Computer analysis of two-dimensional gels

New methods and computer programs are described which enable one to analyze autoradiograms produced by two-dimensional gel electrophoresis. These programs are completely automatic with respect to finding spots resolved by such gels and quantitating the radioactivity in them. Semiautomatic programs have also been developed to match the spot patterns of different autoradiograms, and to follow the synthesis of any individual polypeptide through a series of gels.     

Low-cost two-dimensional gel densitometry

A major obstacle to full utilization of the powerful technique of two-dimensional (2-D) gel electrophoresis is the expense and complexity of quantifying the results. Using an analog-to-digital converter already present in the widely available Commodore 64 or Commodore 128 microcomputer, we have developed a 2-D gel densitometer (GELSCAN) which adds only $20.00 to the cost of the Commodore system (currently around $700.00). The system is designed to work with autoradiograms of 2-D gels. Spots of interest are identified visually and then positioned manually over a light source. A pinhole photoelectric sensor mounted in a hand-held, Plexiglas holder, or "mouse," is briefly rubbed over each spot. Maximum density of the spot is determined and its value is converted to counts per minute via an internal calibration curve which corrects for the nonlinear response of film to radiation. Local spot backgrounds can be subtracted and values can be normalized between gels to adjust for variation in amount of radioactivity applied or in exposure time. Reproducibility is excellent and the technique has some practical as well as theoretical advantages over other more complicated approaches to 2-D gel densitometry. In addition, the GELSCAN system can also be used for scanning individual bands in 1-D gels, quantitation of "dot-blot" autoradiograms and other tasks involving transmission densitometry.     

Radioimagers as an alternative to film autoradiography forin situ quantitative analysis of125I-ligand receptor binding and pharmacological studies

Summary Three radioimagers, the μ-imager, the β-imager and the phosphorimager, were tested as alternatives to quantitative autoradiography on film, for receptor imaging and pharmacologicalin situ quantitative analysis. Two iodinated ligands¹²⁵I-interleukin-1α and¹²⁵I-gonadotropin releasing hormone agonist, were used for receptor characterization in mouse brain and pituitary sections. Due to the high number of the agonist receptors in rat pituitary gland, this tissue was used to compare measurements obtained from digital autoradiograms with classical γ detector determination. This permits the evaluation of radioimager efficiency and absolute quantification. Radioimagers represent an improvement in terms of time of image acquisition. All the radioimagers are more sensitive than film for the detection of low levels of radioactivity. The spatial resolution provided by the μ-imager compares favourably with that obtained on film autoradiograms while digital autoradiograms from the phosphorimager and β-imager did not show precise definition under our experimental conditions. Superimposition of histological structures from the stained sections with radiolabelled areas in the autoradiograms remains, at this time, the unique advantage of film. In conclusion, radioimagers represent an alternative to autoradiography on film or emulsion forin situ quantitative studies on tissue sections. They combine precise imaging forin situ binding studies with easy and direct access to counts in cpm. The improvement in radioimaging technology has, therefore, broughtin situ analysis of iodinated ligand binding to the level of accuracy that is obtained with classical detectors of radioactivity.     

Giemsa Staining of Autoradiograms Prepared with Stripping Film

A method is described in which sections or smears of animal tissue are covered with stripping film, exposed and developed for autoradiograms, and stained with modified Giemsa through the film. Results are consistently satisfactory.     

A pitfall in the computer-aided quantitation of autoradiograms

Computer-aided quantitation of autoradiograms is now available as a result of recent developments in optical scanners and microcomputers. Data expressed as optical density values, however, are based on the unverified assumption that optical density and radioactivity density are linearly correlated. This article demonstrates the need to construct a calibration curve which should be used to calculate radioactivity density values more precisely.     

Application and efficiency of scintillation autoradiography for Drosophila polytene chromosomes

Summary A rapid method of autoradiography using the scintillation cocktail (Toluene and scintillation fluid, Omnifluor) has been described earlier. Its application and efficiency have been tested using both ³H-thymidine and ³H-uridine. The optimum time required for processing the autoradiograms has been found to be 24 h dry exposure followed by 48 h in the scintillation mixture. Detailed analysis of the autoradiograms with ³H-uridine reveals that with the rapid method the 100% level of labelling index is reached by 48 h while with the conventional method the same level is reached by 10 to 12 days of dry exposure. The maximum grain density is reached by 16 to 17 days by the conventional method. While by the rapid method, the maximum grain density is approximately 80% of the control, this grain density is reached by 48 h (plus 24 h of dry exposure) and thereafter forms a plateau. With Toluene alone the grain density never exceeds 20%. The background is also relatively low and less variable in the O-T-processed autoradiograms, as compared to the two controls. These results support that the scintillation fluid plays the key role in augmenting the labelling. Furthermore, although the maximum grain density by the rapid technique is 80% of the control, the grain density obtained by the rapid method gives less coincidence and superimposition of grains.On the other hand, with ³H-thymidine, although all labelling patterns could be resolved, the labelling index (i.e., percent of labelled cells) is about 40% at 48 h (plus 24 h) and about 79.5% at 96 h with the rapid method, as compared to about 30% and 44% with the conventional method at the two time points, respectively. Only with 16–17 days' dry exposure the ³H-thymidine labelling index increases to 67%. The frequency of the initial patterns (DD-2C) which are usually less frequent, has been found to have increased with the rapid method. No difference in grain density of labelling of ³H-thymidine could be detected between the rapid method and the conventional method. The resolution of grains also seems to be better by the rapid method, due probably to smaller size and lack of superimposition of grains. Other applications, advantages and limitations have been discussed.     

The QUEST system for quantitative analysis of two-dimensional gels

The strategies and methods used by the QUEST system for two-dimensional gel analysis are described, and the performance of the system is evaluated. Radiolabeled proteins, resolved on two-dimensional gels and detected using calibrated exposures to film, are quantified in units of disintegrations per minute or as a fraction of the total protein radioactivity applied to the gel. Spot quantitation and resolution of overlapping spots is performed by two-dimensional gaussian fitting. Pattern matching is carried out for groups of gels called matchsets, and within each matchset every gel is matched to every other gel. During the matching process, spots are automatically added to each pattern at positions where unmatched spots were detected in other patterns. This results in enhanced accuracy for both spot detection and for matching. The spot fitting procedure is repeated after matching. Tests show that up to 97% of spots in each pattern can be matched and that fewer than 1% of the spots are matched inconsistently. Approximately 2000 proteins are detected from typical gels. Of these 1600 are high quality spots. Tests to measure the coefficient of variation of spot quantitation versus spot quality show that the average coefficient of variation for high quality spots is 21%. The intensities of the detected proteins range from 4 to 20,000 ppm of total protein synthesis. The QUEST analysis system has been used to build a quantitative database for the proteins of normal and transformed REF52 cells, as presented in the accompanying reports (Garrels, J., and Franza, B. R., Jr. (1989) J. Biol. Chem. 264, 5283-5298, 5299-5312).     

Quantitative evaluation by computer-based video densitometry of film response to different isotopes and fluorographic treatments

Previous investigations of fluorographic methods have generally reported the minimum level of radioactivity that can be detected for a given length of exposure or the amount of radioactivity needed to reach a given optical density value. Even when the full characteristic curve is given, comparisons with other published values are difficult or, if different lengths of exposures are used, impossible. Computer-based video densitometry was used to evaluate different fluorographic methods. Sections of polyacrylamide gels with uniform distributions of either 35S or 125I radioactivity were embedded with different fluors and used to expose X-ray film for various lengths of time. Due to its compatibility with algorithms used in densitometry, the effectiveness of various treatments was evaluated by fitting relevant parameters to a modified Hill-type equation. The two parameters that were affected by treatments were the amount of radioactivity needed to half-saturate the film (ED50, or the midpoint) and the slope at the midpoint. The relationship between length of exposure and film density was investigated. Since the ED50 values for both 35S and 125I were found to vary in a linear fashion with respect to 1/time, one can determine the ED50 for lengths of exposure not reported. A unique feature of this method of evaluating effectiveness of different treatments, therefore, is that it is possible to compare films that were exposed for different lengths of time. The method of presenting data used here overcomes many of the difficulties in comparing different fluorographic and film treatments, is compatible with the needs of computer-based densitometry, and is suggested as a useful way in which to present the relationship among isotopes, fluors, and films.     

A program which automatically quantitates gel electrophoretic autoradiograms

The use of a computer-coupled film scanner to measure and analyze autoradiograms of gel electropherograms is described. A program has been written which fits Gaussian curves to the complex band pattern that constitutes a density profile without the need for estimated parameters in the input. The great majority of the fits are satisfactory. This program, which is written in FORTRAN, runs on a small, inexpensive computer. Another program which approximates a Gaussian least squares fit has been run for comparison; this procedure can also be used to refine occasional unsatisfactory fits. Finally, a program has been written which sums the density profile within specified limits, so that the integrated intensities of bands due to isolated protein components may be found.     

High-performance liquid chromatographic analysis of acylated lipids containing pyrene fatty acids

A high-performance liquid chromatographic method for the separation and quantification of acylated lipids containing pyrene fatty acids is described. The method is adapted from a procedure originally developed for the analysis of tissue lipids (Christie, W. W. (1985) J. Lipid Res. 26, 507-512). Pyrenyl lipid analogs ranging in polarity from cholesteryl ester to lysophosphatidylcholine are completely resolved on a silica column in 50 min by gradient elution with a ternary solvent system. Furthermore, pyrene-labeled triglycerides are resolved according to the number of pyrene fatty acid residues incorporated. Pyrenyl lipids are detected at levels of 10(-13) mol by high-sensitivity fluorescence detection. Accurate quantification of pyrenyl lipids is obtained by correcting peak areas for mobile-phase quenching effects. The close correspondence between chromatograms obtained for the separation of labeled lipids extracted from Hep-G2 cells incubated with either 12-(1-pyrenyl)dodecanoic acid (fluorescence detection) or [1-14C]oleic acid (radioactivity detection) indicates that this HPLC method is equally suitable for analysis of native lipids.     

MICROSCOPIC DETERMINATION OF BONE PHOSPHORUS BY QUANTITATIVE AUTORADIOGRAPHY OF NEUTRON-ACTIVATED SECTIONS

Longitudinal sections of human cortical bone were submitted to thermal neutrons. γ-ray spectra were recorded repeatedly during 15 days following irradiation. They showed that Na²⁴ is predominant as early as 3 hours after activation and that all the γ-emitters have decayed on the 15th day. When the γ-rays have disappeared, β-rays are still produced by the sections. It was proved by the absorption curve in aluminium that all these β-rays are issued from the P³² induced in the sections by activation of P³¹. Therefore autoradiograms registered 15 days after activation reveal the distribution of P³² in the sections. γ-ray spectra and β-ray absorption curves of neutron activated sections of ivory demonstrated a mineral composition similar to that of bone. Autoradiograms of ivory sections activated for various times were used to establish the relation between the optical density of the autoradiograms and the radioactivity in P³². When the bone autoradiograms are compared with the ivory standards of known radioactivity, the optical densities of single osteons (Haversian systems), can be related to their phosphorus contents. Autoradiograms and microradiograms of the same sections were examined side by side. The least calcified osteons, that contain 80 per cent of the calcium of the fully calcified osteons, also contain about 80 per cent of the phosphorus of the fully mineralized osteons. It is concluded that the Ca:P ratio remains constant while mineralization of bone tissue is being completed.     

Uptake of serotonin, 5-hydroxytryptophan and tryptophan by giant serotonin-containing neurones and other neurones in the central nervous system of the snail (Helix pomatia)

Summary Following exposure to tritiated 5-HTP, silver grains were observed over the perikarya of the GSCs (Giant serotonin cells) of Helix pomatia and other known serotonin-containing neurones in light and electron microscope autoradiograms. There was no indication that the 5-HTP was taken up by nerve endings or by non-nervous structures. The distribution of radioactivity was completely different in autoradiograms of tissue exposed to tritiated serotonin. Silver grains, often in very high concentrations, were observed only over certain fine axon branches and processes thought to be nerve endings. Electron microscope autoradiography showed that these processes contained small dense-cored vesicles, morphologically identical to those thought to sequester serotonin in the perikarya of the GSCs. The accumulation of tritiated tryptophan was less specific; all the neurone perikarya showed an accumulation of radioactivity after exposure to this substance.We are grateful to Professor J. F. Lamb for the use of the Scintillation Spectrometer.     

The tissue distribution and the pattern of excretion of [14C]-13-labeled 12, 13-epoxytrichothec-9-ene in mice and rats

The distribution in the mouse tissues of 13-[14C]-12,13-epoxtrichothec-9-ene administered intravenously was determined by whole-body autoradiography and by tracing the radioactivity of the tissues oxidized in an Auto Sample Oxidizer. The appearance of the label in urine and feces was also followed by the tracer technique. The distributions of radioactivity in tissues as determined by the two methods were almost identical. On the autoradiograms of mice killed 10 min after the injection, marked blackening of the film was observed at the sites corresponding to the liver, kidney, and bladder with urine, and much less darkening at other sites. The radioactivities contained in the liver, kidney, urine and small intestine were 13.3, 2.3, 2.6 and 10.2% of the dose, respectively. The labeled toxin was rapidly excreted into urine and feces, 56.0 and 4.9% in 6 hr and 66.7 and 28.0% in 24 hr after injection, respectively. Oral administration of the labeled toxin to mother mice resulted in the appearance of radioactivity in the stomach contents of 7-day suckling mice, thus demonstrating indirectly the secretion of the toxin into the milk. An attempt to show a respiratory route of excretion in rats given the radioactive compound orally or intravenously failed to detect any radioactivity in the expired CO2 collected for 6 hr, suggesting that the 14C in the epoxy ring was intact.     

Computerized quantification of tissue vascularization using high-resolution slide scanning of whole tumor sections.

Assessment of tissue vascularization using immunohistochemical techniques for microvessel detection has been limited by difficulties in generating reproducible quantitative data. The distinction of individual blood vessels and the selection of microscopic fields to be analyzed remain two factors of subjectivity. In this study, we used imaging analysis software and a high-resolution slide scanner for measurement of CD31-immunostained endothelial area (EA) in whole sections of human neuroblastoma xenograft and murine mammary adenocarcinoma tumors. Imaging analysis software provided objective criteria for analysis of sections of different tumors. The use of the criteria on images of entire tumor section acquired with the slide scanner constituted a rapid method to quantify tumor vascularization. Compared with previously described methods, the "hot spot" and the "random fields" methods, EA measurements obtained with our "whole section scanning" method were more reproducible with 8.6% interobserver disagreement for the "whole section scanning" method vs 42.2% and 39.0% interobserver disagreement for the "hot spot" method and the "random fields," respectively. Microvessel density was also measured with the whole section scanning method and provided additional data on the distribution and the size of the blood vessels. Therefore, this method constitutes a time efficient and reproducible method for quantification of tumor vascularization.     

A Small-Scale Method for Screening of Lignin-Degrading Microorganisms

A new method to facilitate rapid screening of lignin-degrading microorganisms was developed. Fungal strains are cultivated in tissue culture plates containing ¹⁴C-ring-labeled dehydrogenation polymerizate (DHP) (synthetic lignin). Evolved ¹⁴CO₂ is trapped in barium-saturated filter paper and is detected by exposing the paper to X-ray film. Analysis of the autoradiograms, carried out by density measurement with an image analysis program, allows for a semiquantitative estimation of the amount of ¹⁴CO₂ evolved. The method is especially useful for screening for new, powerful lignin-degrading strains in both man-made and natural environments. It eliminates the need for special equipment for their cultivation and trapping of ¹⁴CO₂ as well as laborious sample analysis. The method has in this study been used to test three novel fungal isolates and a laccaseless mutant of the basidiomycete Pycnoporus cinnabarinus. Their ligninolytic capacities were compared with those of the potent lignin degrader Ceriporiopsis subvermispora.     

DNA contents of replication without DNA density labeling

A new method for determining the timing of DNA replication in specific regions of the mammalian genome without the use of DNA density labeling and DNA density centrifugation is described. The method is based on determination of average relative DNA copy numbers in specific genomic regions as cells progress through S phase, and "time of replication" for a specific region is described in terms of the cell's DNA content when the region is replicated. DNA is isolated from synchronized populations of G1 and S phase cells, it is slot-blotted at the same DNA concentration(s) for each population, and it is hybridized with 32P-labeled DNA probes that are specific to the regions of interest. Quantitation of the slot blot autoradiograms and flow cytometric analysis allows determination of (a) average relative DNA copy numbers for the regions of interest in synchronized cell populations, and (b) the average total DNA content in each population of synchronized cells. This information and the flow cytometry histograms are then used to calculate the cellular DNA content at which each region of interest is replicated. The results have a precision of less than or equal to +/- 10% of S phase for Chinese hamster (line CHO) rhodopsin, metallothionein II, the 5'-end of dihydrofolate reductase, the telomeric repeated sequence, pHuR-093 (also located near the centromeres in CHO chromosomes), and the c-Ki-ras family.     

Autoradiographic immunoassay (ARIA): a rapid technique for the semiquantitative mass screening of haptens.

A semi-quantitative radioimmunoassay was developed which allows the mass screening of more than 10⁴ samples per day per person. The assay is performed in multiwell test plates and employs charcoal separation of antibody-bound and free antigen fractions. The radioactivity of the charcoal phase is transformed into fluorescent light which exposes a sheet of X-ray film. By this autoradiographic immunoassay (ARIA) technique, both visual and quantitative evaluation of the tests can be accomplished. The applicability of this new method is demonstrated for two haptens, digoxin and nicotine. Correlation coefficients between charcoal radioactivity and film density were found to exceed 0.95. Measuring ranges extend from 0.2 to 50 ng (digoxin) and from 5 to 500 ng (nicotine). Interassay variabilities are about twice as high for ARIA as for radioimmunoassays. By proper choice of the sample dilution used for assays, the screening for high or low antigen concentrations is possible.     

Comparison of dietary and waterborne exposure to benzo a pyrene: bioavailability,tissue disposition and CYP1A1 induction in rainbow trout Oncorhynchus mykiss

Absorption and tissue distribution of benzo\[ a ]pyrene (BaP)-derived radioactivity were studied in juvenile rainbow trout following dietary or waterborne exposure. In order to compare the bioavailability of BaP, the fish were exposed to 1.5 mCi 3H-BaP kg-1 fish, either in the diet or in the water as a 2 days static exposure. Furthermore, tissue levels of BaP-derived radioactivity bound to macromolecules in different tissues were studied in non-induced fish, and in fish induced by additional treatment with unlabelled BaP (corresponding to 5 mg kg-1 fish) in the water. Absorption and tissue distribution of 3H BaP were studied by liquid scintillation counting and whole-body autoradiography. BaPderived radioactivity bound to macromolecules in different tissues was studied by autoradiography of solvent-extracted whole-body sections. The hepatic CYP1A induction was measured as EROD activity. Exposure to unlabelled BaP resulted in a marked induction of hepatic EROD activity in rainbow trout 2 days after the start of the exposure. Significant higher concentrations of radiolabelled compound were observed in waterborne-exposed fish, in contrast to dietary-exposed fish. High concentrations of radiolabelling were observed in the gills, liver, bile, intestines, olfactory organ, kidney and the skin of the waterborne-exposed fish. In the dietary-exposed fish, high levels of radioactivity were observed in the intestines and the bile, whereas lower concentrations were present in the liver. Only traces of radioactive compound were observed in the gills. In contrast to waterborne-exposed fish, no radioactivity was detected in the olfactory organ or skin. In autoradiograms of sections extracted with a series of polar and non-polar solvents, a large fraction of radioactivity was still present in the gills, olfactory organ, liver, kidney, skin and intestinal mucosa of the waterborne-exposed fish, indicating that reactive BaP intermediates formed by CYP1A-mediated metabolism were bound to macromolecules in these tissues.