Electropermeabilization of mammalian cells. Quantitative analysis of the phenomenon.

Transient membrane permeabilization by application of high electric field intensity pulses on cells (electropermeabilization) depends on several physical parameters associated with the technique (pulse intensity, number, and duration). In the present study, electropermeabilization is studied in terms of flow of diffusing molecules between cells and external medium. Direct quantification of the phenomenon shows that electric field intensity is a critical parameter in the induction of permeabilization. Electric field intensity must be higher than a critical threshold to make the membrane permeable. This critical threshold depends on the cell size. Extent of permeabilization (i.e., the flow rate across the membrane) is then controlled by both pulse number and duration. Increasing electric field intensity above the critical threshold needed for permeabilization results in an increase membrane area able to be permeabilized but not due to an increase in the specific permeability of the field alterated area. The electroinduced permeabilization is transient and disappears progressively after the application of the electric field pulses. Its life time is under the control of the electric field parameters. The rate constant of the annealing phase is shown to be dependent on both pulse duration and number, but is independent of electric field intensity which creates the permeabilization. The phenomenon is described in terms of membrane organization transition between the natural impermeable state and the electro-induced permeable state, phenomenon only locally induced for electric field intensities above a critical threshold and expanding in relation to both pulse number and duration.     

Quantitative affinity chromatography.

The objective of this review is to summarize developments in the use of quantitative affinity chromatography to determine equilibrium constants for solute interactions of biological interest. Affinity chromatography is an extremely versatile method for characterizing interactions between dissimilar reactants because the biospecificity incorporated into the design of the affinity matrix ensures applicability of the method regardless of the relative sizes of the two reacting solutes. Adoption of different experimental strategies, such as column chromatography, simple partition equilibrium experiments, solid-phase immunoassay, and biosensor technology, has led to a situation whereby affinity chromatography affords a means of characterizing interactions governed by an extremely broad range of binding affinities--relatively weak interactions (binding constants below 10(3) M(-1)) through to interactions with binding constants in excess of 10(9) M(-1). In addition to its important role in solute separation and purification, affinity chromatography thus also possesses considerable potential for investigating the functional roles of the reactants thereby purified.     

Quantitative studies of the mutagenesis of Toxoplasma gondii.

The induction of mutants resistant to 5-fluorodeoxyuridine (FUDR) was used to measure the efficiency of various physical and chemical mutagens on extracellular and intracellular Toxoplasma gondii. The frequency of resistant mutant was measured by plaque assay in human fibroblast cultures in the presence and absence of FUDR. When considered as a function of lethality, the most efficient mutagenesis was obtained with nitrosoguanidine treatment of extracellular parasites and with ethylmethane sulfonate treatment of actively growing intracellular parasites. Each of these treatments increased the frequency of FUDR-resistant mutants from less than one to more than 200 per million parasites. Ultraviolet irradiation, X-rays, and the alkylating mustard ICR-191 also induced FUDR-resistant mutants in a dose-dependent fashion.     

Quantitative analysis of two-dimensional electrophoretograms.

A method for quantitative analysis of complex film density distributions in autoradiograms is described. The method is intended particularly for measuring the distribution of radioactivity among the proteins resolved by two-dimensional gel electrophoresis but should, of course, be suited to analyzing other two dimensional separations. The film density distribution is first digitized by a high speed rotating drum scanner to generate the image data array that is stored on a magnetic disk. Subsequent analysis involves: 1) data averaging, 2) detection of contours and of their locations, 3) splitting of overlapping spots, 4) conversion of film density to radioactive intensity by means of calibration films, and 5) differentiation and integration to measure the total radioactivity contained in the protein which generates a spot in the autoradiogram. The product of the analysis is a numbered contour map and a table listing coordinates and radioactivity content of each resolved spot. Coordinate transformations for comparison and matching of autoradiograms are also described. A set of utility programs print and graph the data at intermediate stages of the analysis in order to facilitate the checking of procedures and programs.     

Quantitative assessment of the germicidal efficacy of ultrasonic energy.

Propagated (free-field) ultrasonic energy at a frequency of 26 kHz was used to expose aqueous suspensions of bacteria (Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa), fungus (Trichophyton mentagrophytes), and viruses (feline herpesvirus type 1 and feline calicivirus) to evaluate the germicidal efficacy of ultrasound. There was a significant effect of time for all four bacteria, with percent killed increasing with increased duration of exposure, and a significant effect of intensity for all bacteria except E. coli, with percent killed increasing with increased intensity level. There was a significant reduction in fungal growth compared with that in the controls, with decreased growth with increased ultrasound intensity. There was a significant reduction for feline herpesvirus with intensity, but there was no apparent effect of ultrasound on feline calicivirus. These results suggest that ultrasound in the low-kilohertz frequency range is capable to some degree of inactivating certain disease agents that may reside in water. The physical mechanism of inactivation appears to be transient cavitation.     

Quantitative studies of the development of S. cerevisiae mitochondria

The quantitative changes in mitochondria and cytochromes during transition of Saccharomyces cerevisiae from one steady state to another, while growing in continuous culture under controlled environmental conditions, were followed. No Mitochondria, or mitochondria like structures, were detectable in electron micrographs of permanganate-fixed anaerobic cells. Microaerobiosis (3μM dissolved oxygen) was sufficient to visualize mitochondrial profiles and induce cytochromes and their sections had a reduced number of mitochondrial profiles compared with cells grown in limiting glucose. In the presence of ergosterol and Tween 80 mitochondriogenesis, whether induced by aerobiosis or glucose limitation, involved enhanced definition of crystal and outer mitochondrial membranes and increased number of profiles. Where membrane formation was limited, by the absence of aerobiosis involved eytochrome induction and profile visualization, but limited profile Proliferation; the adapted cells consequently contained fewer, but more eytochrome-enriched, mitochondria than cells adapted in the presence of ergosterol and Tween 80. Increase in dissolved oxygen from 3μM to 52μM further enhanced membrane definition and increased the size, but not the number, of mitochondrial profiles. Evidence, obtained by measurement of eytochrome concentration per unit mitochondrial volume and per unit crystal area, support the concept that mitochondriogensis and cytochrome synthesis are not synchronized process and that cytochromes are added to or depleted from the mitochondrial cristae in response to culture conditions.     

Binding of aldolase to actin-containing filaments. Quantitative reappraisal of the interactions.

Previously reported results of equilibrium-partition experiments on the interaction of aldolase with actin-containing filaments [Walsh, Winzor, Clarke, Masters & Morton (1980) Biochem. J. 186, 89-98] have been subjected to a more rigorous theoretical analysis involving consideration of the consequences of cross-linking interactions between enzyme and filament. The experimental results obtained with F-actin-tropomyosin are best described by a model with one binding site per heptameric repeat unit of filament and a value of 39000 M-1 for the site binding constant, k. Similar analyses of the influence of Ca2+ on aldolase binding to F-actin--tropomyosin--troponin substantiate the existence of two equivalent binding sites (k = 14900 M-1) for the enzyme on each repeat unit of the thin filament. The Ca2+-sensitivity of this interaction reflects either a decrease in the strength of aldolase binding to these two sites (k = 8200 M-1) or the elimination of one site.     

Quantitative comparison of the hominoid thalamus. I. Specific sensory relay nuclei.

Studies to date have indicated few differences in sensory perception among hominoids. Sensory relay nuclei in the dorsal thalamus--portions of the medial and lateral geniculate bodies (MGBp, LGBd) and the ventrobasal complex (VB)--in two gibbons, one gorilla, one chimpanzee and three humans were examined for anatomical similarity by measuring and estimating the nuclear volumes, neuronal densities, numbers of neurons per nucleus, and volumes of neuronal perikarya. The absolute volumes of these nuclei were larger in the larger brains; however, with the volume of the dorsal thalamus as a standard, these sensory relay nuclei showed negative allometry. The gibbons had about half as many neurons as did the other hominoids. Although the human VB had slightly more neurons, the numbers of neurons in LGBd and MGBp did not significantly differ between the great apes and humans. The volumetric distribution of the neuronal perikarya were similar among these hominoids. Other thalamic nuclei had much more diverse numbers of neurons and relative frequencies of their neuronal perikarya. The sensory relay nuclei appear to be a group of conservative nuclei in the forebrain. These results suggest that as a neurological base for complex behaviors evolved in hominids, not all parts of the brain changed equally.     

Quantitative comparison of the amygdala in insectivores and primates.

Comparative architectonic studies have resulted in a classification of the amygdaloid complex which differs somewhat from the commonly used classification (first proposed by Humphrey, 1936) by separating the cortical amygdaloid nucleus from the centromedial group and assigning it to the basolateral group, which then forms a cortico-basolateral group. The size changes of these groups and of the nucleus of the lateral olfactory tract (belonging to the centromedial group) and the large-celled part of the basal nucleus (belonging to the corticobasolateral group) have been investigated in representatives of an ascending primate scale. In all structural complexes investigated so far, the small-celled part of the cortico-basolateral group is the most progressive. In descending order of progression there follow: the corticobasolateral group as a whole, the amygdala as a whole, and the large-celled basal nucleus. No clear changes were found in the centromedial group as a whole, whereas the size of the nucleus of the lateral olfactory tract, which represents a small component of this latter group, shows a strong reduction. These differences in the developmental trends point to increasing or decreasing capacities of the functional (limbic and olfactory) systems, to which these structures are related.     

Quantitative distribution of lysosomal hydrolases in the rat nephron.

The activities of N-acetyl-beta, D-glucosaminidase (NAG, EC 3.2.1.30), beta, D-galactosidase (beta-gal, EC 3.2.1.23) and acid phosphatase (ac-Pase, EC 3.1.3.2) were measured in the glomeruli, five segments of the proximal and four segments of the distal tubule of normal male Wistar rats. The activities of NAG and beta-gal are 3- to 5-fold higher in the first part of the proximal tubule than in other segments and very low in glomeruli. We propose that the distribution of these two glycosidases reflects the contribution of the different tubular segments to the reabsorption of glycoproteins. The maximal activity of ac-Pase was found in the straight part of the proximal tubule. It was only 1.5-fold higher than in the distal tubule. Moreover, the activity in glomeruli is rather high. We conclude that ac-Pase is not primarily involved in the handling of reabsorbed molecules.     

Quantitative Determination of Indole-3-Acetaldehyde. II.

The method by Larsen and Klungsöyr (1964) for the quantitative determination of indole-3-acetaldehyde (IAAld) was modified for the purpose of eliminating the need for filtration after oxidation of the IAAld to indole-3-acetic acid (IAA). The essentials of the modified method are as follows: Samples of IAAld or IAA containing 0.015 to 0.15 μmol (ca. 2.5 to 25 μg) dissolved in peroxide-free ether are evaporated to dryness and redissolved in 1.5 ml 0.02 M Ag2SO4. The oxidation is carried out in dim light by adding 0.5 ml 0.12 N NaOH. After 1.5 min, 2 ml of a modified Salkowski reagent are added. The optical density at 525 nm is read on a spectrophotometer after 75 min. The modified Salkowski reagent consists of 100 ml 0.05 M Fe2(SO4)3 in 1.5 N H2SO4; 240 ml H2O; and 160 ml cone. H2SO4 (sp. gr. 1.84). O.D. readings are identical for equal samples of IAAld and IAA (the latter used as a standard) up to 0.08 μmol (O.D. = 0.32). Larger quantities of IAAld may be determined when using pure IAAld as a standard, but at 0.20 μmol the O.D. for IAAld is lower than for IAA (0.69 as against 0.72). Indole-3-acetonitrile, tryptophol, indole-3-carboxylic acid, and indole-3-aldehyde all give O.D. values lower than 0.1 when tested at 0.20 μmol under the same conditions as described for IAAld and IAA.