographic characteristics in solution, as well as with a model system consisting of lipid containing polyacrylamide films. Isolated nuclei are stained with Sudan Black B dissolved in ethylalcohol, only when the dye-solution is at least one month old. Extraction with chloroform-methanol (2:1) before staining resulted in a decrease of 35% in intensity. Treatment with proteolytic enzymes and DNA-se caused a complete disappearance of the staining capacity. The binding of Sudan Black B with phospholipids enclosed in the form of liposomes in modelfilms when stained with this dye in ethylene glycol obeys the law of Lamber-Beer, Proteins were however, also coloured by the dye. The chromatographic experiments showed that the dye is built up from two main and a number of secondary products. The secondary products which increase by aging of the dye-solution, change the spectrophotometric properties of the total dye and show a specific binding. The conclusion was reached that on the basis of a positive reaction with Sudan Black B no definite conclusions can be drawn about the presence of lipids in the interior of the cell nucleus.     

Nuclear remodelling and the developmental potential of nuclear transferred porcine oocytes under delayed-activated conditions

It is still unclear whether nuclear envelope breakdown and premature chromosome condensation are essential for the reprogramming of the donor nucleus following somatic nuclear transfer. To address this, we determined the ability of delayed-activated or simultaneously activated porcine oocytes to undergo nuclear remodelling and development following somatic cell nuclear transfer. A small microtubule aster was observed in association with decondensed chromatin following nuclear transfer, suggesting the introduction of a somatic cell centrosome. In the delayed-activated condition, most fibroblast nuclei divided into two chromosome masses and two pronuclear-like structures following transfer into oocytes. In contrast, fibroblast nuclei in the simultaneously activated condition formed a large, swollen, pronuclear-like structure. Microtubule asters were organised in the vicinity of the nucleus regardless of the number of nuclei. More reconstructed oocytes developed to the blastocyst stage in the delayed-activated condition than in the simultaneously activated condition (p < 0.05). Nine piglets were born from two recipient sows following transfer of delayed-activated reconstructed oocytes, while none developed to full term in the simultaneously activated condition. Fingerprint analysis showed that the PCR-RFLP patterns of the nine offspring were identical to that of the donor pig. These results suggest that the activation of recipient oocytes during nuclear transfer probably relates to the nuclear remodelling process, which can affect the ability of embryos created by somatic cell nuclear transfer to develop.     

Changes in nucleosome structure and histone H3 accessibility : Iodoacetamidofluorescein labelling after treatment with phosphatidylserine vesicles

The accessibility of the sulfhydryl-specific dye 6-iodoacetamidofluorescein (IAF) to H3 histone has been studied in isolated rat liver nuclei and mononucleosomal core particles after treatment with phosphatidylserine (PS) multilamellar vesicles (MLV). In isolated nuclei, despite the enhancement of total RNA synthesis and the massive chromatin decondensation produced by liposomes, the amount of histone H3 which can be labelled with the dye remains essentially the same in PS-treated as in control nuclei. However, when mononucleosomal core particles, treated with PS vesicles, are reacted with IAF, H3 becomes derivatized by the dye, while controls do not. These data provide additional evidence that the metabolic and structural changes observed in isolated nuclei treated with PS MLV, are due mainly to the reported removal of histone H1. Moreover, the experiments reported confirm the usefulness of IAF in studying the changes of nucleosome organization, since PS is able to affect the nucleosomal core configuration in isolated nucleosome particles, derivatizing the buried cysteine groups of H3 histone.     

Nuclear transplantation in the pig embryo: nuclear swelling

The transfer of nuclei from cleavage stage embryos to enucleated activated meiotic metaphase II oocytes results in a reprogramming of the transferred nucleus such that it behaves as a zygotic nucleus. One estimator of nuclear reprogramming is nuclear swelling after nuclear transfer. The diameter of nuclei after nuclear transfer was not found to be dependent upon the amount of cytoplasm transferred with the donor cell or the amount of cytoplasm in the recipient cell. Nuclei from 4-, 8-, and 16-cell stage embryos swelled to a similar diameter after nuclear transfer (26.9, 27.3, and 27.2 microns, respectively) and this was significantly different from the diameter of contemporary donor embryos (18.3, 14.3, and 13.0 microns, respectively). This is a swelling of 47, 91, and 109%, respectively. Since the degree of nuclear swelling does not appear to be related to cytoplasmic volume it is concluded that the components mediating nuclear swelling are not in a limiting supply.     

Some aspects of the value of Sudan Black B in lipid histochemistry

Summary The lipid dye Sudan Black B, as generally used to demonstrate lipids in the interior of the cell nucleus, was studied with regard to its staining properties for isolated nuclei in relation with its chromatographic characteristics in solution, as well as with a model system consisting of lipid containing polyacrylamide films.Isolated nuclei are stained with Sudan Black B dissolved in ethylalcohol, only when the dye-solution is at least one month old. Extraction with chloroform-methanol (21) before stainig resulted in a decrease of 35% in intensity. Treatment with proteolytic enzymes and DNA-se caused a complete disappearance of the staining capacity. The binding of Sudan Black B with phospholipids enclosed in the form of liposomes in modelfilms when stained with this dye in ethylene glycol obeys the law of Lambert-Beer. Proteins were however, also coloured by the dye.The chromatographic experiments showed that the dye is built up from two main and a number of secondary products. The secondary products which increase by aging of the dye-solution, change the spectrophotometric properties of the total dye and show aspecific binding.The conclusion was reached that on the basis of a positive reaction with Sudan Black B no definite conclusions can be drawn about the presence of lipids in the interior of the cell nucleus.