Diffusion of small molecules into medaka embryos improved by electroporation.

BACKGROUND: Diffusion of small molecules into fish embryos is essential for many experimental procedures in developmental biology and toxicology. Since we observed a weak uptake of lithium into medaka eggs we started a detailed analysis of its diffusion properties using small fluorescent molecules. RESULTS: Contrary to our expectations, not the rigid outer chorion but instead membrane systems surrounding the embryo/yolk turned out to be the limiting factor for diffusion into medaka eggs. The consequence is a bi-phasic uptake of small molecules first reaching the pervitelline space with a diffusion half-time in the range of a few minutes. This is followed by a slow second phase (half-time in the range of several hours) during which accumulation in the embryo/yolk takes place. Treatment with detergents improved the uptake, but strongly affected the internal distribution of the molecules. Testing electroporation we could establish conditions to overcome the diffusion barrier. Applying this method to lithium chloride we observed anterior truncations in medaka embryos in agreement with its proposed activation of Wnt signalling. CONCLUSIONS: The diffusion of small molecules into medaka embryos is slow, caused by membrane systems underneath the chorion. These results have important implications for pharmacologic/toxicologic techniques like the fish embryo test, which therefore require extended incubation times in order to reach sufficient concentrations in the embryos.

Hyperthermic potentiation of cisplatin by magnetic nanoparticle heaters is correlated with an increase in cell membrane fluidity.

Magnetic fluid hyperthermia as a cancer treatment method is an attractive alternative to other forms of hyperthermia. It is based on the heat released by magnetic nanoparticles subjected to an alternating magnetic field. Recent studies have shown that magnetic fluid hyperthermia-treated cells respond significantly better to chemotherapeutic treatment compared with cells treated with hot water hyperthermia under the same temperature conditions. We hypothesized that this synergistic effect is due to an additional stress on the cellular membrane, independent of the thermal heat dose effect that is induced by nanoparticles exposed to an alternating magnetic field. This would result in an increase in Cis-diammine-dichloroplatinum (II) (cDDP, cisplatin) uptake via passive transport. To test this hypothesis, we exposed cDDP-treated cells to extracellular copper in order to hinder the human cell copper transporter (hCTR1)-mediated active transport of cDDP. This, in turn, can increase the passive transport of the drug through the cell membrane. Our results did not show statistically significant differences in surviving fractions for cells treated concomitantly with magnetic fluid hyperthermia and cDDP, in the presence or absence of copper. Nonetheless, significant copper-dependent variations in cell survival were observed for samples treated with combined cDDP and hot water hyperthermia. These results correlated with platinum uptake studies, which showed that cells treated with magnetic fluid hyperthermia had higher platinum uptake than cells treated with hot water hyperthermia. Changes in membrane fluidity were tested through fluorescence anisotropy measurements using trimethylamine-diphenylhexatriene. Additional uptake studies were conducted with acridine orange and measured by flow cytometry. These studies indicated that magnetic fluid hyperthermia significantly increases cell membrane fluidity relative to hot water hyperthermia and untreated cells, and hence this could be a factor contributing to the increase of cDDP uptake in magnetic fluid hyperthermia-treated cells. Overall, our data provide convincing evidence that cell membrane permeability induced by magnetic fluid hyperthermia is significantly greater than that induced by hot water hyperthermia under similar temperature conditions, and is at least one of the mechanisms responsible for potentiation of cDDP by magnetic fluid hyperthermia in Caco-2 cells.

Synaptopathy under conditions of altered gravity: changes in synaptic vesicle fusion and glutamate release.

Glutamate release and synaptic vesicle heterotypic/homotypic fusion were characterized in brain synaptosomes of rats exposed to hypergravity (10 G, 1h). Stimulated vesicular exocytosis determined as KCl-evoked fluorescence spike of pH-sensitive dye acridine orange (AO) was decreased twice in synaptosomes under hypergravity conditions as compared to control. Sets of measurements demonstrated reduced ability of synaptic vesicles to accumulate AO ( approximately 10% higher steady-state baseline level of AO fluorescence). Experiments with preloaded l-[(14)C]glutamate exhibited similar amount of total glutamate accumulated by synaptosomes, equal concentration of ambient glutamate, but the enlarged level of cytoplasmic glutamate measuring as leakage from digitonin-permeabilized synaptosomes in hypergravity. Thus, it may be suggested that +G-induced changes in stimulated vesicular exocytosis were a result of the redistribution of intracellular pool of glutamate, i.e. a decrease in glutamate content of synaptic vesicles and an enrichment of the cytoplasmic glutamate level. To investigate the effect of hypergravity on the last step of exocytosis, i.e. membrane fusion, a cell-free system consisted of synaptic vesicles, plasma membrane vesicles, cytosolic proteins isolated from rat brain synaptosomes was used. It was found that hypergravity reduced the fusion competence of synaptic vesicles and plasma membrane vesicles, whereas synaptosomal cytosolic proteins became more active to promote membrane fusion. The total rate of homo- and heterotypic fusion reaction initiated by Ca(2+) or Mg(2+)/ATP remained unchanged under hypergravity conditions. Thus, hypergravity could induce synaptopathy that was associated with incomplete filling of synaptic vesicles with the neuromediator and changes in exocytotic release.

A rapid cell counting method utilising acridine orange as a novel discriminating marker for both cultured astrocytes and microglia.

Cell culture analyses of growth, morphology and apoptosis commonly require counting of different cell types stained with antibodies to discriminate between them. Previously, we reported the use of l-Leucine methyl ester (l-LME) to prepare purified cultures of type 1 astrocytes with minimal microglia, and staining by GFAP and CD antibodies, respectively. Here, we demonstrate a novel use of acridine orange (AO) for rapid discrimination between these cell types using fluorescence microscopy. AO accumulates in the lysosomes and also binds strongly to nuclear DNA and cytoplasmic/nucleolar RNA. Microglia may contain abundant lysosomes due to known roles in homeostasis and immune response. AO staining of lysosomes was tested at a range of concentrations, and 2.5 microg/mL was most suitable. In agreement with previous reports, microglia treated with AO showed very intense yellow, orange or red granular cytoplasmic staining of lysosomes. Microglia contain a substantially higher number of lysosomes than astrocytes, which have a variable amount. We measured the microglia population at 5.14+/-0.50% in mixed cultures. Thus, these results show AO is a novel discriminatory marker, as microglia were easily observed and counted in clumps on top of the monolayer of astrocytes, providing a rapid alternative to time-consuming and costly antibody-based assays.

Review. Acridine orange could be an innovative anticancer agent under photon energy.

Acridine orange (AO) was extracted as a dye from coal tar over a hundred years ago. It has various unique biological activities and has been shown to be a useful fluorescent dye specific for DNA and RNA, a pH indicator, photosensitizer, antitumor and antimalarial drug, and detector of bacteria and parasites. It has recently been found that AO accumulates in musculoskeletal sarcomas and that after illumination of the tumors with visible light or irradiation with low-dose X-rays, the dye rapidly exerts selective cytocidal effect against the sarcoma cells. Therefore, surgery combined with photo- (PDT) or radiodynamic therapy (RDT) with AO (AO-PDT and -RDT) has been applied to human musculoskeletal sarcomas. The results of a clinical study on the outcome of this therapeutic strategy revealed that it yielded better local control and remarkably better limb function than wide resectional surgery. Based on our experimental studies, it was clarified that AO accumulates in acidic organelles or structures, especially lysosomes, depending on the acidity. An enormous number of protons are produced in cancer from lactate or CO2 under hypoxic conditions, which are moved into the extracellular fluid or lysosomes to maintain the intracellularfluid pH. Therefore, AO shows marked accumulation in the acidic lysosomes of cancer cells. Photon energy from visible light or X-rays excites the AO accumulated in lysosomes; the excited AO emits fluorescence and forms activated oxygen from intra-cytoplasmic oxygen. The activated oxygen destroys lysosomes, with the released lysosomal enzymes causing rapid death of the cancer cells. On the other hand, normal cells can exclude AO quickly because they are not acidic. Thus, AO-PDT and AO-RDT exhibit strong and selective cytocidal effect against malignant tumors. In conclusion, we believe that AO-PDT and AO-RDT exhibit selective anticancer cell activity and that AO excited by photon energy has excellent potential as an anticancer agent.

A new water-based topical carrier with polar skin-lipids.

A new water-based topical formulation is presented that aims at providing good penetration properties for both lipophilic and hydrophilic drugs with as small a disturbance of the skin barrier function as possible. The formulation contains dispersed lipids in a ratio resembling that of human skin. The capacity to deliver is addressed in this first study while the mild effect on skin will be presented later. Three variations of the lipid formulation were investigated by use of pigskin in vitro diffusion cell. The hydrophilic 5(6)-carboxyfluorescein (CF) and the lipophilic acridine orange 10-nonyl bromide (AO) were used as model drug substances. The results showed that the delivery properties of the new formulation exceeded that of the references (vaseline and xanthan gum gel). The effect was largest for lipophilic AO where all lipid matrix formulations were superior in amount detected in the skin. The results for the hydrophilic CF were also promising. Especially efficient was the lipid formulation containing the non-ionic adjuvants tetra ethylene glycol monododecyl ether and polyoxyethylene 23 dodecyl ether. The additional in vivo study suggests that the used in vitro model has qualitative bearing on relevant in vivo situations.

Acridine orange used for photodynamic therapy accumulates in malignant musculoskeletal tumors depending on pH gradient.

BACKGROUND: Intra-operative photodynamic therapy has been applied with acridine orange (AO-PDT) to human musculoskeletal sarcomas for the past 4 years, resulting in a low local recurrence rate, within 10%, after intra-marginal tumor resection and excellent limb function. However, it is still unclear why acridine orange (AO) specifically accumulates in tumor cells, especially in malignant tumor cells. The purpose of this study was to clarify the mechanism of AO accumulation in malignant musculoskeletal tumors. MATERIALS AND METHODS: Sixty-two musculoskeletal tumors, including 35 malignant and 27 benign tumors, were studied. Using freshly resected tumor material, the extracellular pH (pHe) was measured and the fluorescence intensity of AO accumulated in the tumors was measured by an image analyzer after ex vivo exposure to 1.0 microg/ml AO, followed by blue excitation. In the in vitro study, bafilomycin A1 was exposed to LM 8 mouse osteosarcoma cells, in order to inhibit V-ATPase, subsequently causing a decrease in the pHgradient (deltapH) between the intracellular pH (pHi) and extracellular pH (pHe) or between the pHi and the pHe. AO accumulation and the cytocidal effect of AO were evaluated. RESULTS: The results of the in vivo study, using human materials freshly resected at surgery, revealed that the pHe of the malignant musculoskeletal tumors was significantly lower than that of the benign tumors and normal muscles or adipose tissues and also showed that the AO fluorescence intensity of the malignant musculoskeletal tumors was significantly stronger than that of the benign tumors and normal muscles or adipose tissues. The results also revealed that the AO fluorescence intensity negatively correlated with the pHe in tumors and normal tissues. The in vitro study showed that bafilomycin A1 inhibited the accumulation of AO in acidic organelles, such as lysosomes, and that the cytocidal effect of AO-PDT was also remarkably inhibited. DISCUSSION: Based on results of the in vivo and in vitro studies, it is suggested that malignant musculoskeletal tumors have a large deltapH between the pHi and the pHe or between the pHi and the vaculolar pH and also that a large ApH increases AO accumulation in tumors. We, therefore, believe that AO-PDT may be more effective in highly malignant musculoskeletal tumors than low grade ones, because of their acidity. CONCLUSION: AO accumulation in musculoskeletal tumors was dependent on the ApH between the pHi and the pHe, or between the pHi and the vacuolar pH.

SYBR Green I-induced fluorescence in cultured immune cells: a comparison with Acridine Orange.

Using fluorescence microscopy, we explored the ability of cultured immune cells to take up aqueous SYBR Green I (SGI). SYBR Green I, a highly sensitive fluorescent nucleic acid stain, which preferentially binds to dsDNA over ssDNA or RNA with little background fluorescence from unbound molecules. A time course study over 2h using final dilutions of SGI of 1:10,000 and 1:100,000 at 22 and 37 degrees C, revealed the dye quickly entered the cells, stained mitochondrial DNA then nuclear DNA, and SGI-induced green fluorescence increased over time. As staining progressed, heterochromatin appeared as more intense green fluorescent lines, patches and circles against the lower fluorescence of the nucleoplasm. The lower fluorescence from the nucleoplasm indicated SGI also bound to areas of euchromatin. Similar progressive uptake experiments were carried out with the permeant DNA dye Acridine Orange (AO) to provide insight into staining patterns and mode of uptake. Statistical analysis of cells prestained with SGI then tested with Trypan Blue for changes in membrane permeability, revealed no significant difference between controls and treatment for each temperature. It appears that SGI does not compromise cells for up to 2 h following initial exposure.

Neural stem cell transplantation in cold lesion: a novel approach for the investigation of brain trauma and repair.

We developed a new neural transplantation protocol for the investigation of the repair of brain trauma. Cortical lesion was induced by touching a cold (-60 degrees C) metal stamp to the dura over the forelimb motor cortex of adult rats. The procedure caused a localized lesion and the animals developed a significant motor deficit, which was monitored throughout the protocol. Six days later the animals received embryonic neural stem cells in the penumbra of the lesion. The donor cells were freshly isolated from E14 rat embryos, had a high viability, and expressed the stem cell marker nestin. A further 6 days later the survival and differentiation of the grafted cells were investigated by immunohistochemistry. The majority of the surviving grafted cells were found in the lesion and they did not express lineage-specific markers. Only 10% of all surviving transplanted cells were located in the penumbra. These cells had an astrocytic phenotype and expressed glial fibrillary acidic protein. A few cells expressed neural or oligodendrocytic markers. In conclusion, we established a novel neural transplantation protocol, which focuses on cortical brain trauma. The model is a combination of surgical, neurological and histological approaches, all adapted to each other to make a reliable and reproducible experimental model.

Intracellular distribution of the fluorescent dye nonyl acridine orange responds to the mitochondrial membrane potential: implications for assays of cardiolipin and mitochondrial mass.

Cardiolipin, a polyunsaturated acidic phospholipid, is found exclusively in bacterial and mitochondrial membranes where it is intimately associated with the enzyme complexes of the respiratory chain. Cardiolipin structure and concentration are central to the function of these enzyme complexes and damage to the phospholipid may have consequences for mitochondrial function. The fluorescent dye, 10 nonyl acridine orange (NAO), has been shown to bind cardiolipin in vitro and is frequently used as a stain in living cells to assay cardiolipin content. Additionally, NAO staining has been used to measure the mitochondrial content of cells as dye binding to mitochondria is reportedly independent of the membrane potential. We used confocal microscopy to examine the properties of NAO in cortical astrocytes, neonatal cardiomyocytes and in isolated brain mitochondria. We show that NAO, a lipophilic cation, stained mitochondria selectively. However, the accumulation of the dye was clearly dependent upon the mitochondrial membrane potential and depolarisation of mitochondria induced a redistribution of dye. Moreover, depolarisation of mitochondria prior to NAO staining also resulted in a reduced NAO signal. These observations demonstrate that loading and retention of NAO is dependant upon membrane potential, and that the dye cannot be used as an assay of either cardiolipin or mitochondrial mass in living cells.

Photodynamic inactivation with acridine orange on a multidrug-resistant mouse osteosarcoma cell line.

Overcoming multidrug resistance (MDR) is an urgent issue to improve the prognosis of osteosarcoma patients. In this study, we undertook to clarify the effect of photodynamic therapy (PDT) with acridine orange (AO) on the MDR mouse osteosarcoma (MOS / ADR1) cell line, by comparing the outcome with the effect on a chemosensitive osteosarcoma (MOS) cell line. Cultured cells of MOS and MOS / ADR1 cell lines were exposed to AO at various concentrations for various times, followed by long- or short-term (10 or 1 min) illumination with blue light (466.5 nm) for excitation. Living cells were counted by means of the trypan blue exclusion test. The results showed that AO rapidly bound to DNA, RNA and lysosomes of living MOS and MOS / ADR1 cells and also that most tumor cells in both cell lines died rapidly (viability ratio to untreated cells: 1/1000) within 48 h under conditions of continuous or 15-min flash exposure to AO at concentrations above 1.0 microg/ml plus 10-min illumination with blue light. Even after flash exposure to AO at concentrations above 1.0 microg/ml plus 1-min illumination, the viability of MOS/ADR1 cells decreased to a viability ratio of less than 1/ 1000 within 72 h. Based on these results, we concluded that AO with photo-excitation has a strong cytocidal effect, not only on chemosensitive mouse osteosarcoma cells, but also on MDR mouse osteosarcoma cells. These results suggested that photodynamic therapy with AO may be a new approach to treating MDR human osteosarcomas.

Intracellular binding sites of acridine orange in living osteosarcoma cells.

There have been many reports concerning the intracellular binding sites of acridine orange (AO), although the actual localization of AO in living cells remains controversial. This study was undertaken to clarify the intracellular localization of AO in living mouse osteosarcoma cells by cytochemical staining. A mouse osteosarcoma cell line (MOS) was cultured and continuously exposed to 0.5 microgram/ml of AO. The intracellular localization and stainability of AO the living tumor cells was morphologically detected by a high resolution fluorescence microscope. To detect the intracellular microstructure, cytochemical staining with rhodamin 123 for mitochondria, acid phosphatase for lysosome, Sudan-black for fat vesicle and toluidine blue for glucosaminoglycan were performed using fixed cells. The results showed that both the nucleus and cytoplasm of tumor cells at 10 minutes after exposure to 0.5 microgram/ml of AO emitted green fluorescence, which was especially intense in the nucleolus, but not brilliant in the nucleus and was granular orange to red fluorescence in the perinuclear particles. This stainability of AO was different from that of rhodamin 123, Sudan-black or toluidine blue, but similar to that of acid phosphatase. Based on these results, we conclude that the green fluorescence may have derived from AO binding to double stranded RNA, not to DNA, and that orange fluorescence may have derived from aggregated AO binding to lysosome.

Role of P-glycoprotein in human natural killer-like cell line-mediated cytotoxicity.

Natural killer (NK) cells express the highest amount of P-glycoprotein (Pgp), a product of the multidrug resistance (MDR) 1 gene, among lymphoid cells, and our previous studies demonstrated that Pgp is required for NK cell-mediated cytotoxicity. In this study we examined the role of Pgp in NK cell-mediated cytotoxicity using a human NK-like cell line, i.e., YTN cells and two MDR reversing agents, nicardipine and its structural analog, AHC-93. These two agents inhibited the Pgp function (rhodamine-123 excretion) as well as cell-mediated cytotoxicity, confirming that Pgp is critical for NK cell-mediated cytotoxicity. As revealed by video-rate ultraviolet laser-scanning confocal microscopy, AHC-93 did not inhibit the increase in the intracellular calcium concentration upon binding to target cells, whereas nicardipine did, as reported previously. These two reagents relocated acridine orange dye from lysosomes to the cytoplasm at concentrations similar to those required for the inhibition of cell-mediated cytotoxicity. These results suggest that Pgp is directly or indirectly involved in pH regulation in lysosomes, but not in calcium homeostasis.

The pH-sensitive dye acridine orange as a tool to monitor exocytosis/endocytosis in synaptosomes.

We introduce the use of the pH-sensitive dye acridine orange (AO) to monitor exo/endocytosis of acidic neurotransmitter-containing vesicles in synaptosomes. AO is accumulated exclusively in acidic v-ATPase-dependent bafilomycin (Baf)-sensitive compartments. A fraction of the accumulated AO is rapidly released (fluorescence increase) upon depolarization with KCl in the presence of Ca2+. The release (completed in 5-6 s) is followed by reuptake to values below the predepolarization baseline. The reuptake, but not the release, is inhibited by Baf added 5 s prior to KCl. In a similar protocol, Baf does not affect the initial fast phase of glutamate release measured enzymatically, but it abolishes the subsequent slow phase. Thus, the fast AO release corresponds to the rapid phase of glutamate release and the slow phase depends on vesicle cycling. AO reuptake depends in part on the progressive accumulation of acid-loaded vesicles during cycling. Stopping exocytosis at selected times after KCl by Ca2+ removal with EGTA evidences endocytosis: Its T(1/2) was 12 +/- 0.6 s. The K(A)+, channel inhibitors 4-aminopyridine (100 microM) and alpha-dendrotoxin (10-100 nM) are known to induce glutamate release by inducing the firing of Na+ channels; their action is potentiated by the activation of protein kinase C. Also these agents promote a Ca2+-dependent AO release, which is prevented by the Na+ channel inhibitor tetrodotoxin and potentiated by 4beta-phorbol 12-myristate 13-acetate (PMA). With alpha-dendrotoxin, endocytosis was monitored by stopping exocytosis at selected times with EGTA or alternatively with Cd2+ or tetrodotoxin. The T(1/2) of endocytosis, which was unaffected by PMA, was 12 +/- 0.4 s with EGTA and Cd2+ and 9.5 +/- 0.5 s with tetrodotoxin. Protein kinase C activation appeared to facilitate vesicle turnover.

RNA sequestration to pathological lesions of neurodegenerative diseases.

Cytoplasmic RNA species have been identified recently within neurofibrillary tangles and senile plaques of Alzheimer's disease brain. To determine whether RNA sequestration is a common feature of other lesions found in progressive neurodegenerative disorders, acridine orange histofluorescence was employed, alone or in combination with immunohistochemistry and thioflavine-S staining to identify RNA species in paraffin-embedded brain tissue sections. Postmortem samples came from 39 subjects with the following diagnoses: Alzheimer's disease, amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam, corticobasal degeneration, diffuse Lewy body disease, normal controls, multiple system atrophy, Parkinson's disease, Pick's disease, progressive supranuclear palsy, and Shy-Drager syndrome. RNAs were detected in neurofibrillary tangles and neuritic senile plaques as well as in Pick bodies. However, Lewy bodies, Hirano bodies, and cytoplasmic glial inclusions did not contain abundant cytoplasmic RNA species. These observations demonstrate the selective localization of RNA species to distinct pathological lesions of neurodegenerative disease brains.

Macrophage cytoplasmic vesicle pH gradients and vacuolar H+-ATPase activities relative to virus infection.

A number of viruses replicate in macrophages, some having an obligate requirement for a macrophage host. This raised the question concerning the role of the macrophage endosomal/lysosomal compartment during such infections. Both lysosomotropic weak bases, amantadine and chloroquine, which interfere with endosomal/lysosomal pH gradients, and the macrolide antibiotic bafilomycin A1, which interferes with vacuolar H+-ATPase, inhibited African swine fever (ASF) virus replication in porcine macrophages. This inhibition was reversible: replenishment of bafilomycin, but not amantadine or chloroquine, maintained the inhibition. The characteristics of the inhibition, and the capacity of virus to escape and re-commence replication, related to the capacity of each drug to interfere with the endosomal/lysosomal proton pump. These results demonstrate that the virus actually requires macrophage endosomal/lysosomal activity for its replication. Therein, vacuolar H+-ATPase activity is particularly critical for successful virus replication, which is interesting considering the importance of this for endosomal/lysosomal activity and macrophage function.

Mitochondrial function in oncogene-transfected rat fibroblasts isolated from multicellular spheroids.

Two mitochondrion-specific fluorochromes, 10-N-nonyl acridine orange (NAO) and rhodamine 123 (Rh123), were used to determine the mechanism responsible for alterations in energy metabolism of transformed rat embryo fibroblast cells isolated from different locations within multicellular spheroids. Accumulation of Rh123 depends on intact mitochondrial membrane potential, whereas NAO is taken up by mitochondria independently of their function and thus represents mitochondrial distribution only. A reproducible selective dissociation procedure was used to isolate cells from different locations within the spheroids. After isolation, cells were simultaneously stained with one mitochondrial stain and the DNA dye Hoechst 33342, and several parameters, including cell volume, were monitored via multilaser-multiparameter flow cytometry. Our data clearly show a decrease in the uptake of Rh123 in cells from the periphery to the inner regions of the tumor spheroids, reflecting a persistent alteration in mitochondrial function. However, NAO staining experiments showed no reduction in the total mitochondrial mass per unit cell volume. Because cells were exposed to stain under uniform conditions after isolation from the spheroid, these data indicate the downregulation of mitochondrial function is associated with cell quiescence rather than a transient effect of reduced nutrient availability. This result, which is in accordance with data from two other cell lines (EMT6 and 9L), might reflect a general phenomenon in multicellular spheroids, supporting the hypothesis that quiescent cells in the innermost viable spheroid layer stably reduce their mitochondrial function, presumably to compensate for lower nutrient supply and/or decreased energy demand.

Flow cytometric analysis of the early phases of apoptosis by cellular and nuclear techniques.

The early events occurring during apoptosis at the plasma membrane, chromatin, and mitochondrial levels were investigated in freshly isolated irradiated human lymphocytes, growth factor-deprived cultured human lymphocytes, and dexamethasone (DEX)-treated murine thymocytes. In intact, unfixed cells, evaluation of the light scatter properties and of DNA stainability with ethidium bromide (EB) allowed a cell subset suggestive for initial apoptosis to be identified. The apoptotic nature of these cells was confirmed by cell sorting in irradiated human lymphocyte model. EB could not be substituted for by propidium iodide, indicating that the nature of DNA probe used is of major importance for detecting initial apoptotic changes. Because mitochondria are thought to represent a primary target during apoptosis, we measured the uptake of mitochondria transmembrane potential sensitive (Rhodamine 123) and nonsensitive (10-nonyl-acridine-orange) probes concomitantly with EB uptake. Cells starting apoptosis had an enhanced incorporation of both mitochondria dyes, which in combination with EB identified several cell subsets. This suggests that complex alterations in mitochondrial structure and functioning occur in the early stages of apoptosis. To investigate phenomena occurring at the chromatin level in similar phases of apoptosis, irradiated human lymphocytes and DEX-treated murine thymocytes were disrupted and DNA stainability assessed in nuclear suspensions. A transient increase in DNA stainability, i.e., the appearance of distinct hyperdiploid peaks in the human model and a generalised upward shift of the G0/1 peak in the murine model, was observed in the early phases of apoptosis concomitantly with specific alterations in light scattering properties. These findings suggest that chromatin texture is altered in early apoptosis and affects DNA stainability.

A color image analysis method for assessment of germination based on differential fluorescence staining of bacterial spores and vegetative cells using acridine orange.

Color fluorescence image analysis of acridine orange (AO) stained germinating Bacillus subtilis var. niger bacteria revealed a cell population initially dominated by small green spores followed by the emergence of at least three additional discernible subpopulations in response to stimulation with D-glucose. These subpopulations were small, round or oblong red cells; intermediate to large metachromatic cells; and large red rods. Large green rods were rarely observed. An increase in red emissions (i.e., putative RNA synthesis) was sometimes seen as early as 90 min after exposure to D-glucose and uptake of AO at room temperature. This may represent either metabolic recovery from quiescence or RNA synthesis associated with germination. In the absence of D-glucose, or using autoclaved bacteria in the presence of glucose, no relative increase in the red signal was observed despite hours of observation. Digital image analysis was used for relative measurement of red, green and blue signals and to correlate the size of various subpopulations with their fluorescence color emissions over time. Image analysis demonstrated a trend toward increasing size and red emission in the presence of glucose. The average red emission was found to be a good discriminator of the various subpopulations, while the average green emission was approximately equal among the subpopulations making it a poor discriminator. These data suggest that AO staining might be used for rapid computer-assisted discrimination of spores vs. vegetative cells.

Anticholinesterasic drugs: tacrine but not physostigmine, accumulates in acidic compartments of the cells.

Tacrine (THA) and physostigmine (PHS) have been used in Alzheimer's disease therapy for their anticholinesterasic activity. Here we show that THA is taken up in rat lysosomes in an energy-dependent manner, and that it is also accumulated in acidic compartments of rat thymocytes and neuroblastoma cells. A concentration of THA less than 1 mM dissipated the pH gradient (delta pH) in all the above mentioned in vitro systems. On the contrary higher concentrations of PHS (1-2 mM) were ineffective. The accumulation of THA in acidic organelles of the cell may be relevant for the pharmacological action of THA in Alzheimer's treatment.