Using monoclonal antibodies directed against different cytoplasmic isoforms of hsp70 proteins, namely, the constitutive hsc73 and the inducible hsp72 isoforms, we found that one isoform related to hsc73 was present in Euglena gracilis. This hsc73-like protein is expressed with a higher rate of synthesis in cells growing under heat shock than in control cells. Moreover, in cadmium-resistant cells, cultured at normal growth temperature, the rate of synthesis of this protein is constitutively increased. These results indicate that a heat-shock protein related to hsc73 is present in an ancestral eukaryote, Euglena gracilis, and that this protein may be constitutive and stress inducible as well.

Antibodies directed against rat α1-acid glycoprotein (AGP) recognize a 70 kDa antigen, designated JB70, present in extracts of achlorophyllousEuglena graciliscells as well as in their culture medium. By using 2-dimensional electrophoresis, JB70 appears to be composed of two acidic polypeptides. Additionally, Northern blot analysis reveals the presence inE. graciliscells of a 2.3 kb mRNA hybridizing with a cDNA probe specific for rat AGP mRNA. Moreover, elevated mRNA levels are detected in dexamethasone-treatedE. graciliscells, indicating a response to this inducer similar to that observed for hepatic AGP. These results strongly suggest that polypeptides closely related to hepatic rat AGP are expressed inE.graciliscells. They also indicate that, like other gene families implicated in natural defense processes such as heat-shock protein and metallothionein genes, the AGP gene appears to be conserved down to this early diverging eucaryote.

Six monoclonal antibodies directed against different epitopes of bovine retinal arrestin recognized a single polypeptide in extracts of achlorophyllous Euglena gracilis cells. This polypeptide had an apparent molecular weight slightly lower: 45kDa vs. 48 kDa, and a more basic isoelectric point compared to that of bovine visual arrestin. It was located in an insoluble cytoplasmic fraction. Immunofluorescence assays show a cytoplasmic punctuated pattern suggesting a cluster distribution or a linkage to some cell structure. The presence of arrestine-like molecules in achlorophyllous Euglena gracilis cells suggests that these proteins might be involved in a peculiar step of chemical signal transduction processes.

2-5A-Dependent RNase (RNase L), an important component of the 2-5A pathway, is directly implicated in the molecular mechanism of interferon action. We have cloned and sequenced following immunoscreening, a full-length cDNA that encodes the RNase L inhibitor (RLI), Northern blot analysis from a variety of human tissues revealed that two transcript forms (3.8 kb and 2.4 kb) are ubiquitously expressed but differences in levels of expression suggest a tissue-specific regulation, The RLI gene was localized to locus 4q31 by in situ hybridization indicating that this gene and other enzymes of the 2-5A pathway are not organized in cluster in the human genome.

To determine whether cellular resistance to a given stressor is related to induction of specific stress-proteins, responses of two adapted Euglena gracilis cell lines, one adapted to cadmium, the other adapted to pentachlorophenol, were analyzed. Our experiments showed that two sets of heat-shock proteins (hsps) were constitutively overexpressed in each cell line: while hsp90, hsp70, hsp55, and hsp40 were induced in cadmium-resistant cells, only hsp40 was induced in pentachlorophenol-adapted cells.

This work is a preliminary characterization of two adapted Euglena gracilis cell lines, one to cadmium and the other to pentachlorophenol. Growth curve analyses indicate that tolerance to one pollutant did not protect against the second pollutant. These suggest that metabolic pathways that are induced by one pollutant are specific for this pollutant. This specificity is detectable at the level of gene expression.

The heat-shock response of Euglena gracilis was studied by cell labeling at both the normal growth temperature (23 degrees C) and an elevated temperature (35 degrees C). Analysis of the labeled proteins by two-dimensional polyacrylamide gel electrophoresis indicated that the rate of synthesis of two polypeptides p55 (55 kDa) and p40 (40 kDa) increased in cells labeled at the highest temperature studied. These polypeptides are also overexpressed in Cadmium-resistant Euglena gracilis cells labeled at the normal growth temperature (23 degrees C). On the basis of these results, p55 and p40 appear to be heat-shock proteins involved in some steps of the acquired Cd-resistance process in Euglena gracilis cells.

This report describes the preliminary characterization of a novel antigen reactive with a murine monoclonal antibody designated B1N produced in our laboratory. This antibody (IgM) reacts in IFI with mammals and also insect cells, by staining in a speckled fashion the nucleus of these cells. Immunoblotting analysis of Hela and murine D55 nuclear extracts revealed a polypeptide with an apparent molecular weight of 120kD (p120). In this work we demonstrated that: 1. this polypeptide appeared in human peripheral blood lymphocytes only when they were induced to proliferate in vitro after phytohemagglutinin stimulation; 2. this polypeptide was no longer detected in D55 resting cells, following serum deprivation; 3. the MAb B1N specifically revealed the nucleus of proliferating cells on frozen sections of uterine tissue. These data strongly suggest that the p120 nuclear antigen expression is associated with the proliferation state of cells.

We have characterized a new antibody specificity in a panel of sera from dogs developing systemic lupus erythematosus (SLE) or clinically related autoimmune disorders. This antibody stains in a speckled fashion the nucleus of cells of different mammalian origins. The target antigen is a basic (pI 9.2) nuclear polypeptide with an apparent molecular weight of 43 kDa (p43) which is detected in various mammalian cell nuclei. p43, as studied in HeLa cells, appears to be cell cycle-independent. It is released from nuclei by salts (0.5 M NaCl or 0.25 M ammonium sulfate). Upon subfractionation of nuclear components, p43 is found in the fraction containing HnRNPs and is recovered in immunoprecipitates obtained with 4F4 monoclonal antibody to HnRNP C proteins. Immunoelectron microscopy revealed that p43 is concentrated over the dense chromatin periphery and interchromatin granule clusters. Another important feature of p43 is its ability to specifically bind wheat germ agglutinin lectin but not concanavalin A nor Ulex europaeus I, supporting the notion that p43 is a glycoprotein bearing an N-acetyl-glucosamine moiety. Consistent with this result, a radio-active p43 band is specifically immunoprecipitated by canine anti-p43 autoantibodies from HeLa cells metabolically labeled with [14C]glucosamine. Finally, anti-p43 antibodies do not immunoprecipitate SnRNA, indicating that p43 has no apparent association with SnRNPs.

This report describes the preliminary characterization of a novel antigen reactive with a murine monoclonal antibody designated B1N produced in our laboratory. This antibody (IgM) reacts in IFI with mammals and also insect cells, by staining in a speckled fashion the nucleus of these cells. Immunoblotting analysis of Hela and murine D55 nuclear extracts revealed a polypeptide with an apparent molecular weight of 120 kD (p120). In this work we demonstrated that: 1. this polypeptide appeared in human peripheral blood lymphocytes only when they were induced to proliferate in vitro after phytohemagglutinin stimulation; 2. this polypeptide was no longer detected in D55 resting cells, following serum deprivation; 3. the MAb B1N specifically revealed the nucleus of proliferating cells on frozen sections of uterine tissue. These data strongly suggest that the p120 nuclear antigen expression is associated with the proliferation state of cells.

Monoclonal antibody (mAb) 83D4 was generated using formol-fixed paraffin-embedded human breast carcinoma tissue as the immunogen. Previous studies demonstrated that it was reactive with breast carcinoma tissues, but not with normal breast. The antigen identified by mAb 83D4 was detected, using ELISA, in MCF7 breast carcinoma cell line membrane extracts, in primary breast and colon carcinoma tissue extracts and in pleural effusion fluid from patients with metastatic breast cancer. No reactivity with 83D4 was found in either human milk fat globule membranes or skimmed milk. 83D4 reactive antigen was found to be a heterogeneous high molecular weight (MW) protein (apparent Mr:300-400 to over 1000 kDa) by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The antigen was purified from MCF7 cells, breast and colon carcinomas and effusion fluid, by perchloric acid solubilisation followed by immunoaffinity chromatography with 83D4. The immunopurified antigen from MCF7 cells and pleural effusion fluid was further analysed by gel filtration and ion-exchange chromatography, which confirmed the high MW and indicated the charge heterogeneity of the reactive molecules. The 83D4 reactive antigen strongly bound to wheat-germ agglutinin and weakly to peanut lectin. No binding was found with lentil lectin or concanavalin A. Antigenic activity was strongly reduced by trypsin and subtilysin digestion and by treatment with sodium periodate, but it was not affected by neuraminidase. These results imply the glycoprotein nature of the 83D4-defined antigen and the involvement of carbohydrate, but probably not sialic acid, in the epitope. Purified 83D4 antigen did not display reactivity for mAb HMFG-1, directed against a polymorphic epithelial mucin, PEM, using ELISA, but bound mAb CC49 and weakly mAb B72.3, antibodies which define a tumour associated glycoprotein, TAG-72. Moreover CC49 and 83D4 showed similar reactivity pattern in immunoblotting assays. A double determinant radioimmunoassay confirmed that 83D4 antigen carries epitopes for mAb B72.3 and CC49. Competition radioimmunoassays clearly distinguished the 83D4 defined epitope from those recognised by B72.3 and CC49, demonstrating that antibody 83D4 identifies a unique epitope. It is suggested that the antigens identified by mAb 83D4 and by mAb B72.3 and CC49 may form part of the same family of carcinoma associated glycoproteins.

We report a new antibody specificity in 15 sera recovered from a group of dogs developing systemic lupus erythematosus (SLE) or clinically related disorders. This antibody stains in a speckled fashion the nucleus of human Hep-2 cells. Immunodiffusion tests with saline extracts of rabbit thymus showed that all 15 sera generate a common precipitation line which crosses the lines from reference sera to Sm, SS-A/ro, SS-B/La, and RNP antigens. The target nuclear antigen is a 40 kD polypeptide (p40). An important property of p40 resides in its ability to bind specifically Wheat Germ Agglutinin lectin but not Concanavalin A, supporting the notion that the antigen is a glycoprotein bearing a N-acetylglucosamine moiety.

In the course of a systematic screening of sera from dogs suffering from autoimmune disorders, three sera were shown by indirect immunofluorescence to characteristically label the nucleoli and nucleoplasm of human cell lines (Hep-2 and HeLa). This pattern of staining persisted throughout the cell cycle, except for mitosis when the fluorescence was localized in extrachromosomal areas. By immunoblotting nuclear and subnuclear fractions, three polypeptides of 110,000, 95,000, and 45,000 Da apparent molecular weight were identified, which reacted with all three sera. By means of affinity purification, it was shown that an antibody specific for any one of the three proteins also reacts with the two others. This antigenic cross-reactivity suggested regions of structural homology shared by the three proteins. Indeed, treatment of nucleoli with high concentrations of DNase I containing residual proteolytic activity resulted in the disappearance of the 110- and 95-kDa proteins and the concomitant appearance of a doublet of 45-kDa proteins. Subnuclear localization studies indicated that all three polypeptides were located in both nucleoli and nucleoplasm. Significantly, the 110-kDa protein differs from the major nucleolar protein, nucleolin, by its electrophoretic mobility in two-dimensional gels, its location in nucleoli and in nucleoplasm, its absence in nucleolar organizer regions of chromosomes, and its differential solubility of DNase I. Therefore, the three antigenically related species reported in this study constitute a new class of nucleolar proteins.

The sera of two patients with autoimmune disorders recognize in HeLa cell extracts two proteins with apparent molecular masses of 37,000 (p37) daltons and 32,000 daltons (p32). These proteins are non covalently associated with 5S RNA and sediment as 7-10 S particles in sucrose density gradients. Both proteins are antigenetically related to TFIIIA, a previously described protein of Xenopus laevis, which is known as a 5S RNA transcription factor and occurs in oocytes as a noncovalent complex with 5S RNA. Like TFIIIA, HeLa cell proteins p37 binds in vitro to 5S RNA and to cloned 5S RNA genes. These results suggest that protein p37 fulfils in HeLa cells a function similar to that of TFIIIA in amphibian oocytes, ie control of 5S RNA transcription.

The myeloproliferative leukemia virus (MPLV) is a new acute leukemogenic, nonsarcomatogenic retroviral complex that is generated during the in vivo passage of a molecularly cloned Friend ecotropic helper virus. Examination of viral RNA expression in MPLV-producing cells revealed the presence of two distinct molecular species that hybridized with a long terminal repeat or an ecotropic env-specific probe but not with a xenotropic mink cell focus-forming virus env-specific probe derived from a spleen focus-forming virus: an 8.2-kilobase species corresponding to a full-length Friend murine leukemia virus (F-MuLV) and a deleted species with a genomic size of 7.4 kilobases. This deleted virus was biologically cloned by limiting dilutions and single cell cloning in Mus dunni fibroblasts. Three nonproducer clones with normal morphologies and containing one single integrated copy of the deleted virus were superinfected with F-MuLV, Moloney murine leukemia virus, Gross murine leukemia virus, mink cell focus-forming virus (HIX), or the amphotropic 1504 murine leukemia virus. All pseudotypes caused macroscopic and microscopic abnormalities in mice that were similar to those seen in the parental stock. A comparison of the physical maps of F-MuLV and MPLV, which was deduced from the restriction enzyme digests of unintegrated proviral DNAs, indicated that the MPLV-defective genome (i) is probably derived from F-MuLV, (ii) has conserved the F-MuLV gag and pol regions, and (iii) is deleted and rearranged in the env region in a manner that is clearly distinct from that of Friend or Rauscher spleen focus-forming viruses.