Inferences on RNA synthesis rates during mitosis from flow cytofluorimetric RNA histograms.

Flow cytofluorimetric techniques with acridine orange staining have been used to study RNA levels of cells during and immediately after mitosis. It can be inferred from the histogram data that, of the mitotic cells, those in telophase have the highest RNA content, and that the rate of synthesis increases rapidly between anaphase and telophase. These inferences correlate with results obtained from parallel labeled precursor studies.

Effect of actinomycin-D on the staining of whole cells with acridine orange.

Flow cytofluorometric techniques have been used to investigate the interference of actinomycin-D with the staining of acridine orange in whole cells. The results show that reduction in fluorescence intensity will only occur at actinomycin-D concentrations greater than 10(3) ng/ml in our system, providing the drug is washed out prior to staining.

The ability of male and female clinicians to effectively test knee extension strength using manual muscle testing.

It has been suggested that the accuracy of manual muscle testing is dependent on examiner strength. Our purpose was to relate male and female clinicians' upper extremity strength to their ability to challenge the quadriceps and detect weakness in patients using manual muscle testing. Quadriceps muscles of seven men and 12 women with postpoliomyelitis were tested manually by a male and female clinician while forces were recorded with a hand-held dynamometer. Patients' maximal isometric knee extension force was recorded with a Lido dynamometer and clinicians' maximal vertical push force was recorded with the hand-held dynamometer. Manual muscle testing forces, patient maximum quadriceps forces, and examiner push forces were compared with repeated measures analysis of variance. Female examiners' maximal vertical push force (235.7 +/- 54.3 N) was not significantly different from either female or male patients' maximal quadriceps force (166.8 +/- 66.7 N and 341.6 +/- 123.7 N) but was only 60% and 40% of the isometric knee extension forces generated by a group of normal women and men. Male examiners were significantly stronger (357.0 +/- 93.4 N) than the female but not the male patients and produced 90% and 60% of the normal isometric quadriceps forces for women and men. Examiners gave appropriate grades in 30 of 38 tests. Examiner strength limits detection of moderate quadriceps weakness with manual resistance. Most of the muscle test grades, however, were appropriate, given the examiner's upper extremity strength. Clinicians using manual muscle testing should determine their maximal vertical push force and the extent of weakness they can detect.

Correcting two-dimensional kinematic errors for chick embryonic movements in ovo.

In motor behavior studies of chick embryos in ovo, kinematic recording is limited to a single camera system and produces kinematic data that are distorted if out-of-plane movements are not considered. CONVERT is a rule based algorithm designed to calculate 3D limb movements given 2D kinematic data. CONVERT's calculations are based on a stationary reference point, limited translation of the chick embryo's trunk point, a multi-linked model of the body, and approximate limb segment lengths. Simulations indicate CONVERT calculates joint movements from 2D data with a maximum error of 6 degrees compared to a maximum error of 79 degrees if out-of-plane considerations are ignored. The approach used to correct two-dimensional kinematic measurement errors can be readily applied to other experimental conditions that restrict video recording to single camera systems.

Fluorescence discrimination between diploid cells on their RNA content: a possible distinction between clonogenic and non-clonogenic cells.

Flow cytofluorimetric techniques, using acridine orange fluorescence to measure RNA and DNA simultaneously in EMT6 cells, have been employed to discriminate between three diploid DNA populations in vivo on the basis of their RNA content. Cells with the lowest RNA levels seem to be in the process of disintegration. Cells with the highest RNA levels correspond to those with the highest plating efficiency, and those with intermediate RNA levels are those with the lowest plating efficiency. In vitro studies have shown that log-phase cells have higher RNA levels than cells in the late plateau phase of growth.

Nucleic acid profile of the EMT6 cell cycle in vitro.

Simultaneous RNA and DNA estimations were carried out during the cell cycle of EMT6/M/CC cells growing in vitro following synchronization by mitotic selection. The determinations were performed with a flow cytofluorimeter on individual cells stained with acridine orange. It was found that the RNA content increased during G1 then remained virtually constant between early and mid S phase, but a second increase occurred during late S. The rate of uptake of tritiated uridine paralleled these changes in RNA levels, and it was also found that the rate of uptake in metaphase and anaphase was virtually zero, but a rapid increase occurred in telophase. The increase in DNA during S was approximately linear, and the intermitotic phase and cycle durations were very similar to previously reported results.

Kinematic analysis of wing and leg movements for type I motility in E9 chick embryos.

Based on studies using direct observation methods, type I motility, the first motility pattern to emerge in chick embryos, is characterized as random, uncoordinated movement. Yet, electromyographic (EMG) studies indicate that leg muscles are recruited in orderly patterns of alternating flexor and extensor activity during type I motility. It has been suggested that this apparent paradox may be attributable to perturbations arising during movement in ovo under buoyant conditions. It is also possible that direct observation methods are insufficient to detect the extent of coordination between body parts during type I motility. To address the apparent discrepancy between random features reported in observational studies and reliable features reported in EMG studies, embryos were video recorded continuously for 60 min at embryonic day 9 and criteria were established to obtain homogeneous samples of motility for kinematic analysis of synchronous wing and leg movements. Limited to a single camera attached to a stereomicroscope, methods were developed to correct for out-of-plane movements of the ipsilateral wing and leg. Also, amniotic fluid was extracted from the egg in some recordings to test the possibility that movement under buoyant conditions may mask coordinated movement. Extended sequences of activity were digitized and analyzed. Results indicated that within a limb (wing or leg), direction and timing of excursions at adjacent joints co-varied and limb excursions were characterized by reliable patterns of alternating flexion and extension consistent with EMG studies.(ABSTRACT TRUNCATED AT 250 WORDS)

A pragmatic approach to the analysis of DNA histograms with a definable G1 peak.

A method for DNA histogram analysis is described that depends only on the simple assumption that the data are normally distributed and a requirement that a G1 peak is present. A probability density function was derived from the assumption that extracted the S-phase component from the whole histogram. The model was tested with simulated data, and good agreement between predicted and known proportions in G1, S, and G1 + M was found. Good agreement was also found between duplicates of experimentally derived data. Some systematic errors are present in the analysis of certain types of histograms. However, these result in small errors when compared with biological and experimental variation and are less than the average of variation and are less than the average of algorithms in current use. The program required only two queued requests, those of the start and the end channels over which the analysis is to be performed. The algorithms perform rapidly on a microcomputer with only 28K addressable memory. Only two failures occurred in over 350 analyses and the method can be used for drug- and radiation-perturbed populations as well as with unperturbed.

Different esterase activities of exponential and plateau phases of EMT6 cells monitored by flow cytofluorimetry.

The reaction rates of enzymes hydrolysing fluorescein diacetate have been studied in populations of intact tissue-culture EMT6, cells using flow cytofluorimetric techniques. It was found that the activity of these enzymes increased in plateau phases and that this correlated inversely with plating efficiency. Highly abnormal substrate-dependent reaction velocity kinetics were found in 14-, 21-, 28- and 35-day cultures.

Effect of cell density on intracellular adriamycin concentration and cytotoxicity in exponential and plateau phase EMT6 cells.

The relationship between cell number and available Adriamycin (ADM) has been investigated in EMT6 cells. Results have shown that the ratio between cell number and total available ADM is important in determining in vitro ADM uptake and surviving fraction. Having established this effect, the sensitivity of exponentially growing and plateau phase EMT6 cells to ADM was investigated. ADM was assayed by extraction followed by spectrofluorimetry and also by flow cytometry (FCM); both methods were found to give the same ratio of intracellular ADM between exponentially growing and plateau phase cells. We found that for a given exposure dose plateau phase cells were more sensitive than exponentially growing cells. For the same dose per cell, plateau cells take up more ADM than exponentially growing cells. But for a given intracellular ADM concentration exponentially growing cells have a lower surviving fraction than plateau phase cells. We conclude that the surviving fraction is dependent on the proliferative state of the cells and in order to draw that conclusion it is important to relate the ADM effect on cells in vitro to the total ADM available to each cell.

Reliability of measurement of tempo-spatial parameters of gait after stroke using GaitMat II.

OBJECTIVE: (1) To find the inter-rater and intra-rater reliability of raters using GaitMat II to measure tempo-spatial gait parameters of stroke patients. (2) To find if reliability is related to abnormality of footprint patterns. DESIGN: A prospective agreement study. SETTING: A movement analysis laboratory. SUBJECTS: Nineteen adults who were at least six months after stroke and able to walk 4 metres with or without assistance participated as subjects in this study. There were five raters. METHODS: Subjects completed at least two walks along GaitMat II. The raw footprint data were analysed independently by each of the five raters. RESULTS: Mean intraclass correlation coefficients (ICCs) for each rater within subject ranged from 0.8398 to 0.9318. However eight of the 17 parameters tested showed significant differences between raters (p = 0.0029 to p < 0.0001). When the most inexperienced rater was excluded, differences between experienced raters (n = 4) was found to be statistically insignificant for all parameters. The association of the mean score and standard deviation of experienced raters was significant for nine parameters (r values 0.5087 to 0.9459), i.e., as walking pattern became more abnormal, variation in scoring between raters increased. CONCLUSIONS: GaitMat II may have acceptable inter-rater reliability if raters have experience of gait analysis but disagreement may increase when stroke patients exhibit more abnormal gait patterns.

Flow cytometric fluorescence emission spectrum analysis of Hoechst-33342-stained DNA in chicken thymocytes.

Hoechst-33342-stained chicken thymocytes were analysed simultaneously on two fluorescence wavelength bands (green and violet) in our custom-built flow cytometer, and two major subsets were identified. In one subset (33% of the total) the emission spectrum remained constant with time, with little change in the respective green and violet fluorescence intensities. In the other subset (42% of the total) the green fluorescence increased during staining, resulting in a considerable change in the green-to-violet ratio, due to a change in the "shape" of the fluorescence emission with time. The data indicate that two binding sites, or two types of binding at the same site, exist in DNA for this dye and that these have different binding energies and, consequently, different fluorescence emission properties.