Francisco J. Cañizares

Changes in intracellular sodium, chlorine, and potassium concentrations in staurosporine-induced apoptosis

Ion gradients across the plasma membrane, fundamentally K+, play a pivotal role in the execution phase of apoptosis. However, little is known about other monovalent anions (Cl−) or cations (Na+) in apoptosis. In addition, the relationship between changes in total ion composition and morphological and biochemical events are poorly understood. We investigated simultaneous changes in sodium (Na), chlorine (Cl), and potassium (K) concentrations in stauroporine‐induced apoptosis by quantitative electron probe X‐ray microanalysis (EPXMA) in single cells. Apoptotic cells identified unequivocally from the presence of chromatin condensation in backscattered electron images were characterized by an increase in intracellular Na, a decrease in intracellular Cl and K concentrations, and a decrease in K/Na ratio. The ouabain‐sensitive Rb‐uptake assay demonstrated a net decrease in Na+/K+‐ATPase activity, suggesting that increases in Na and decreases in K and the K/Na ratio in apoptotic cells were related with inhibition of the Na+/K+‐ATPase pump. These changes in diffusible elements were associated with externalization of phosphatidyl serine and oligonucleosomal fragmentation of DNA. This alteration in ion homeostasis and morphological hallmarks of apoptosis occur in cells that have lost their inner mitochondrial transmembrane potential and before the plasma membrane becomes permeable.

Improvement of the antioxidant and hypolipidaemic effects of cowpea flours (Vigna unguiculata) by fermentation: results of in vitro and in vivo experiments

BACKGROUND The antioxidant capacity and hypolipidaemic effects of Vigna unguiculata, as well as their potential improvement by different fermentation and thermal processes were studied using in vitro and in vivo methods. RESULTS Phenolic content and reducing capacity of legume acetone extract were significantly increased by different fermentation processes, and by the thermal treatment of fermented legume flours. TBARS inhibiting capacity was increased by fermentation but not by thermal treatment. A higher ability to decrease Cu(2+)/H2O2-induced electrophoretic mobility of LDL was found in fermented when compared to raw legume extracts, and a higher protective effect on short term metabolic status of HT-29 cells was found for raw and lactobacillus-fermented Vigna followed by naturally fermented Vigna extracts. Significant improvements in plasma antioxidant capacity and hepatic activity of antioxidant enzymes were observed in rats that consumed fermented legume flours when compared to the untreated legume or a casein-methionine control diet. In addition, liver weight and plasma levels of cholesterol and triglycerides were also positively affected by untreated or naturally fermented Vigna. CONCLUSION V. unguiculata has demonstrated its potential as a functional food with interesting antioxidant and lipid lowering properties, which can be further augmented by fermentation processes associated or not to thermal processing.

ELECTRON PROBE X-RAY MICROANALYSIS OF CULTURED EPITHELIAL TUMOUR CELLS WITH SCANNING ELECTRON MICROSCOPY

Three methods have been used to prepare cultured cells for electron probe X-ray microanalysis (EPXMA): (1) analysis at the subcellular level of freeze-dried ultrathin cryosections with scanning transmission electron microscopy (STEM); (2) analysis at the cellular level of whole freeze-dried cells with STEM; and (3) analysis at the cellular level of whole freeze-dried cells with scanning electron microscopy (SEM) (for a review see: Wroblewski and Roomans, 1984; Wroblewski and Wroblewski, 1993; Warley, 1994). However, EPXMA of whole freeze-dried cells with SEM has some disadvantages. This method requires that cultured cells be adapted to growth on a thick substrate such as plastic or glass coverslips (Zierold and Schafer, 1988), graphite discs (Abraham et al., 1985; Larsson et al., 1986) and microcarrier beads (Hall et al., 1992). In addition, a suitable washing procedure to remove the extracellular medium must be found because the deposition of culture medium on the cell surface after freezing and freeze-drying may interfere with X-ray spectra from the cell. Moreover, it is difficult to perform absolute quantitative analyses of elemental content, since the substrate may contribute to the background, decreasing the peak-to-background (P/B) ratio (Roomans, 1981). We present here a simple method to study the intracellular concentrations of elements in whole cultured epithelial tumour cells by EPXMA with scanning electron microscopy.

Gentamicin Ototoxicity in Otoconia: Quantitative Electron Probe X-ray Microanalysis

Chronic gentamicin ototoxicity was evaluated in the otolithic membrane of adult OF1 mice at the otoconial layer of the saccule and utricle by quantitative electron probe X-ray microanalysis of Ca and K. The otolithic membranes were plunge-frozen and freeze-dried. The analysis was carried out with an energy dispersive detector using the peak-to-back-ground ratio method and different inorganic salts of Ca and K as standards to calibrate the microprobe. Ca and K in the otoconia are related via a linear function in both the saccule and the utricle. This association is not maintained after exposure to gentamicin, which suggests that this aminoglycoside antibiotic interferes with the Ca-K equilibrium in the otoconia. A dose of 200 mg/kg gentamicin twice a day for 5 days did not affect Ca in the mineral phase of the otoconia, but did increase K in both saccular (p < 0.05) and utricular (p < 0.01) otoconia. These increases in K may reflect a modification in the composition of the endolymph, resulting from cellular damage at the plasma membrane.

X-ray microanalytical determination of P, S and K concentrations in the gelatinous membrane of the utricle

Electron probe X-ray microanalysis was used to determine the concentrations of P, S and K (Cp, CS, CK) in the gelatinous membrane of the mouse utricle. The otolithic membranes were plunge-frozen in liquid N2, freeze-dried and carbon-coated. Quantitative analysis was carried out with an energy dispersive detector using the peak-to-background ratio method and different concentrations of KH2PO4 and K2SO4 salts dissolved in dextran solutions to calibrate the microprobe. P, S and K were measured and their concentrations plotted as bar graphs to study the frequency distributions. Regression analysis revealed a dependence between the concentrations of P and K (CK = 1454.10 - 2.83 CP, r = -0.68745, p < 0.05), and P and S (CS = 43.18 + 0.23 CP, r = 0.66949, p < 0.05); however, no correlation was found between CK and CP (r = -0.25424). The findings obtained in the present study show an inverse relationship between P and K ions, and direct relationship between P and S in the gelatinous membrane of the utricle.

Standards for quantification of elements in the otolithic membrane by electron probe X-ray microanalysis: Calibration curves and electron beam sensitivity

An absolute quantitative standardization technique has been developed to measure Ca and K weight fractions (WF) in the otolithic membrane of the saccule and utricle by scanning electron microscopy and electron probe X‐ray analysis using the peak‐to‐background (P/B) ratio method. Microcrystalline salt standards were used to calibrate Ca and K Kα P/B or Y = (P/B) · Z2/A (Z = atomic number; A = atomic weight) against WF at 10, 15, 20 and 25 kV accelerating voltage. The effect of voltage on the calibration, plotting the coefficient of correlation (r) as a function of voltage, was not dependent on the voltage in the range 10–25 kV for Ca standards. K standards were also independent when P/B was corrected for Z2/A. Background counts in the otoconia (Bo) were obtained at 5, 25, 50, 100, 200 and 500 s and used to test the electron beam sensitivity of saccular and utricular otoconia. Bo was not dependent on the spectra acquisition time, with the exception of Bo under Kα K peak in the saccule at 10 kV. Ca and K WF were determined at 10, 15, 20 and 25 kV in the saccule and utricle, showing similar values regardless of the voltage used. This method of calibration offers several advantages, such as stability, homogeneity, known composition of the standards, high reproducibility at different voltages even without Z2/A correction and the similarity between the otoconia and crystal standards. We recommend the application of this method for other elements and biomineral systems.

Electron probe microanalysis of gentamicin-induced changes on ionic composition of the vestibular gelatinous membrane

Gentamicin-induced changes in ionic composition in the otolithic membrane of adult OF1 mice were evaluated in the gelatinous layers of the saccule and utricle by quantitative electron probe X-ray microanalysis. The otolithic membranes were plunge-frozen and freeze-dried to prevent the redistribution of elements. Quantitative analysis was carried out with an energy dispersive detector using the peak-to-background (P/B) ratio method and different salts dissolved in dextran as standards to calibrate the P/B ratio against the concentration of the elements P, S and K in the microprobe. Gentamicin selectively decreased the concentrations of P (P < 0.001) and S (P < 0.01) in the gelatinous membrane of the saccule, and had no effect in the utricle. The concentration of K also increased in the utricular gelatinous membrane (P < 0.05). The mechanism of ototoxicity in the gelatinous membrane is unknown, but the ability of aminoglycosides to block calcium channels may induce disturbances in the ionic equilibrium of the endolymphatic fluid, and thus affect the biochemical composition of the gelatinous membrane. This technique can be useful to evaluate the distribution of ions in the process of drug-induced ototoxicity.

Changes in Intracellular Sodium, Chlorine, and Potassium Content in Hematopoietic Cells after Hypotermic Storage

Hematopoietic precursor cells (HPCs) hold tremendous potential in the emerging field of cell-based therapies. The purpose of this therapy is to replace, repair or enhance the biological function of damaged tissue or organs. This can be achieved by the transplantation of cells in sufficient number and quality to restore function. Transplantation of HPCs requires the ability to preserve cells. Cell preservation technology involves both hypothermic methods for short-term storage and cryopreservation for long-term storage. However, both methods they are not exempt to induce cell damage and therefore to diminish the engraftment kinetics. Oxidative stress, mechanical injury due to ice crystal formation, altered physical properties of cellular structures, osmotic injury, and disturbed ion homeostasis are responsible for cell damage. However, in spite of the not clear correlation with capacity of engraftment, quality control assays currently used are based on methods revealing the loss of integrity of plasma membrane or ex vivo expansion capacity through clonogenic assays. Based on these premises, we propose to utilize the elemental composition as an indicator of cell viability and quality previous to cell transplantation.

Ultrastructural and Intracellular Elemental Composition Analysis of Human Hematopoietic Cells During Cold Storage in Preservation Solutions

Hematopoietic cell transplantation is a medical therapeutic procedure which aims to reconstitute the hematopoietic activity of bone marrow. Stem cells from bone marrow, mobilized peripheral blood or umbilical cord blood cells are used for these transplants. These transplant modalities require hypothermic storage and cryopreservation. However, cellular injury still occurs after extended cold storage. Despite large number of studies, however, optimal non-frozen clinical storage conditions for hematopoietic cells have not been established. Optimization of cell preservation protocols to maintain the viability and quality of hematopoietic progenitor cells has been an important task for tissue banks, and several attempts have been made to create alternative solutions for preservation of cells for transplantation. Evaluation of the storage solutions has been carried out using morphological techniques, studies of cell viability, functional studies as cellular ATP levels as well as by clinical assessment. However, few studies determining changes in electrolyte composition of cells during cold storage have been carried out [1]. In this study, we propose electron probe X-ray microanalysis (EPXMA) as a gold standard method for evaluation of the effect of preservation solutions on the intracellular elemental composition of cells for transplantation. For this, we evaluated the time-related elemental changes during cold storage in tissue culture medium in comparison with EuroCollins and lactated Ringer’s solutions. In addition, we examined the time-related ultrastructural changes of hematopoietic cells in preservation solutions.