Study of propidium iodide binding to DNA in intact cells by flow cytometry.

We studied the in situ binding of propidium iodide to DNA in fixed human lymphocytes, using flow cytometry. Experimental data of fluorescence emission vs dye concentration and vs cell concentration were obtained. Data were interpreted by means of two different mathematical models specific for the staining reaction, and the binding parameters were obtained by "best-fitting" of the data. A model based on two classes of binding sites with different affinity constants gave the most satisfactory fitting. The accessibility of the in situ chromatin turned out to be reduced with respect to the non in situ accessibility for ethidium bromide as reported in the literature. The present study shows the usefulness of the flow-cytometric technique for probing DNA structure in intact cells.     

Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.     

GEDAE-LaB: A Free Software to Calculate the Energy System Contributions during Exercise

Purpose

The aim of the current study is to describe the functionality of free software developed for energy system contributions and energy expenditure calculation during exercise, namely GEDAE-LaB.

Methods

Eleven participants performed the following tests: 1) a maximal cycling incremental test to measure the ventilatory threshold and maximal oxygen uptake (V˙O₂max); 2) a cycling workload constant test at moderate domain (90% ventilatory threshold); 3) a cycling workload constant test at severe domain (110% V˙O₂max). Oxygen uptake and plasma lactate were measured during the tests. The contributions of the aerobic (A_MET), anaerobic lactic (LA_MET), and anaerobic alactic (AL_MET) systems were calculated based on the oxygen uptake during exercise, the oxygen energy equivalents provided by lactate accumulation, and the fast component of excess post-exercise oxygen consumption, respectively. In order to assess the intra-investigator variation, four different investigators performed the analyses independently using GEDAE-LaB. A direct comparison with commercial software was also provided.

Results

All subjects completed 10 min of exercise at moderate domain, while the time to exhaustion at severe domain was 144 ± 65 s. The A_MET, LA_MET, and AL_MET contributions during moderate domain were about 93, 2, and 5%, respectively. The A_MET, LA_MET, and AL_MET contributions during severe domain were about 66, 21, and 13%, respectively. No statistical differences were found between the energy system contributions and energy expenditure obtained by GEDAE-LaB and commercial software for both moderate and severe domains (P > 0.05). The ICC revealed that these estimates were highly reliable among the four investigators for both moderate and severe domains (all ICC ≥ 0.94).

Conclusion

These findings suggest that GEDAE-LaB is a free software easily comprehended by users minimally familiarized with adopted procedures for calculations of energetic profile using oxygen uptake and lactate accumulation during exercise. By providing availability of the software and its source code we hope to facilitate future related research.     

Chiral discrimination by ligand-exchange chromatography: A comparison between phenylalaninamide-based stationary and mobile phases

The copper(II) complexes of two new diastereomeric ligands, N²-(R)- and N²-(S)-2′-hydroxypropyl-(S)-phenylalaninamide [(R, S)-1 and (S, S)-1], have been used as additives to the eluent in high-performance liquid chromatography (HPLC) reversed phase for the chiral separation of DNS-amino acids. The aim was that of comparing the separation process obtained by the chiral eluent with that obtained by an analogous bonded stationary phase containing (S)-phenylalaninamide, previously studied [CSP-(S)-Phe-NH₂]. The affinity of the ternary complexes for the C₁₈ column was determined by adsorption experiments in HPLC. It was shown that the two systems (chiral eluent, chiral stationary phase) work according to different mechanisms. Ternary complex formation in solution was studied by fluorescence spectroscopy. It was shown that chiral separation with the Cu(II) complexes added to the eluent was determined by the relative affinities of the ternary complexes for the column-stationary phase rather than by their stabilities in solution. With CSP-(S)-Phe-NH₂ the separation is accounted for by the relative stabilities of the ternary complexes, which depends mainly on the “allowed” geometry of the complex and on the steric repulsion of the amino acid side chain with the spacer. © 1996 Wiley-Liss, Inc.     

Kinetic heterogeneity of an experimental tumour revealed by BrdUrd incorporation and mathematical modelling

In the present paper we propose a method of analysis of the cell kinetic characteristics of in vivo experimental tumours, that uses DNA-BrdUrd flow cytometry data at various times after the bromodeoxyuridine (BrdUrd) injection and mathematical modelling. The model of the cell population takes into account the cell-cell heterogeneity of the progression rate across cell cycle phases within the tumour, and assumes a strict correlation between the durations of S and G2M phases. The model also allows for a nonconstant DNA synthesis rate across S phase. In addition, the measurement process is modelled, considering the possibility of nonimpulsive labelling and providing a representation of the time course of the bivariate DNA-BrdUrd fluorescence distribution. Sequential DNA-BrdUrd distributions were obtained in vivo from a human ovarian carcinoma transplanted in mice and, for comparison, in vitro from a cell line of the same origin. From these data, that included the fractional density and the mean BrdUrd-fluorescence of BrdUrd-positive cells as a function of the DNA-fluorescence, kinetic parameters such as the potential doubling time (T _pot) and the mean and variance of the transit times in S and G2M phases, were estimated. This study revealed the presence of a substantial heterogeneity in S and G2M phases within the in vivo cell population and of a lower heterogeneity in the in vitro population. Moreover, our analysis suggests a nonnegligible effect of the BrdUrd pharmacokinetics in the in vivo cell labelling.     

Renal excretion and saline intake during post-stress immobilization period in rats

An experiment in which the rats access either to 0.5% or 1.5% saline was designed in order to further characterise the relationship between sodium intake and renal excretion after acute immobilization stress. A saline solution for 3 days was provided to the rats previous to the experimental day. On that day, after finishing acute immobilization stress, all variables under observation were measured every 6 h for 24 h. These periods were denominated as follows: T1 (12.00 to 18.00 h), T2 (18.00 to 24.00 h), T3 (24.00 to 06.00 h) and T4 (06.00 to 12.00 h). Acute immobilization stress reduced sodium renal excretion in both T1 and T2. Sodium intake in acute immobilization stress rats was lower than in control rats during all observed periods, while the urine volume was only reduced in the stressed animals in T1. These results were similar in both saline solution concentrations. A good correlation was observed between sodium intake and sodium excretion in control rats having access to either 0.5% or 1.5% saline as well as in stressed rats having access to 0.5% saline, this correlation was not observed in stressed rats with 1.5% saline. This suggests that stress impaired the renal capability of rats to handle high sodium but not a slight sodium overload. The inability of the kidney to excrete sodium may be critical to reduce sodium intake after acute immobilization stress.     

Different Stress-Induced Calcium Signatures Are Reported by Aequorin-Mediated Calcium Measurements in Living Cells of Aspergillus fumigatus

Aspergillus fumigatus is an inhaled fungal pathogen of human lungs, the developmental growth of which is reliant upon Ca²⁺-mediated signalling. Ca²⁺ signalling has regulatory significance in all eukaryotic cells but how A. fumigatus uses intracellular Ca²⁺ signals to respond to stresses imposed by the mammalian lung is poorly understood. In this work, A. fumigatus strains derived from the clinical isolate CEA10, and a non-homologous recombination mutant ΔakuB ^KU80, were engineered to express the bioluminescent Ca²⁺-reporter aequorin. An aequorin-mediated method for routine Ca²⁺ measurements during the early stages of colony initiation was successfully developed and dynamic changes in cytosolic free calcium ([Ca²⁺]_c) in response to extracellular stimuli were measured. The response to extracellular challenges (hypo- and hyper-osmotic shock, mechanical perturbation, high extracellular Ca²⁺, oxidative stress or exposure to human serum) that the fungus might be exposed to during infection, were analysed in living conidial germlings. The ‘signatures’ of the transient [Ca²⁺]_c responses to extracellular stimuli were found to be dose- and age-dependent. Moreover, Ca²⁺-signatures associated with each physico-chemical treatment were found to be unique, suggesting the involvement of heterogeneous combinations of Ca²⁺-signalling components in each stress response. Concordant with the involvement of Ca²⁺-calmodulin complexes in these Ca²⁺-mediated responses, the calmodulin inhibitor trifluoperazine (TFP) induced changes in the Ca²⁺-signatures to all the challenges. The Ca²⁺-chelator BAPTA potently inhibited the initial responses to most stressors in accordance with a critical role for extracellular Ca²⁺ in initiating the stress responses.     

Response of Tumor Spheroids to Radiation: Modeling and Parameter Estimation

We propose a spatially distributed continuous model for the spheroid response to radiation, in which the oxygen distribution is represented by means of a diffusion-consumption equation and the radiosensitivity parameters depend on the oxygen concentration. The induction of lethally damaged cells by a pulse of radiation, their death, and the degradation of dead cells are included. The compartments of lethally damaged cells and of dead cells are subdivided into different subcompartments to simulate the delays that occur in cell death and cell degradation, with a gain in model flexibility. It is shown that, for a single irradiation and under the hypothesis of a sufficiently small spheroid radius, the model can be reformulated as a linear stationary ordinary differential equation system. For this system, the parameter identifiability has been investigated, showing that the set of unknown parameters can be univocally identified by exploiting the response of the model to at least two different radiation doses. Experimental data from spheroids originated from different cell lines are used to identify the unknown parameters and to test the predictive capability of the model with satisfactory results.