Two di-leucine-based motifs account for the different subcellular localizations of the human endothelin-converting enzyme (ECE-1) isoforms.

Endothelin-converting enzyme (ECE-1) is a type II integral membrane protein which plays a key role in the biosynthetic pathway of the vasoconstricting endothelins. Three ECE-1 isoforms, differing by their N-terminal cytoplasmic tails, are generated from a single gene. When expressed in CHO cells, they display comparable enzymatic activity but whereas ECE-1a is strongly expressed at the cell surface, ECE-1b is exclusively intracellular and ECE-1c presents an intermediate distribution. In the present study these different localizations were further described at the ultrastructural level, by electron microscope immunocytochemistry. To characterize the motifs responsible for the intracellular localization of ECE-1b we constructed chimeric proteins and point mutants. Two di-leucine-based motifs, contained in the N-terminal part of ECE-1b, were thus identified. One of these motifs (LV), displayed by both ECE-1b and ECE-1c, accounts for the reduced surface expression of ECE-1c as compared to ECE-1a. Mutation of both motifs (LL and LV) induces a very strong appearance of ECE-1b at the cell surface indicating that their presence in the N-terminal extremity of ECE-1b is critical for its exclusively intracellular localization.

Major histocompatibility complex class II (Ia-like) antigens on chicken embryo fibroblasts: effect of transformation by Rous sarcoma virus.

In this study, three monomorphic monoclonal antibodies to chicken MHC class II molecules (B-L) were tested for reactivity in normal and RSV-transformed embryo fibroblasts. The immunocytochemical staining, the cell-bound ELISA assay, and the immunoprecipitation analysis showed that all three antibodies reacted with the B-L (Ia-like) molecules on normal cells of different genotypes. Conversely, the expression of these antigens was not detected in fibroblasts cultured from feather follicles of adult birds. The level of expression of B-L molecules as well as the class II specific RNA increased consistently after transformation of the cells by the SR-RSV and infection with the avian leukosis virus RAV-1. Analysis of genomic DNA by the Southern blot technique, performed after digestion with several restriction endonucleases, showed that the restriction pattern of B-L genes was not altered in cells transformed by Rous sarcoma virus.

Inhibition of HLA class I antigen and mRNA expression induced by Rous sarcoma virus in transformed human fibroblasts.

Cells from various human nonlymphoreticular neoplasms show reduced HLA class I antigen expression. In this report, a system of human fibroblasts transformed by an avian retrovirus has been employed to investigate the mechanism of this phenomenon. Rous sarcoma virus has been used to transform in vitro human dermal fibroblasts, and clonal cell lines have been established from these cultures. In all the clones studied the integration of the provirus induced a reduction of cell-surface HLA-A, -B, -C framework antigen and beta 2-microglobulin expression when compared to levels for the respective parental fibroblasts. The reduction was correlated with a diminished intracellular synthesis of these molecules. Uninfected cells derived from an osteogenic sarcoma exhibited a reduced expression comparable to that of dermal diploid fibroblasts obtained from the same donor and transformed by Rous sarcoma virus. RNA gel blot analysis of total cellular RNA and of poly(A)+ cytoplasmic RNA showed a markedly decreased amount of HLA class I transcripts in the transformed cells. Southern blot study of genomic DNAs digested with several restriction endonucleases showed that the banding patterns of the HLA genes were not altered in the cells harboring the Rous sarcoma provirus. Our data are consistent with the hypothesis that the Rous sarcoma provirus that does not seem to be linked to the major histocompatibility complex class I gene superfamily may negatively control HLA gene expression.

Integration and expression of provirus in human cells transformed by avian sarcoma virus.

Previously, human diploid fibroblasts from some donors infected in vitro by avian sarcoma virus (ASV) were transformed and found, by electron microscopy, to produce small numbers of virus particles that were infectious by bioassay; also, a line of human osteosarcoma cells infected with ASV developed additional characteristics of transformation and released a small number of infectious virus particles. In this study the complete proviral sequence was shown to be integrated in the genome of these cells. The env-related proteins gp85 and gp37 and the gag-related proteins pr76, pr60, and p19 can be detected in cytoplasmic extracts of ASV-infected human cells. Comparable amounts of pp60v-src were found in human and avian cells infected with ASV. The associated kinase activity in infected human cells was dramatically increased as compared to that of uninfected controls; the enzyme had the same cation and substrate requirements as those from ASV-transformed avian cells. Replicating particles from infected human cells were purified and were significantly modified compared to those from avian hosts as shown by a) higher specific gravity, b) the presence of RSV gag-related but not env-related antigens, and c) the fact that the virus-associated reverse transcriptase preferred the divalent cations Mn2+ and Fe2+ over Mg2+.

Expression of major histocompatibility complex antigens (H-B) on haemopoietic cells, lymphoid cells, and chick embryo fibroblasts from congenic lines of chickens.

The expression and distribution of chicken major histocompatibility antigens (H-B) was examined by electron microscopy. Using an indirect method of ferritin and peroxidase labelling with monospecific heteroantisera and with monoclonal antibodies, the H-B alloantigens could be localized at the plasma membrane of various cell types. The antigenic binding sites were quantified on mature erythrocytes, erythropoietic cells, heterophils, lymphoid cells of both thymus and bursa origin, and on cultured normal and Rous sarcoma virus-transformed chick embryo fibroblasts. The degree of labelling was higher in mature normoblastic erythrocytes and lymphoid cells than in other haemopoietic cells and fibroblasts when the heterospecific antisera were used. A larger number of antigenic binding sites was labelled by the monomeric monoclonal antibodies than by the polyclonal antisera. The distribution and aggregation of the specific label on the plasma membrane of cells other than erythrocytes, however, differed in the fixed and unfixed cells.

Presence of actin in oncornaviruses.

A 43K protein present in avian myeloblastosis virus has been identified as actin by 2D gel electrophoresis and peptide mapping proteolysis. Electron microscopy of chicken embryo fibroblasts infected with different pseudotypes of oncornaviruses treated with anti-actin antibody showed positive staining at the level of the virions especially on buds. Our results indicate that this actin is unlikely to have been artefactually absorbed at the virion surface during its preparation. It may therefore play a possible role in the budding of enveloped virions.

[Transformation of diploid human fibroblasts and viral production after infection by avian retroviruses]. / Transformation de fibroblastes diploïdes humains avec production virale après infection par des rétrovirus aviaires.

Among the cultures established from skin biopsies of 128 donors, two were transformed by RSV and produced virus. One out of 6 cellular lines developed from six human sarcomas was likewise transformed and also produced virus.

Nucleolar ultrastructure and transcription of preribosomal RNA in Rous sarcoma virus-transformed chick embryo fibroblasts treated with alpha-amanitin.

Uninfected and Rous sarcoma virus (RSV)-transformed chick embryo fibroblasts (CEF) were exposed to various concentrations of alpha-amanitin for different lengths of time. At a concentration of 4 micrograms alpha-amanitin/ml, RSV-transformed cells were shown to maintain a normal rate of transcription of all classes of RNA, whereas in uninfected cells transcription was reduced to a very low level. These observations cannot be accounted for by a difference in the penetration rate of alpha-amanitin through the plasma membrane. Investigation of the ultrastructure showed that the degree of nucleolar fragmentation induced by alpha-amanitin was comparable in both types of cells. Persistence of transcription with concomitant nucleolar fragmentation in alpha-amanitin-treated RSV-transformed CEF is not in accord with the hypothesis that nucleolar integrity is required for preribosomal RNA transcription.

Immunoelectron microscopic localization of Rous sarcoma virus structural protein p27 in infected chicken embryo fibroblasts.

The immunoperoxidase technique was employed to localize, at the ultrastructural level, protein Pr76 (a precursor of the 'gag' gene protein of avian leukemia and sarcoma viruses) in chick embryo cells infected with RSV-RAV-2 virus. The protein was confined to free and membrane-bound ribosomes. This was often particularly conspicuous in the perinuclear region. No staining was found in the ergastoplasmic cisternae, in the Golgi apparatus, or in the nucleus. This pattern of localization is compatible with previous studies where the protein was searched for in various cell subfractions.

Recent data and significance of intracisternal tubular inclusions in human and animal cells.

In 24 out of 60 patients with nasopharyngeal carcinoma (NPC) the cells were shown to contain a tubulo-reticular network in the dilated cisternae of the ergastoplasm which corresponds to the entity described previously as "intracisternal-tubular inclusion" (ITI). The significance of such inclusions is discussed in different pathological conditions and the literature is reviewed. This is the first report of such inclusions in nasopharyngeal carcinoma.

[Morphology of ribosomal RNA in chicken fibroblasts]. / Morphologiè des ARN ribosomiques de fibroblastes de poulets.

It is shown with the electron microscope that the 28 S RNA component of the ribosomal RNA extracted from Chicken fibroblasts contains secondary structures which are not present in the 18S component.