Ellen Rasch

Altered structure and composition of retinal cells in dark-reared mammals☆

Abstract Retinas of cats, hooded rats, and chimpanzees reared in total darkness from birth were compared with those from controls matched for age and reared in light with normal day and night variation. Microphotometric measurement of azure B binding was used to estimate ribonucleic acid (RNA) concentration in individual ganglion cells. Relative protein levels were judged visually after staining with fast green or the Millon reaction. There was a marked lowering of cytoplasmic and nucleolar RNA levels in neurons from the multipolar, bipolar, and receptor cell layers of retinas from dark-reared cats. Protein levels were also appreciably lowered in these subjects, and a significant reduction of mean nucleolar volume and in cytoplasmic cross-sectional area of ganglion cells was found. The inner plexiform layer was consistently thinner in retinas from cats reared in darkness, although there were no apparent differences in thickness of cell body layers, nor in frequencies of the several cell types within any one particular layer. Comparable but less extensive data are presented for hooded rats. Markedly depressed RNA and protein levels were also found in retinal ganglion cells of two dark-reared chimpanzees, associated with degeneration of the ganglion cell layer. The data suggest that adequate light stimulation is a major variable controlling the development of normal ribonucleoprotein levels in cells of the mammalian retina.

KINETICS OF HYDROLYSIS DURING THE FEULGEN REACTION FOR DEOXYRIBONUCLEIC ACID A REEVALUATION

Using thin films of Feulgen-stained chicken blood cells, we have found that the course of deoxyribonucleic acid (DNA) hydrolysis with 5 N HCl at room temperature over a period of 24 hr can be represented by equations based upon a simple kinetic model involving branched convergent reactions. In addition to its use for analysis of the Feulgen hydrolysis curves generated from avian erythrocytes and lymphocytes, the model has also been applied to previously published data on human lymphocytes. Use of the model to determine appropriate conditions for validating stoichiometry of the DNA-Feulgen reaction is discussed, with particular emphasis directed to possible implications for DNA-Feulgen cytophotometry of a two-compartment substrate moiety.

Microphotometric analysis of the cytochemical Millon reaction.

The cytochemical Millon reaction has been studied with test protein systems and with several plant and animal tissues. Absorption curves of the Millon chromophores have been analyzed and compared with natural ultraviolet protein absorption. The findings indicate the validity of applying the Millon reaction as a quantitative cytochemical method in studies of the nucleoprotein complexes of cells, but only when proper corrections for non-specific light loss and other variables have been applied. Under these conditions, a roughly linear relationship was found between natural ultraviolet protein absorption at 280 mµ and the intensity of the Millon chromophore at 500 mµ. A scheme of intermediate products and competing reactions coincident to formation of Millon chromophores has been proposed, based on changes in absorption spectra during color development in test protein systems.

The DNA content of trophozoites and cysts of Giardia lamblia by microdensitometric quantitation of Feulgen staining and examination by laser scanning confocal microscopy.

We investigated direct measurement of the DNA content of the parasitic intestinal flagellate Giardia lamblia through quantitation by Feulgen microspectrophotometry and also by visualization of Feulgen-stained DNA chromosomes within dividing cells by laser scanning confocal microscopy. Individual trophozoites of Giardia (binucleate) contained 0.144 +/- 0.018 pg of DNA/cell or 0.072 pg DNA/nucleus. Giardia lamblia cysts (quadranucleate) contained 0.313 +/- 0.003 pg DNA or 0.078 pg DNA/nucleus. The genome size (C) value per nucleus ranged between 6.5-7.1 x 10(7) BP for trophozoites and cysts, respectively. Confocal microscopic examination of Giardia trophozoites undergoing binary fission revealed five chromosome-like bodies within each nucleus. Further information about genome size and DNA content within different Giardia species may help to clarify the pivotal role of these primitive eukaryotic cells in evolutionary development.

Applications of microcomputer technology to cytophotometry.

Lack of appropriate software support for microprocessor program development has previously limited the applications of such technology in the field of microspectrophotometry. This paper describes our use of a Vickers M86 integrating microdensitometer coupled through a custom-designed interface circuit to a Processor Technology SOL III microcomputer. W e have developed a series of interactive, user-oriented programs for DNA-Feulgen cytophotometry with this instrument to allow automatic storage of data in files on floppy disks and instant retrieval of sets of measurements for statistical processing. The same data files can also be used to generate graphic displays in the form of bar histograms or plots of linear regressions on a video monitor and to produce hardcopy output of data files and graphic displays through the use of a high speed DIABLO printer.

A VERTICAL MICROSYSTEM FOR DISCONTINUOUS ELECTROPHORESIS OF INSECT TISSUE PROTEINS USING THIN SHEETS OF POLYACRYLAMIDE GEL

Proteins extracted from individual pairs of salivary glands or other larval tissues of Sciara coprophila (Diptera) were separated in a vertical microsystem for discontinuous electrophoresis using thin sheets of polyacrylamide gel cast in multiple layers of varying pore size. After electrophoresis at 150 volts for 40 min, gels were stained (a) for total proteins with Coomassie brilliant blue, (b) for glycoproteins with the periodic acid-Schiff reaction, (c) for lipoproteins with Sudan black B or (d) for nonspecific esterases with fast blue RR as coupler and α-naphthol acetate as substrate. Sequential application of these reactions to individual gel sectors permitted direct comparisons of protein profiles for 15-20 different samples of tissue extracts carried on a single gel sheet in adjacent lanes and thus subjected to identical conditions of electrophoresis. Representative photographs and densitometric scans are presented to show the suitability of thin gel sheets for autoradiography and for both qualitative and quantitative evaluation of tissue-specific differences in patterns of protein banding found for salivary gland cells, the gastric ceca, or the hemolymph of individual Sciara larvae sacrificed at particular stages of fourth instar development. Innovative details of methodology are presented, including the use of a microspectrophotometer to scan electropherograms of insect proteins and several types of human blood serum.

DISCONTINUOUS ELECTROPHORESIS OF PEPSIN AND PEPSINOGEN IN THIN SHEETS OF POLYACRYLAMIDE GEL

Electrophoretic separation of discrete protein bands from solutions of crystalline pepsin or pepsinogen was accomplished in a system incorporating pH discontinuity and using standard, glass microslides to support thin (0.25-mm) sheets of 15% polyacrylamide gel. Adherence of gel films to a glass support during fixation and staining with crystal violet not only prevented distortion of gels as a result of swelling, but also provided preparations that could be readily scanned with a microspectrophotometer. A number of discrete peptide bands were identified from different samples of purified pepsin. Among the several heterogeneous components found in commercially available samples of crystalline pepsinogen, there was a characteristic impurity, the mobility of which was directly comparable to that of pepsin run under the same conditions of electrophoresis. Specific details of methodology are presented.

Liver Nucleoproteins in Vitamin B12 Deficiency.

Summary Cytochemical studies on liver tissue from rats fed vit. B12-dencient or supplemented diets for 6 weeks have indicated the following: (1) Total protein showed no change in concentration, but a marked increase in amount per cell, correlated with significant increases in mean cytoplasmic volume for livers of deficient animals. (2) RNA, determined with Azure B binding at pH 4.0, showed a decrease in concentration, but an increase in amount per cell. (3) DNA, determined photometrically on Feulgen-stained nuclei was unchanged in amounts per single tetraploid nucleus when compared with B12-supplemented controls. (4) The average amount of DNA per nucleus, when calculated from data on polyploid class frequencies, averaged 6% less than controls, associated with a higher frequency of diploid nuclei.