Immunolocalization of basic fibroblast growth factor and its receptor in adult goldfish retina

The neural retina of teleost fish can regenerate following surgical or neurotoxic lesions. As a first attempt to uncover the factors important for the regenerative response, we used immunocytochemistry to demonstrate the presence of basic fibroblast growth factor (bFGF) and its receptor in the goldfish retina. The bFGF-immunoreactivity was present throughout the retina, but was most intense in photoreceptor cells, especially cones, and Muller glia. Immunoreactivity for the bFGF receptor was strongest in the axon terminals of photoreceptors, both rods and cones. This pattern of immunolocalization is especially interesting since the proliferating cells that are thought to be responsible for generating the neural regenerate are located among the photoreceptor axon terminals. These proliferating cells have been identified as rod precursors because in the intact retina they give rise only to rod photoreceptors. When the neural retina is damaged, however, rod precursors are thought to be the source of proliferating neuroepithelial cells responsible for generating the retinal regenerate. The role played by bFGF in normal neurogenesis, cell differentiation, and/or neuronal regeneration in the fish retina has yet to be determined.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30286/1/0000688.pd

Characterization of cytosolic forms of CTP: choline-phosphate cytidylyltransferase in lung, isolated alveolar type II cells, A549 cell and Hep G2 cells

The subcellular forms of cytidylyltransferase (EC 2.7.7.15) in rat lung, rat liver, Hep G2 cells, A549 cells and alveolar Type II cells from adult rats were separated by glycerol density centrifugation. Cytosol prepared from lung, Hep G2 cells, A549 cells and alveolar Type II cells contained two forms of the enzyme. These species were identical to the L-Form and H-Form isolated previously from lung cytosol by gel filtration. Liver cytosol contained only the L-Form. Rapid treatment of Hep G2 cells with digitonin released all of the cytoplasmic cytidylyltransferase activity. The released activity was present in both H-Form and L-Form. The molecular weight of L-Form was determined from sedimentation coefficients and Stokes radius values to be 97 690 +/- 10 175. Thus, the L-Form appears to be a dimer of the Mr 45 000 catalytic subunit. The [function of (italic small f)] [function of (italic small f)] [deg] value of 1.5 indicated that the protein molecule has an axial ratio of 10, assuming a prolate ellipsoid shape. The estimated molecular weight of the H-Form was 284 000 +/- 25 000. The H-Form was dissociated into L-Form by incubation of cytosol at 37[deg]C. Triton X-100 (0.1%) and chlorpromazine (1.0 mM) also dissociated the H-Form into L-Form. Western blot analysis indicated that both forms contained the catalytic subunit. An increase in Mr 45 000 subunit coincided with the increase in cytidylyltransferase activity in L-Form, which resulted from the dissociated of H-Form. The L-Form was dependent on phospholipid for activity. The H-Form was active without lipid. Phosphatidylinositol was present in the H-Form isolated from Hep G2 cells. The phosphatidylinositol dispersed when the H-Form was dissociated into L-Form. Phosphatidylinositol and phosphatidylglycerol cause L-Form to aggregate into a form similar to H-Form. Phosphatidylcholine/oleic acid (1:1 molar ratio) and oleic acid also aggregated the L-Form. Phosphatidylcholine did not produce aggregation. We conclude that the H-Form is the active form of cytidylyltransferase in cytoplasm. The H-Form appears to be a lipoprotein consisting of an apoprotein (L-Form dimer of the Mr 45 000 subunit) complexed with lipids. A change in the relative distribution of H-Form and L-Form in cytosol would alter the cellular activity and thus may be important in the regulation of phosphatidylcholine synthesis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27634/1/0000010.pd

Expression of rod and cone visual pigments in goldfish and zebrafish: A rhodopsin-like gene is expressed in cones

The primary purpose of the present study was to determine whether a rhodopsin-like gene, which has been postulated to represent the green cone pigment in several species, is in fact expressed in cone photoreceptors instead of rods. The expression patterns of rod opsin and blue and red cone opsins were also examined in both goldfish and zebrafish retinas using colorimetric in situ hybridization. The results demonstrate that the rhodopsin-like gene is expressed in green cones, as predicted. A subset of small cones that do not hybridize with these cRNA probes are tentatively identified as ultraviolet receptors. The results also demonstrate that opsin message in cones is restricted to the perinuclear region, whereas in rods, it is both perinuclear and adjacent to the ellipsoid.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30780/1/0000433.pd

CTP : phosphocholine cytidylyltransferase in rat lung: relationship between cytosolic and membrane forms

The purpose of these studies was to determine the properties of the membrane-bound cytidylyltransferase in adult lung and to assess the relationship between the microsomal enzyme and the two forms of cytidylyltransferase in cytosol. Microsomes, isolated by glycerol density centrifugation, contained significantly less cytidylyltransferase than microsomes isolated by differential centrifugation (11.6 +/- 3.2 vs. 30 +/- 11 nmol/min per g lung). The released activity was recovered as H-form cytidylyltransferase. Cytidylyltransferase activity was not removed from microsomes by washing of the microsomal pellet with homogenizing buffer. Triton X 100 extracted all of the cytidylyltransferase from microsomes. The extracted activity was similar to H-form. Chlorpromazine dissociated microsomal enzyme to L-form. Chlorpromazine has been shown previously to dissociate H-form to L-form. These results suggested that microsomal cytidylyltransferase existed in a form similar if not identical to cytosolic H-form. In vitro translocation experiments demonstrated that the L-form of cytidylyltransferase was the species which binds to microsomal membranes. Triton X 100 extraction of microsomes from translocations experiments removed the bound enzyme activity. Glycerol density fractionation indicated that the activity in the Triton extract was H-form cytidylyltransferase. We concluded that the active lipoprotein form of cytidylyltransferase (H-form) is the membrane-associated form of cytidylyltransferase in adult lung; that it is formed after the L-form binds to microsomal membranes and that cytosolic H-form is released from the membrane.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28515/3/0000312.pd