Bcl-2 overexpression protects photooxidative stress-induced apoptosis of photoreceptor cells via NF-kappaB preservation.

We recently showed that photooxidative stress on cultured photoreceptor cells results in down-modulation of NF-kappaB activity which then leads to apoptosis of cultured 661W photoreceptor cells. In an effort to further delineate the mechanism of photoreceptor cell death, we sought to determine the effects of Bcl-2 overexpression on cell survivability. Wild-type 661W cells were transfected with the plasmid construct pSFFV-neo-Bcl-2 and several clones were isolated. All clones demonstrated increased Bcl-2 mRNA and protein levels, with the B4 clone exhibiting the greatest enhancement. On exposure to visible light the B4 cells were protected from undergoing apoptosis when compared with the mock transfected cells, as ascertained by TUNEL apoptosis assay and formazan based estimation of cell viability. The Bcl-2 overexpressing cells also maintained a higher Bcl-2/Bax ratio, suggesting that this ratio is important in protection from photooxidative stress. Electrophoretic mobility shift assays for NF-kappaB demonstrated higher activity in both nuclear and cytosolic fractions of the B4 photoreceptors compared with the 661W wild-type cells at all light exposure time points. Furthermore, the findings of the gel shift assays were further supported by immunocytochemistry for NF-kappaB which revealed that protein levels of the RelA subunit of NF-kappaB were protected in the nucleus as well as in the cytoplasm of Bcl-2 overexpressing B4 cells exposed to light compared to the 661W cells. These results suggest that Bcl-2 overexpression protects NF-kappaB protein levels and activity in the nucleus, indicating that preservation of NF-kappaB binding activity in the nucleus may be essential for photoreceptor cells to survive photooxidative damage induced apoptosis.

Amplification of the Golgi complex in MDCK cells secreting human growth hormone.

MDCK cells were transfected with pXGH5, a plasmid containing the human growth hormone (hGH) gene, and permanently expressing cell lines were selected. Clone 3A cells, which secrete quantities of hGH through both apical and basolateral surfaces, were examined in detail. Immunofluorescence analysis using anti-hGH antibody revealed bright perinuclear staining coinciding with the area delineated by anti-p52 kDa protein (a resident Golgi protein) antibody. There appeared to be less Golgi-specific fluorescence in untransfected cells. This difference correlated with an increased amount of 52 kDa in the clone 3A cells. Morphometric analysis was performed on electron micrographs of clone 3A and untransfected cells using the fractionator to estimate average number of Golgi stacks per cell, and values were statistically analyzed. It was found that clone 3A cells contained 3.3 and untransfected cells 1.6 stacks (P < or = 0.005), respectively. When clone 3A cells were placed into defined medium, the synthesis and secretion of hGH declined 4-fold, and the number of Golgi stacks also decreased to the untransfected level within seven days. The number of Golgi stacks per untransfected cell was not affected by the presence of exogenous hGH, indicating that Golgi amplification was directly related to secretory demand. Generation times and cell volumes were identical for both cell types under all growth conditions. In addition, the kinetics of protein secretion from radiolabelled cells demonstrated that clone 3A cells generally secrete lower amounts of endogenously synthesized apical proteins than do untransfected cells, while basolateral secretion remains the same. In both cases hGH comprised only about 10% of total secretory proteins, so that the increase in total protein secretion did not seem to warrant the two-fold elaboration of Golgi by 3A cells. But there might be significant amounts of hGH which traverse the Golgi to end up in lysosomes, rather than being secreted, leading to Golgi amplification.

Routing of a secretory protein to the endocytic compartment in transfected Madin Darby canine kidney cells.

Transfected Madin Darby canine kidney (MDCK) cells (3A) expressing human growth hormone (hGH) contain twice as many Golgi stacks as untransfected cells. How MDCK cells, lacking a regulated pathway, deal with (over)expression of a protein hormone, or any exogenous protein, has not been examined in detail. Since hGH constituted 10% of total secreted proteins, it was not apparent why Golgi amplification was needed, unless some enters a nonsecretory compartment. Studies were undertaken to determine hGH fate. By using an inhibitor of protein synthesis, or by analyzing pulse labeled immunoprecipitated hGH, 20-30% of hGH was shown to remain intracellular even after 4 h. That portion might be localized in the endosome/lysosome compartment, because it is post-Golgi. Immunoelectron microscopy with antibodies against hGH, clathrin, and cathepsin D demonstrated clathrin and hGH colocalized, as did hGH and cathepsin D. The latter were found in large vesicles, but no hGH appeared in lysosomes, due to its degradation. Analysis of isolated lysosome/endosomes revealed vesicles containing both hGH and cathepsin D, but more containing only cathepsin D. Endocytosis studies suggested the 3A basolateral endosomal compartment may be more capacious than normal. Thus, 3A Golgi amplification resulted in an expanded endosome compartment to accommodate secretory protein (over)expression.

A method for using low-temperature embedding media for electron microscopy of cells grown on microporous supports.

Attempts were made to embed Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose microporous supports in Lowicryl, a medium designed for the retention of antigenicity for electron microscopic immunocytochemical studies. It was found that the membrane fragmented during the prescribed embedding procedure leading to loss of the cell sample. This necessitated the formulation of a new combination of fixative and dehydration agents which would allow: (i) preservation of membrane and cellular integrity; (ii) infiltration and embedding in Lowicryl; and (iii) detection of a specific antigen in thin sections. The cellular monolayer and organelle profiles were best preserved with glutaraldehyde fixation at room temperature followed by ethanol dehydration. Since the latter was carried out at temperatures attainable with an ice-salt bath, there was no need for a special ultralow-temperature apparatus. This procedure was applied to a MDCK cell clone that consisted of stable secretors of human growth hormone (hGH), as a result of being transfected with a plasmid containing the hGH gene. Thus, it was demonstrated that hGH could be detected by immunogold labeling of thin sections and localized to specific cellular structures. The procedure developed in this report is applicable to cells grown on two other supports and may be extended further.

Ultrastructural and immunocytochemical characterization of cultured cells from rat molar stellate reticulum.

By scanning electron microscopy, the cultured cells were stellate and had numerous filipodia--characteristics of stellate reticulum cells in vivo. By transmission electron microscopy, they contained bundles of intermediate filaments, numerous mitochondria, large electron-dense granules and desmosomes--all features of the stellate reticulum in vivo. Moreover, the stellate reticulum was the only region of the enamel organ in vivo that contained large, electron-dense granules. By immunocytochemistry, the cultured cells contained cytokeratins, confirming their epithelial nature, and stellate reticulum cells in vivo and in vitro did not have an EGF receptor. Thus, these combined ultrastructural and immunocytochemical findings suggest that the cell culture was of stellate reticulum.

The effects of proteins secreted by fibroblasts from patients with cystic fibrosis on hamster tracheal explants.

Hamster tracheal explants have been used to assay for mucosecretory activity in media taken from cultures of fibroblasts isolated from patients with cystic fibrosis (CF). Cystic fibrosis and normal sera were first used to establish optimal conditions for mucus release in the hamster tracheal ring assay. Unless protein levels were maintained at 5% serum concentration or greater there was loss of cilia, nonspecific mucus accumulation, and extensive epithelial damage to the luminal surface. Likewise, it was shown that exposure of the explants to unconcentrated conditioned media from CF (GM 770, 768, 1348, 142) or normal (GM 3349, 38) cultured fibroblasts for 1, 6, or 12 h resulted in the same type of damage and this was due to low protein levels. When the protein concentration of the conditioned media was increased with fetal bovine serum, the morphological integrity of the explants was maintained, demonstrating that there was no apparent difference between CF and normal fibroblast-secreted proteins in ability to induce mucus release. The ciliary inhibitory capacity of CF serum-derived or fibroblast-derived factor had been reported to require IgG for activity. However, addition of IgG to high molecular weight (VoP10) or low molecular weight (VeP10) secreted proteins had no apparent effect on stimulating secretion. In conclusion, it is possible that CF fibroblasts do not secrete a protein that has the mucostimulatory effect and thus these cells may not be suitable for studying the CF-related activity.

Effects of cystic fibrosis serum on calcium influx and secretion using isolated tracheal epithelial cells.

Hamster trachea epithelial (HTE) cells were shown to respond to 20% cystic fibrosis serum (CFS) by secreting twice as much protein as in the presence of 20% normal human serum (NHS). Serum from obligate heterozygotes (HHS) produced an intermediate effect. A peak of Ca2+ entry into the HTE cells occurred about 30 min after exposure to 20% CFS, followed by a slow decline to basal levels. In contrast, 20% NHS did not cause an influx of Ca2+ and HHS produced an influx to about half that of CFS. Increasing concentrations (5-30%) of pooled NHS had no effect on HTE cell Ca2+ uptake or secretion, but pooled CFS and HHS caused progressive increases in Ca2+ influx and protein secretion from 10 to 25% sera. The CFS-induced Ca2+ influx and secretion were about twice those of HHS throughout the range of serum concentrations tested, suggesting the presence of a modulatory influence in HHS. When EGTA was used to chelate extracellular Ca2+ in the presence of CFS, Ca2+ influx was prevented and there was no stimulation of secretion. Ionophore A23187 allowed Ca2+ entry into HTE cells in the presence or absence of serum and a heightened level of secretory activity followed. The time course of Ca2+ influx under the influence of CFS was shown to correspond to the efflux of Na+ from the cells. Also verapamil, a Ca2+ channel blocking agent, inhibited CFS-induced Ca2+ influx by 50% at 10(-5)M and prevented secretion. Thus, it appears that CFS, but not NHS, contains an agent which stimulates Ca2+ uptake into HTE cells by means of a Ca2+ channel and/or Na+-Ca2+ exchange mechanism, and that increased intracellular Ca2+ levels then trigger secretion. The intermediate HTE cell response to HHS suggests that half of the CFS stimulatory agent is present as would be expected in a gene dose effect, lending support for a genetic basis for CF.

Secretory activity of hamster tracheal explants and isolated tracheal epithelial cells and the effects of cystic fibrosis serum.

Serum from cystic fibrosis patients has been shown by scanning electron microscopy to cause release of large quantities of mucus from the cultured tracheal rings of 3-4-month-old male Golden Syrian hamsters. In order to study this phenomenon on single cells, an epithelial (HTE) cell culture has been established from the hamster tracheal rings using the cell rescue method of Goldman and Baseman (1980a, In Vitro, 16:313). The cells were demonstrated to be epithelial by histochemical staining and immunofluorescent detection of laminin. Proteins secreted by HTE cells were partially characterized and shown to consist, at least in part, of acidic glycoproteins. The proteins were precipitated by addition of buffered alcian blue (AB) to the cell-free medium under conditions in which all of a polyanionic protein [3H]-labeled mucin, was precipitated without carrier. [14C] galactosamine-labeled AB precipitate was beta-eliminated and, after neutralization and centrifugation, the material in the supernatant was sized by chromatography on a calibrated Bio-Gel P2 column. The label eluted with a molecular weight close to a disaccharide. HTE cells pulse-labeled for 1.0 hr with [3H] leucine or [14C] galactosamine secreted increasing amounts of labeled glycoprotein during the chase. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography of labeled AB precipitates revealed three major bands, two with molecular weights greater than 100 kd. Secretion was stimulated by retinoate (50% increase), but not by retinol. Exposure of HTE cells to whole sera from cystic fibrosis patients resulted in heightened secretion rates as compared to results obtained with normal sera. Heterozygote sera produced secretion rates intermediate between the two extremes.

Immunohistochemical localization of laminin within the mouse.

Laminin, a noncollagenous glycoprotein, was observed in the mouse ovary using a direct immunofluorescence technique. Laminin was localized within the basal lamina underlying growing and atretic ovarian follicles, blood vessels, and the germinal epithelium. In addition, laminin was also present at the periphery of individual corpora lutea cells. The presence of laminin in the basal lamina underlying ovarian follicles may be important for the passage of material between the follicle cells and the connective tissue stroma. In addition, its location around the corpora lutea cells may reflect an involvement in cell to cell contact as well as intercellular communication.

Glycosyl acceptors in intact and permeabilized normal and cystic fibrosis fibroblasts.

Using cell permeabilization, a technique which allows addition of exogenously supplied radiolabeled sugar nucleotides to serve as direct glycosyl donors, oligosaccharide biosynthesis was examined in fibroblasts obtained from normal and cystic fibrosis (CF) subjects. Incubation of logarithmically growing cells with either radiolabeled leucine or xylose has indicated that there was a difference in the synthetic rate between the cell types. Protein synthesis in normal cells made permeable with 50 micrograms/ml lysolecithin (LL) was demonstrated to be absent, and could not be induced to take place by adding exogenous components, including energy sources and amino acids, normally required for protein synthesis. Thus radiolabeled sugars were being added to peptide acceptors which were already present at the time of LL addition. Both permeable and intact fibroblasts were exposed to labeled UDP-xylose, UDP-galactose, and UDP-glucuronic acid, all donors of mucopolysaccharide precursors. The uptake of xylose into protein was the same for both normal and CF cells, but permeable CF fibroblasts incorporated statistically greater amounts of sugar from UDP-galactose and UDP-glucuronic acid. Intact CF cells were also labeled using these two sugar nucleotides. Trypan blue exclusion indicated CF and normal fibroblasts were equally intact. This and the fact that preincubation of CF cells with the appropriate cold sugar nucleotide eliminated the differences in incorporation between the normal and CF cells suggested that CF fibroblasts had more cell surface acceptor than the normal cells.

Relationships between nucleic Acid synthetic patterns and encystment in aging unagitated cultures of acanthamoeba castellanii.

Changes in the levels of DNA and RNA syntheses have been studied in unagitated cultures of Acanthamoeba castellanii during the phases of logarithmic multiplication (LM) and population growth deceleration (PGD). Pulse-labeling experiments show that the rate of DNA synthesis decreases at the same time that DNA per cell is known to drop by 50%. The drop in DNA content has been explained by demonstrating with hydroxyurea that the majority of LM amebas can replicate once when DNA synthesis is inhibited and, therefore, must be in G(2), whereas the PGD amebas cannot multiply in the presence of inhibitor and, therefore, must be in G(1). The inhibition of DNA synthesis in LM or PGD cells has been shown to induce encystment. The rate of RNA synthesis, as illustrated by pulse-labeling experiments, increases 25% in late LM-early PGD while RNA per cell increases 75%. The rate of synthesis then decreases 65%. The majority of accumulated RNA has been demonstrated to be ribosomal by disc electrophoresis. By using actinomycin D at different stages during the RNA build-up, the ability of the amebas to encyst has been shown to depend on the presence of this RNA. The observations on DNA and RNA are discussed with respect to the occurrence of cysts in the cultures during PGD.