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We have modified an existing technique in order to perform DNA analysis by flow cytometry (FCM) of corneal epithelium from the mouse, rat, chicken, rabbit, and human. This protocol permitted an investigation of human corneal scrapings from several categories: normal, aphakic bullous keratopathy (ABK), keratoconus (KC), Fuch's dystrophy, edema, epithelial dysplasia, and lipid degeneration. No abnormal characteristic cell-kinetic profile was detected when averaged DNA histograms were compared statistically between the normal and either ABK, KC, edema, or Fuch's dystrophy groups. Abnormal DNA histograms were recorded for cell samples that were taken 1) from three individuals who had epithelial dysplasia and 2) from one individual diagnosed with lipid degeneration. The former condition was characterized by histograms that had a subpopulation of cells with an aneuploid amount of DNA or had higher than normal percentages of cells in the S and G2 + M phases of the cell cycle. Corneal cells from the patient who had lipid degeneration had an abnormally high percentage of cells in the G2 + M phases of the cell cycle. The availability of accurate DNA flow cytometric analysis of corneal epithelium allows further studies on this issue from both experimental and clinical situations.

~ ~The patterns of [.3HI-leucine and [32Pl-phosphate incorporation of proteins extracted with varying molarities of sodium chloride were analyzed from nuclei physically sorted from six fluorescence windows after propidium iodine staining of the GO + GI and (& + M phases of the Chinese hamster ovary (CHO) cell cycle. Eight hundred nanograms of protein were used in each electrophoretic analysis obtained from 200,000 nuclei, a portion of the sample, from each window. Autoradiography was performed in a two-dimensional polyacrylamide gel ultra-microelectrophoresis apparatus (UMEA) designed and fabricated in this laboratory. There was a net reduction and/or loss of ['HI-leucine-and ['2P]-phosphate-labeled protein regions from the autoradiographs occurring primarily inThe refinement of two-dimensional polyacrylamide gel electrophoresis (PAGE) has contributed significantly to the knowledge and the elaboration of gene products during cellular development and differentiation. In disciplines such as biochemistry, cell biology, teratology, etc., a need has arisen for methods to analyze single cells (eggs), small numbers of simple populations (e.g., cell cycle compartments, blastulae), and larger but relatively small numbers of complex populations (e.g., organs, embryos). The adaptation of a two-dimensional PAGE system for the analysis of minute amounts of protein from small samples has been developed (13), and the sensitivity was shown to be far greater than conventional electrophoresis techniques. For example, several hundred protein regions have been observed from a single Drosophila egg with resolution as low as 3 pg of protein (13).The objectives of the present study were, first, to assess the potential of micro-sampling for the collection of relatively small numbers of nuclei (200,000) from winthe 6 2 + M phase. Two phosphorylated proteins that were stage specific were observed in partitions of the Gz + M phase. The use of isolated proteins and the coelectrophoresis of these markers demonstrated the similarity in mobility of a number of proteins seen in the autoradiographs of proteins extracted with high and low salt molarities and implied they are synonymous. Coelectrophoresis indicated that a substantial number of high molecular weight proteins that decreased or disappeared at late stages of Gz + M and early mitosis were composed, in part, of nucleolar proteins.

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