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  • 1
    Electronic Resource
    Electronic Resource
    Expression of kinesin heavy chain isoforms in retinal pigment epithelial cells (1995)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 31 (1995), S. 66-81 
    Details
    ISSN: 0886-1544
    Keywords: microtubule motor proteins ; immunolocalization ; RT-PCR ; Northern/Southern blots ; microinjection ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: To examine the possible role of kinesin in pigment granule migration in the retinal pigment epithelium (RPE) of teleosts, we investigated the expression and distribution of kinesin heavy chain (KHC) in RPE. Blots of fish RPE lysates probed with two well-characterized antibodies to KHC (H2 and HD) displayed a prominent band at 120 kD. A third KHC antibody (SUK4) recognized a band at 118 suggesting the presence of two KHC isoforms in teleost RPE. Reverse transcriptase-polymerase chain reaction (RT-PCR) of mRNA from RPE using primers homologous to conserved regions of the KHC motor domain resulted in the homologous to conserved regions of the KHC motor domain resulted in the identification of two putative KHC genes (FKIF1 and FKIF5) based on partial amino acid sequences. Previous studies had demonstrated a requirement for microtubules in pigment granule aggregation in RPE. In addition, the reported microtubule polarity orientation in RPE apical projections is consistent with a role for kinesin in pigment granule aggregation. Immunofluorescent localization of KHC in isolated RPE cells using H2 revealed a mottled distribution over the entire cell body, with no detectable selective association with pigment granules, even in cells fixed while aggregating pigment granules. Microinjected KHC antibodies had no effect on pigment granule aggregation or dispersion, although each of the three antibodies has been shown to block kinesin function in other systems. Thus we found no evidence for KHC function in RPE pigment granule aggregation. However, the two KHC isoforms may participate in other microtubule-dependent processes in RPE.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970310108
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  • 2
    Electronic Resource
    Electronic Resource
    Stimulation of DNA replication by growth factor and hormones in Swiss 3T3 cells: Comparison of the rate of entry into S phase with in vitro DNA synthesis and DNA polymerase α activity (1988)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 134 (1988), S. 57-66 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: An approach to the investigation how growth factors and hormones regulate mammalian cell proliferation is to study the activity of enzymes involved in DNA replication. Quiescent cultures of Swiss mouse 3T3 cells were stimulated with prostaglandin F2α insulin, and/or hydrocortisone for a time at which less than 50% of the cells had initiated DNA synthesis. Such cells were lysed with a Ca+ +-containing hypotonic buffer and incubated with a nucleotide mixture including [3H]thymidine-triphosphate for 1 hr at 37°C. The amount of radioactive label incorporated into the trichloroacetic acid (TCA)-precipitate and the percentage of labeled nuclei correlated with the in vivo stimulation. Analysis of radioactively and density-labeled DNA in sucrose and CsC gradients indicated that the incorporation of label reflected semiconservative replication. DNA polymerase activities were assayed in supernatants from whole-cell lysates prepared with a hypotonic buffer not containing Ca+ +. Using various templates, it was shown that the increase in activity of DNA polymerase α correlated with the percentage of cells in S phase upon the different stimulation, while DNA polymerase β activity after various times of stimulation showed that this activity increased only when cells began to enter S phase, regardless of the combination of growth factor and hormones.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041340107
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