Endocytosis and de novo expression of major histocompatibility complex encoded class I molecules: kinetic and ultrastructural studies

The endocytic pathway and expression of the major histocompatibility complex encoded class I molecule H-2Kk was investigated in murine fibroblasts. Internalization of H-2K molecules did not occur constitutively. Endocytosis of the molecules was induced by addition of multivalent ligands such as rabbit anti-mouse immunoglobulin serum or protein A-bearing liposomes to cells pretreated with anti-H-2Kk antibodies. The complete removal of H-2K molecules took about 5 h at 37 degrees C and was not inhibited by the lysosomotropic agent NH4Cl or the protein synthesis inhibitor cycloheximide. When targeted liposomes that contained carboxyfluorescein at a self-quenched concentration were directed against H-2K molecules, the cells became highly fluorescent after 30 min: a consequence of carboxyfluorescein release from the liposomes. This process was inhibited by NH4Cl but not by cycloheximide, suggesting internalization of H-2K molecules into acidic intracellular compartments. The endocytic pathway of liposomes directed against H-2K molecules and the subcellular compartments involved in this process were investigated with targeted liposomes containing horseradish peroxidase. By electron microscopy, the endocytic process was shown to start very rapidly (1-2 min) and involved uncoated cell surface invaginations. The cytoplasmic uncoated vesicles fused together into larger vacuoles containing concentrated liposomes and by 1 h, liposomes began to be destroyed in lysosomal compartments. Within 4 h, 90% of liposomes were lysed inside the cell. The fate of radiolabeled anti-H-2K antibody was also investigated. Degradation of the antibody occurred only when cross-linked with a second layer of antibody, beginning after 2 h and becoming more pronounced after 20 h of incubation. The original cell surface abundance of H-2K molecules was reestablished after 5 to 7 h. During this time neither NH4Cl nor cycloheximide had any effect on the cell surface expression of the molecule. However, after a second cycle of internalization, cells incubated with cycloheximide no longer expressed these molecules. These results suggested that H-2K molecules were not recycled back to the surface after internalization but were degraded in lysosomal compartments together with their ligand. Preexisting molecules, already present in intracellular pools, were expressed to replace them. By immunoprecipitation of metabolically labeled intracellular and surface H-2K molecules, we observed an intracellular pool of H-2K of about 70 to 80% of the total cellular H-2K.     

Changes in cellular gene expression in rat thyroid epithelial cells transformed by the Kirsten murine sarcoma virus

We have exploited a recently characterized system of rat thyroid epithelial cells transformed by the wild-type (wt) and a temperature-sensitive (ts) mutant strain of the Kirsten murine sarcoma virus (Ki-MSV) in order to study the effects of the K-ras oncogene on the gene expression of differentiated thyroid epithelial cells. By using cDNAs isolated from normal thyroid glands as probes, we were able to identify three sets of cellular sequences whose expression is influenced by the v-K-ras oncogene. The first set of genes is irreversibly repressed by transformation with both the wt and the ts viruses. The second set of genes is repressed in the ts-Ki-MSV-transformed cells but not in the same cells grown at the nonpermissive temperature. A third set of genes is present at higher levels at the nonpermissive temperature than at the permissive temperature. This system has allowed us to isolate and characterize a number of cDNA clones belonging to each of these three sets of genes. These specific cDNAs are suitable probes to study phenotypical changes during transformation of epithelial cells.     

A site-specific recombination function in Staphylococcus aureus plasmids.

All known small staphylococcal plasmids possess one or two recombination sites at which site-specific cointegrate formation occurs. One of these sites, RSA, is present on two small multicopy plasmids, pT181 and pE194; it consists of 24 base pairs of identity in the two plasmids, the "core," flanked by some 50 base pairs of decreasing homology. Here we show that recombination at RSA is recA independent and is mediated by a plasmid-encoded, trans-acting protein, Pre (plasmid recombination). Pre-mediated recombination is site specific in that it occurs within the core sequence of RSA in a recA1 host. Recombination also occurs between two intramolecular RSA sites. Unlike site-specific recombination systems encoded by other plasmids, Pre-RSA is not involved in plasmid maintenance.     

Influence of Hydrocortisone on Chick Embryo Retina Development

Treatment of chick embryos in ovo with hydrocortisone-21-phosphate (a single dose of 150 micrograms) caused a marked reduction of retinal thymidine kinase activity 24 h later. The inhibitory effect was highest (65-70%) in 8-10-day-old embryos and declined with age, disappearing after day 15. It was accompanied by a reduction in thickness of the retinal layers. Adrenocorticotropic hormone (ACTH) treatment (10 micrograms daily for 2 days) also produced an age-dependent inhibitory effect on retinal thymidine kinase, whereas treatment with a single dose of 200 micrograms of metopirone, a compound that prevents the 11 beta-hydroxylation of steroid molecules in the adrenal glands, impeded the decrease in thymidine kinase activity that normally occurs in chick embryo retina after day 9 of development. In addition, metopirone prevented the inhibition exerted by ACTH on thymidine kinase activity but had no effect on the action of hydrocortisone.     

Quantitative immunocytochemical staining for recombinant tissue-type plasminogen activator in transfected Chinese hamster ovary cells

Tissue-type plasminogen activator (tPA) is a serine protease which cleaves plasminogen to its active form, plasmin. tPA plays a physiologic role in hemostasis, wound healing, and embryogenesis. Therapeutically, recombinant human tPA is used as a thrombolytic in myocardial infarction. Although production of therapeutic quantities of tPA in Chinese hamster ovary (CHO) cells transfected with the human gene for tPA is practical, production costs remain high. One important factor which determines the ultimate cost of tPA (or any other recombinant protein expressed in mammalian cells) is its production level on a per cell basis. We have used postembedding immunocytochemical staining with colloidal gold to study the subcellular localization of tPA in CHO cells expressing recombinant tPA (rCHO) in an effort to understand the factor(s) which might limit secretion. Staining for tPA was evaluated visually and by morphometric analysis and was specific and reproducible. Serially passaged rCHO showed no significant change in staining density over 31 serial passages. Staining density was greatest over dilated cisternae of the rough endoplasmic reticulum and nuclear envelope. Golgi stacks and large acid phosphatase-positive vacuoles (probably lysosomes) were also heavily stained. Staining of lysosomal vacuoles suggested that rCHO might be degrading nascent tPA. Incubation of rCHO with 125I-tPA showed that the cells were not internalizing tPA from the media. These results suggest that rCHO fail to secrete a portion of the tPA they synthesize and that it is degraded in lysosomes. This observation may have important implications on the choice of expression systems for efficient production of large quantities of recombinant proteins.     

Hypothalamic-pituitary somatotropic function in prepubertal hypothyroid rats: effect of growth hormone replacement therapy.

The effect of thyroid hormone deficiency and growth hormone (GH) treatment on hypothalamic GH-releasing hormone (GHRH)/somatostatin (SS) concentrations, GHRH/SS mRNA levels, and plasma GH and somatomedin-C (IGF-I) concentrations were studied in 28- and 35-day-old rats made hypothyroid by giving dams propylthiouracil in the drinking water since the day of parturition. Hypothyroid rats, at both 28 and 35 days of life, had decreased hypothalamic GHRH content and increased GHRH mRNA levels, unaltered SS content and SS mRNA levels, and reduced plasma GH and IGF-I concentrations. Treatment of hypothyroid rats with GH for 14 days completely restored hypothalamic GHRH content and reversed the increase in GHRH mRNA, but did not alter plasma IGF-I concentrations. These data indicate that, in hypothyroid rats, the changes in hypothalamic GHRH content and gene expression are due to the GH deficiency ensuing from the hypothyroid state. Failure of the GH treatment to increase plasma IGF-I indicates that the feedback regulation on GHRH neurons is operated by circulating GH and/or perhaps tissue but not plasma IGF-I concentrations. Presence of low plasma IGF-I concentrations would be directly related to thyroid hormone deficiency.     

Cellular localization of bovine pancreatic trypsin inhibitor and related molecular forms in bovine lung

Summary In addition to bovine pancreatic trypsin inhibitor (BPTI), three BPTI-related molecular forms (isoinhibitors I, II and III) were isolated from bovine lung by affinity chromatography on immobilized trypsin and subsequently purified by Fast Protein Liquid Chromatography. These inhibitors are identical to the isoinhibitors previously isolated from bovine spleen. Their localization in bovine lung was studied by immunohistochemical techniques, using two different immunoglobulin preparations, selectively recognizing BPTI or the other molecular forms.BPTI-related immunoreactivity was found to be restricted to isolated cells, often identified as mast cells by Toluidine Blue staining. In contrast, isoinhibitor-related immunoreactivity, which also occurs in the mast cells, is present in a number of other cell types. These types include: (i) the smooth muscle cells of different calibre vessels, (ii) the ciliated cells of the bronchial epithelium and the related mucus, and (iii) many cells at alveolar level.Comparison of these data with previous results obtained for bovine spleen suggest multiple physiological roles for these inhibitors.