Biomedical subjects

E Y Kam

Publications and source records attributed to E Y Kam.

5 recordsLinked to original sources

PubMed1987

Permeance of Novikoff hepatoma gap junctions: quantitative video analysis of dye transfer.

Fluorescent dyes are commonly used to study permeable (gap) junctions, but only rarely have quantitative values for junctional dye permeability been determined. In the present study, junctional permeance (PA, i.e., the product of the junctional permeability coefficient, P, times the junctional area, A) to Lucifer Yellow CH (LY) has been obtained for pairs of Novikoff hepatoma cells. Dye was microinjected into one cell and the subsequent transfer monitored by a SIT camera and recorded on video tape. The intensities of fluorescence in the injected and "recipient" cell were measured using a Digisector (Microworks) digitizing board and an Apple II Plus computer to analyze the video records. These changes in intensity, along with an estimate of volume of the spherical cells, were used to calculate the junctional permeance (PA) of cell pairs according to Fick's diffusion equation. Junctional permeances show considerable variation ranging from 0.08 X 10(-11) to 27.0 X 10(-11) cm3/sec. Using the mean PA and a previous estimate of the mean number of junctional channels per interface in the Novikoff cultures, a value for diffusion coefficient of LY through gap junctions is calculated to be about 1.4 X 10(-6) cm2/sec. There is a general proportionality between mean PA and cell volume for hepatoma cell pairs of a certain size range. Such a relationship between cell volume and junctional capacity suggests one source of variation of PA. Other possible sources, e.g., related to position in the cell cycle, are discussed.

Animals↗

PubMed1986

A radioimmunocytological quantitative method for the rapid detection of ras oncogene p21 protein in mammalian cells.

In order to provide a sensitive and quantitative detection method of ras p21 at the cytological level, the monoclonal antibody Y13 259 and iodinated protein A were used to locate the ras protein in various mammalian cell lines. The subsequent autoradiograph can be analysed by a computer-assisted system which showed in these reported experiments that the relative levels of p21 detected in these cells corresponded to results obtained earlier using conventional biochemical methods.

Animals↗

PubMed1985

Transformation of mammalian cell by iontophoretic pricking or iontophoretic microinjection.

Thymidine kinase negative mouse (LATK-) and hamster (BHKTK-) cells were transfected with a recombinant plasmid (pTK1) carrying the thymidine kinase gene of HSV-1 by iontophoretic pricking or iontophoretic microinjection techniques. Transfection frequencies were measured using short term survival and long term transformation assays and were found to be at the level of 10 to 15% and 1 to 2% respectively for mouse or hamster cells transfected with pTK1. The presence of covalently linked transcriptional enhancers from SV40 or Moloney MSV increased long term transformation frequencies approximately 10-fold. Transfection by iontophoretic pricking of non-tumorigenic mouse (NIH3T3) or hamster (BHKC13) cells with a recombinant (pAGT1) carrying the human T24 Ha-ras1 oncogene and the bacterial aminoglycoside phosphotransferase (aph) gene as a selectable marker resulted in transformation of these cells. Southern blot hybridization analyses demonstrated the presence of circular, integrated or rearranged donor DNA molecules in the transformed cells. These gene transfer techniques and iontophoretic pricking in particular should be useful for the transfection of a variety of cell lines and markers.

Animals↗

PubMed1984

Computer-assisted grain counting for autoradiography.

The two programs described here are designed to be used on the System III Image Analyser which partially automate the counting process of autoradiography and present the data in histogram form. The system has the advantages of low cost, accuracy and reproducibility. Although the programs are developed for grain counting, they can be easily adapted to perform other particle counting processes using the same system.

Autoradiography↗

PubMed1983

Junctional transfer in cultured vascular endothelium: I. Electrical coupling.

Vascular endothelial cultures are composed of flat, polygonal monolayer cells which retain many of the growth, metabolic and physiological characteristics of the intimal endothelium. However, intercellular gap and tight junctions, which are thought to perform important roles in normal intimal physiology, are reduced in complexity and extent in culture. We have used electrophysiological techniques to test confluent (3- to 5-day) primary cultures of calf aortic (BAEC) and umbilical cord vein (BVEC) endothelium for junctional transfer of small ions. Both cell types are extensively electrically coupled. The passive electrical properties of the cultured cells were calculated from the decrease in induced membrane potential deflections with distance from an intracellular, hyperpolarizing electrode. Data analyses were based on a thin-sheet model for current flow (Bessel function). The generalized space constants (lambda) were 208.6 microns (BAEC) and 288.9 microns (BVEC). The nonjunctional (6.14 and 8.72 X 10(8) omega) and junctional (3.67 and 3.60 X 10(6) omega) resistances were similar for the BAEC and BVEC, respectively. We detected no statistically significant differences in the resistance estimates for the two cell types. In vivo ultrastructural studies have suggested that aortic endothelium has more extensive gap junctions than venous endothelium. We have found that these ultrastructural differences are reduced in culture. The lack of any significant difference in electrical coupling capability suggests that cultured BAEC and BVEC have functionally similar junctional characteristics.

Animals↗