[Changes in mitochondrial function induced by dequalinium precede oxidative stress and apoptosis in the human prostate cancer cell line PC-3].

Mitochondria play central roles in diverse physiological and pathological conditions associated with cell survival and death. Delocalized lipophilic cations, such as dequalinium (DQA), are accumulated in cancer cells attracted by the highly negative mitochondrial transmembrane potential of these cells. DQA showed a potent anticancer activity in cells from different malignancies. Here, we report the effect of DQA on PC-3 prostate cancer cells. Incubation with DQA at concentrations between 1.5 and 100 microM from 24 to 48 h decreases cell viability. The decrease in cell viability together with a loss of mitochondrial transmembrane potential induced an increase in reactive oxygen species production and cell death via caspase-3 dependent apoptotic pathway. QA was shown to cause moderate to strong cell death in a time and concentration dependent manner, causing a most advantageous effect at a concentration of 10 microM applied for a long 48 h time period, which might be a consequence of the kinetics of intracellular DQA accumulation in mitochondria, but also of the mechanisms of DQA-induced cell death. This data shows DQA as a promising agent against the human prostate cancer PC-3 cell line, activating the caspase-3 dependent apoptotic pathway. This fact might be beneficial for possible future applications in cancer therapy.

Influence of oxidation and crosslinking on oxygen binding properties of mouse erythrocytes.

Different chemical treatments for mouse erythrocyte modification has been used. Oxidation treatments with Ascorbate/Fe(3+), a system able to react with intracellular proteins, produced a displacement of the O(2) binding equilibrium curve to a higher affinity behaviour with loss of the haemoglobin cooperativity for oxygen binding. Incubation of mouse erythrocytes with diamide showed that at low reagent concentration (0.8 mM) no modification on oxygen binding equilibrium curves was observed. At higher reagent concentration (2.0 mM), an increased affinity and a disappearance of the cooperative behaviour can be observed. Additionally, crosslinking reactions on mouse erythrocytes with band 3 crosslinkers seemed to affect oxygen binding properties when used at a crosslinker concentration of 5 mM. Oxyhaemoglobin levels in crosslinked and diamide-treated erythrocytes are similar to those found in control cells. In contrast, ascorbate/Fe(3+) treatments produced an increment in the proportion of methaemoglobin, decreasing the oxyhaemoglobin levels in these oxidized erythrocytes.

Molecular crowding and viscosity as determinants of translational diffusion of metabolites in subcellular organelles.

The role of molecular crowding and viscosity on the apparent translational diffusion coefficient (ADC) of small metabolites was investigated in different subcellular organelles using the pulse-field gradient spin-echo 1H NMR technique. ADCs of metabolites with increasing radius of gyration (0.7 A < RG < 4.5 A) were measured in the cytoplasm of rat or chicken erythrocytes, in the nucleus of chicken erythrocytes, and in isolated rat liver mitochondria. Metabolite ADCs in these systems were compared with the corresponding ADCs determined in model solutions of increasing bulk viscosity but different molecular crowding. For solutions having the same viscosity, metabolite ADCs decreased with increasing concentration of cosolutes. This effect is adequately described by the modified Stokes-Einstein relationship, ADC = k/RG (1 + 2.5Phi), where k is a constant for a given temperature and Phi is an obstruction factor reporting the fractional volume of solution occupied by cosolutes, a measure of the molecular crowding in the solution. Cytoplasmic values of Phi for metabolites of different sizes did not depend exclusively on metabolite RG but on additional factors including the chemical nature of the metabolite, the presence of diffusional barriers, and metabolite-specific binding sites. In the case of water, nuclear Phi values approached those of the extracellular space while mitochondrial Phi values were significantly higher than those of the cytoplasm. Taken together, these results reveal important differences in molecular crowding within the different subcellular compartments, suggesting considerable diffusional heterogeneity for small metabolites within the different intracellular organelles.

A comparative study by a single chromatographic procedure of glycolytic regulatory kinase isozymes in rat erythroid cells as a function of differentiation-maturation process.

The isozymes of three glycolytic regulatory kinases: hexokinase, phosphofructokinase and pyruvate kinase are fractionated by a single ion exchange chromatographic procedure on DEAE-cellulose. Enriched-erythroblast bone marrow cells showed two heterogeneous peaks, each consisting of two overlapping peaks: one major and one minor peak, but only two isozymes were observed in reticulocytes and erythrocytes. Phosphofructokinase showed multiple isozymic forms in the three cell populations, but while in erythroblasts the main one eluted in the last fractions, in reticulocytes and erythrocytes it eluted in the early fractions. Pyruvate kinase showed a main early activity peak with a shoulder in erythroblasts, reticulocytes and erythrocytes but the response to the allosteric effectors (fructose-1,6-bisphosphate and ATP) suggests the presence of different pyruvate kinase isozymes in reticulocytes and erythrocytes.

Surface and metabolic properties of microcytic and macrocytic human anaemic red blood cells detected in polymer aqueous two-phase systems.

Microcytic and macrocytic red blood cells from anaemic patients have been fractionated as a function of cell surface properties by the countercurrent distribution technique using charge-sensitive dextran/poly(ethylene glycol) aqueous two-phase systems. As deduced from the fractionation profiles, microcytic cells constitute a heterogeneous cell population with decreased surface charge properties while. macrocytic cells constitute a homogeneous cell population with behaviour similar to that of the control red blood cells. The specific activity of pyruvate kinase, an age-dependent enzyme, did not change along microcytic red blood cells fractionation profiles, suggesting that such cells have altered ageing properties. However, pyruvate kinase specific activity decreases from the left- to the right-hand side of the fractionation profile of macrocytic red blood cells, indicating that these cells follow the normal ageing process. Bisphosphoglycerate mutase specific activity did not change along the fractionation profile of any cell population under study, thus providing 2,3-bisphosphoglycerate during the life-span of the red blood cells from anaemic patients.

Oxygenation capacity of hypotonized and crosslinked rat erythrocytes.

Rat erythrocytes subjected to hypotonic-isotonic dialysis, or crosslinking with bifunctional reagents (glutaraldehyde and dimethyl suberimidate hydrochloride) show a high percentage of methemoglobin and decreased oxyhemoglobin content which implies a low oxygen carrying capacity. Such modified cells maintain reversible oxygen binding properties although, they present a high hemoglobin oxygen affinity (low P50) and a diminished cooperativity in binding oxygen to hemoglobin (low n). These results suggest a reduced capacity of liberating oxygen to tissues under low PO2. Changes produced in erythrocytes can not be restored even in the presence of energy (ATP), reduced glutathione and 2,3-bisphosphoglyceric acid during the dialysis process or after crosslinking/permeabilizing treatment.

A single partitioning step in aqueous polymer two-phase systems reduces hypotonized rat erythrocyte heterogeneity.

Rat carrier erythrocytes prepared by hypotonic dialysis (80 mOsm/kg) are a heterogeneous cell population that can be fractionated into two well-defined cell subpopulations by a single partition step, in charge-sensitive dextran-poly(ethylene glycol) aqueous two-phase systems. One subpopulation (65% of total cells) has a decreased cell surface charge and is partitioned at the interface in a single step and then fractionated by counter-current distribution as a low-G subpopulation. The other subpopulation (35% of total cells) has charge surface properties more like those of the untreated control rat erythrocytes. These last cells are partitioned in the top phase in a single step and then fractionated by counter-current distribution as a high-G subpopulation. Partitioning is more effective in reducing cell heterogeneity in hypotonized rat erythrocyte populations than is density separation in Ficoll-paque which only separates a small less dense cell subpopulation (5% of total cells), with the most fragile cells, from a larger and more dense cell subpopulation (95% of total cells), with a mixture of fragile and normal cells. This simple cell separation procedure quickly reduces carrier erythrocyte heterogeneity in a single partitioning step so it can be used to prepare cells for in vivo studies.

Heterogeneity of hypotonically loaded rat erythrocyte populations as detected by counter-current distribution in aqueous polymer two-phase systems.

Carrier rat erythrocytes loaded with exogenous substances ([125I] carbonic anhydrase) by hypotonic-isotonic dialysis become heterogeneous cell populations that can be fractionated using the counter-current distribution (CCD) technique. Two well-defined low- and high-partition ratio, G, subpopulations are obtained in charge-sensitive dextran-polyethylene glycol two-phase systems. The low-G subpopulation, which contains the most fragile and surface-altered cells, as deduced from their osmotic fragility curves and partition behaviour, respectively, presents a high amount of exogenous substance incorporated (134.6 cpm/10(6) cells). The high-G subpopulation, that contains cells similar to the control or isotonically dialyzed cells presents a lower amount of exogenous substance incorporated (69.8 cpm/10(6) cells). Cells in this high-G subpopulation seem to be fractionated, like the controls, according to ageing as suggested by the decline of the pyruvate kinase specific activity from the left- to the right-hand side of the CCD profile.

Surface properties of crosslinked erythrocytes as studied by counter-current distribution in aqueous polymer two-phase systems.

The bifunctional imidoester dimethyl suberimidate hydrochloride can stabilize rat red blood cells (RBCs) by membrane protein crosslinking, and in that way they can be used as carrier systems for exogenous substances. Counter-current distribution fractionation in charge-sensitive dextran-polyethyleneglycol two-phase systems has been used to detect slight changes in surface charge in stabilized cells. A decrease in the surface charge of crosslinked RBCs and an apparent masking of the age-related cell surface properties have been found to result from the protein crosslinking. Digitonin treatment used to permeabilize crosslinked RBCs produces a significant decrease of the cell surface charge while the age-related surface properties do not seem to be modified by the treatment.

Transferrin binding capacity as a marker of differentiation and maturation of rat erythroid cells fractionated by counter current distribution in aqueous polymer two-phase systems.

Rat bone marrow cell populations, containing different proportions of erythroid cells, have been fractionated by counter-current distribution in the non-charge-sensitive dextran/polyethyleneglycol two-phase systems on the basis of hydrophobic cell surface properties. Cell fractions with a low distribution coefficient, which contain non-erythroid cells and early erythoblasts, showed a low transferrin binding capacity and a low haemoglobin/cell ratio whereas cell fractions with a high distribution coefficient, which contain intermediate-late erythroblasts and mature red cells, showed an elevated transferrin binding capacity and the highest haemoglobin/cell ratio. These results support transferrin binding capacity as a good marker parameter for the erythroid bone marrow cell differentiation and maturation processes.

Biochemical characterization of human erythrocytes fractionated by counter-current distribution in aqueous polymer two-phase systems.

The fractionation of normal human erythrocytes by counter-current distribution (CCD) in charge-sensitive dextran-polyethylene glycol two-phase systems was confirmed and extended to red blood cells from heterozygous beta-thalassaemic patients. The differences between the distribution profiles of normal (homogeneous) and abnormal (heterogeneous) red blood cells reflect their different surface-charge properties. As suggested by the decline of membrane sialic acid released after neuraminidase treatment and the specific activities of two age-dependent enzymes (membrane acetylcholinesterase and intracellular pyruvate kinase) in the distribution profiles (from the left- to the right-hand side fractions), the fractionation seems to be according to red blood cell age. A constancy of the 2,3-bisphosphoglycerate level was observed in ageing red blood cells.

Surface properties of crosslinked and crosslinked-permeabilized erythrocytes as studied by partitioning in aqueous polymer two-phase systems.

Non-charge- and charge-sensitive dextran/polyethyleneglycol two-phase systems have been used to study the surface properties of red blood cells stabilized by crosslinking with dimethyl suberimidate and permeabilized with digitonin. While crosslinked red blood cells show similar hydrophobic- and charge-related surface properties as control cells, the digitonin treatment changes their surface properties. The changes in hydrophobicity are related to the cell total lipid and cholesterol content while the changes in the charge are related to the sialic acid released by neuraminidase.

Rat bone marrow erythroid cell fractionation by counter current distribution in non-charge-sensitive two-phase systems.

Counter-current distribution in non charge-sensitive aqueous poly(ethylene glycol)-dextran two phase systems allows the fractionation of rat bone marrow cells into two broad cell subpopulations with different distribution coefficients in a relatively short time. Morphological identification and enzymatic studies suggest that erythroid cells are mainly present in the subpopulation with the higher distribution coefficient. The distribution coefficient and, therefore, surface hydrophobicity of these cells, apparently increase in parallel with an increase in their degree of differentiation and maturation.

Changes in glycolytic enzyme activities in aging erythrocytes fractionated by counter-current distribution in aqueous polymer two-phase systems.

Human and rat erythrocytes were fractionated by counter-current distribution in charge-sensitive dextran/poly(ethylene glycol) two-phase systems. The specific activities of the key glycolytic enzymes (hexokinase, phosphofructokinase and pyruvate kinase) declined along the distribution profiles, although the relative positions of the activity profiles were reversed in the two species. These enzymes maintained their normal response to specific regulatory effectors in all cell fractions. No variations were observed for phosphoglycerate kinase and bisphosphoglycerate mutase activities. Some correlations between enzyme activities (pyruvate kinase/hexokinase, pyruvate kinase/phosphofructokinase, pyruvate kinase/pyruvate kinase plus phosphoglycerate kinase, pyruvate kinase/bisphosphoglycerate mutase and phosphoglycerate kinase/bisphosphoglycerate mutase ratios) were studied in whole erythrocyte populations as well as in cell fractions. These results strongly support the fractionation of human erythrocytes according to cell age, as occurs with rat erythrocytes.

Settling-time dependence of rat bone marrow cell partition and counter-current distribution in charge-sensitive aqueous two-phase systems. Relationship with the cell partitioning mechanism.

Differences in the settling-time dependence of single and multiple cell partitions have been found between heterogeneous (bone marrow cells) and homogeneous (erythrocytes) populations when using charge-sensitive dextran-poly(ethylene glycol) aqueous two-phase systems. The cell populations were partitioned using both single test-tube experiments and multiple thin-layer counter-current distribution. Lengthening the settling time, to favour phase separation, and decreasing the upper phase volume are more effective in fractionation by the counter-current distribution of heterogeneous cell populations than increasing the interfacial tension, although all three were employed to speed phase settling. On the basis of these results, the original cell partitioning mechanism proposed for non-charge-sensitive systems has been extended to charge-sensitive systems.

Partitioning behaviour of rat bone marrow cells in aqueous two-phase systems. Dependence of cell partition on the interfacial tension and electrical potential difference between the phases.

The partitioning of heterogeneous cell populations of rat bone marrow, in comparison with that of homogeneous populations of erythrocytes, has been studied in aqueous two-phase systems containing increasing concentrations of dextran-poly-(ethylene glycol) (to increase the interfacial tension) and/or decreasing phosphate concentrations (to decrease both the electrical potential difference between the phases and the interfacial tension). At any polymer and/or phosphate concentration tested the partition is lower for bone marrow cells, reflecting their lower surface charge and hydrophobicity.

Fractionation of bone-marrow cells by counter-current distribution in aqueous polymer two-phase systems. Relation between settling time and the efficiency of separation.

The fractionation of heterogeneous populations of rat and human bone-marrow cells has been studied by counter-current distribution in a charged 5% dextran-4% poly(ethylene glycol) two-phase system. The subfractionation into two broad populations has been achieved at a low top/bottom phase volume ratio by increasing (up to 20 min) the settling time allowed for the phases to be separated after each mixing step. No effect of this parameter on a homogeneous population of erythrocytes has been observed. However, heterogeneous cell separations can be improved by exploiting different phase settling times.