The transmembrane domain of murine alpha-mannosidase IB is a major determinant of Golgi localization.

Murine alpha1,2-mannosidase IB is a type II transmembrane protein localized to the Golgi apparatus where it is involved in the biogenesis of complex and hybrid N-glycans. This enzyme consists of a cytoplasmic tail, a transmembrane domain followed by a "stem" region and a large C-terminal catalytic domain. To analyze the determinants of targeting, we constructed various deletion mutants of murine alpha1,2-mannosidase IB as well as alpha1,2-mannosidase IB/yeast alpha1,2-mannosidase and alpha1,2-mannosidase IB/GFP chimeras and localized these proteins by fluorescence microscopy, when expressed transiently in COS7 cells. Replacing the catalytic domain of alpha1,2-mannosidase IB with that of the homologous yeast alpha1,2-mannosidase and deleting the "stem" region in this chimera had no effect on Golgi targeting, but caused increased cell surface localization. The N-terminal tagged protein lacking a catalytic domain was also localized to the Golgi. In the latter case, when the stem region was partially or completely removed, the protein was found in both the ER and the Golgi. A chimera consisting of the alpha1,2-mannosidase IB N-terminal region (cytoplasmic and transmembrane domains plus 10 amino acids of the "stem" region) and GFP was localized mainly to the Golgi. Deletion of 30 out of 35 amino acids in the cytoplasmic tail had no effect on Golgi localization. A GFP chimera lacking the entire cytoplasmic tail was found in both the ER and the Golgi. These results indicate that the transmembrane domain of alpha1,2-mannosidase IB is a major determinant of Golgi localization.

Synthesis and structure-activity relationships of 2-pyrazinylcarboxamidobenzoates and beta-ionylideneacetamidobenzoates with retinoidal activity.

The structure-activity relationships of two series of novel retinoids (2-pyrazinylcarboxamidobenzoates and beta-ionylideneacetamidobenzoates) have been investigated by evaluating their ability to induce differentiation in both human promyelocytic leukemia (HL60) cells and mouse embryonal carcinoma (P19) cells. The most active compound (ED50 = 8.3 x 10(-9) M) of the 2-pyrazinylcarboxamidobenzoates is 4-[2-(5,6,7,8-tetrahydro-5,5,8, 8-tetramethylquinoxalyl)carboxamido]benzoic acid (9u), while the most active analogue of the beta-ionylideneacetamidobenzoates is 4-[3-methyl-5-(2',6',6'-trimethyl-1'-cyclohexen-1'-yl)-(2E, 4E)-pentadienamido]benzoic acid (10a, ED50 = 3.2 x 10(-8) M). Our studies identify an absolute requirement for the carboxylic acid moiety on the aromatic ring to be para relative to the amide linkage for activity. Benzoate substitutions in the ortho position relative to the terminal carboxylate (9d,k,r) are well-tolerated; however, a methoxy substituent meta relative to the terminal carboxylate gives rise to only weakly active analogues (9x). Conformational studies (NMR, X-ray crystallography) of the 2-pyrazinylcarboxamidobenzoates indicate that the preferred conformation exhibits a trans-amide bond and an internal hydrogen bond between the quinoxaline N1 and HN amide which locks the torsional angle between C2 and CO in the s-trans conformation. N-Methylation (9y) results in loss of activity. Studies indicate that there is now a cis-amide bond present which redirects the carboxylate toward the pharmacophoric gem-dimethyl groups. The distance between the gem-dimethyl group and the terminal carboxylate appears to be too short to activate the retinoid receptor. N-Methylation in the beta-ionylideneacetamidobenzoate series (10c) also results in the formation of a cis-amide bond and loss of activity.

Trophic actions of ciliary neurotrophic factor on murine embryonic carcinoma cells.

Recombinant rat CNTF (ciliary neurotrophic factor) at picomolar concentration prevents the death of P19 murine embryonic carcinoma cells that usually follows upon withdrawal of serum from the culture medium. For prolonged survival of P19 cells in serum-free medium, insulin must also be present. In the presence or absence of serum, CNTF stimulates the differentiation of P19 cells, inducing the formation of neurites and synthesis of neurofilament. The results of radioautographic studies with radioiodinated CNTF indicate the presence of high-affinity binding sites on P19 cells. Equilibration of P19 cells with [125I]CNTF followed by incubation with cross-linking reagents reveals evidence for at least two putative receptors of approximately 78 and approximately 167 kDa.

Two distinct pathways of interferon induction as revealed by 2-aminopurine.

Activation and repression of IFN gene expression are controlled primarily at the transcriptional level. In order to elucidate some aspects of the induction mechanism of the IFN genes, we examined the effects of different treatments on IFN production in L929 cells, a well-characterized system, and in primary spleen cells. Our results indicate that 2-Aminopurine (2-AP) inhibits type I IFN (IFN-alpha and IFN-beta) induction in L929 cells but not in spleen cells. In L929 cells, 2-AP inhibited the induction of the MuIFN-beta promoter and of promoters containing tetrahexamer and PRDII sequences linked to a reporter gene. Inhibition of activation of the inducible factors binding to the MuIFN-beta promoter and sub-elements was also observed. In contrast, factors binding to the MuIFN-beta promoter are present constitutively in spleen cell nuclei and their activity is not inhibited by 2-AP. These results suggest that 2-AP inhibits IFN-beta gene induction in L929 cells through blocking of activation of the inducible DNA-binding factors which interact with the IFN-beta promoter.

A developmentally regulated octamer-binding activity in embryonal carcinoma cells which represses beta-interferon expression.

We have previously described ECIF-1, a DNA-binding factor present in nuclear extracts of murine embryonal carcinoma cells which specifically recognizes a region within the human beta-interferon promoter. We show that the promoter region located between -112 and -93 is sufficient for this binding activity, which is not due to binding of interferon-regulatory factor 1 or 2. By mutational analysis of the ECIF-1 site, it was determined that the central nucleotides which are critical for binding contain an octameric motif: ATTTACAT. The binding activity of ECIF-1 with its cognate site within the beta-interferon promoter decreases upon differentiation concurrently with the onset of interferon inducibility. Furthermore, by using an in vitro transcription assay with deleted promoter elements of the beta-interferon gene, we show that undifferentiated P19 nuclear extracts contain a repressing activity which depends on the presence of the ECIF-1 site. This repression is not observed using nuclear extracts from differentiated P19 cells. Comparison of the binding activity of this octamer site with others previously shown to be active in embryonal carcinoma cells reveals similarities and differences in the spectrum of proteins binding there.

An embryonic DNA-binding protein specific for a region of the human IFN beta 1 promoter.

Embryonal carcinoma (EC) cells are unable to make interferon in response to inducing agents. This block disappears after differentiation. We have found that nuclear extracts from undifferentiated P19 EC cells contain a DNA-binding activity which specifically recognizes a region within the human interferon-beta 1 promoter. This activity is absent from differentiated cell types, both of EC and non-EC origin. The binding of the factor in undifferentiated EC cells leads to dramatic changes in the overall protein binding pattern of the interferon promoter as compared with differentiated cells, and may be responsible for repression of the endogenous interferon-beta gene prior to differentiation.

Epitopes of carcinoembryonic antigen defined by monoclonal antibodies prepared from mice immunized with purified carcinoembryonic antigen or HCT-8R cells.

A library of 18 monoclonal antibodies (MAbs) reactive with purified carcinoembryonic antigen (CEA) has been prepared. The specificity of these MAbs was tested and they have been separated into nine subgroups, each recognizing a different region of the CEA molecule. Seven MAbs from four of the groups also react with the nonspecific cross-reacting antigen. Some of the MAbs are directed against conformational determinants: three of the MAb groups bind poorly to sodium dodecyl sulfate-treated CEA, while five of the groups are not reactive with reduced and alkylated CEA. Three of the groups react with purified CEA but not with the cell surface CEA of HCT-8R cells, while the other groups react with both forms. The MAbs were tested for binding to fragments of CEA obtained by chemical cleavage and the groups of MAbs were found to react with different subsets of such fragments.

Characterization of monoclonal antibodies to carcinoembryonic antigen with increased tumor specificity.

Nine monoclonal antibodies reacting with carcinoembryonic antigen (CEA) were produced after immunization of mice with either purified CEA or a CEA-producing human cell line. Their specificities were assessed by immunohistochemistry on tissue sections of neoplastic and nonneoplastic lesions. These monoclonal antibodies have different patterns of tissue reactivity. Two of them, D14 and B18, were found to have a high degree of specificity for colonic carcinoma and did not react with formalin-fixed paraffin-embedded sections of normal colon with standardized staining conditions. Most cases of noncolonic adenocarcinomas and normal epithelial structures were not stained by these two monoclonal antibodies. The specificity of the monoclonal antibodies was further investigated immunochemically using intact, reduced, and alkylated or chemically fragmented CEA. Liquid phase radioimmunoassays and antibody competition immunoenzymatic assays confirmed that the antibodies recognize different epitopes of CEA. These data support the concept of CEA heterogeneity and the reactivity of the D14 and B18 monoclonal antibodies with colonic adenocarcinomas indicates that they are useful immunohistochemical probes.

A high-affinity oestrogen-binding protein in cockerel liver cytosol.

In contrast with several earlier reports, cytosol from cockerel liver contains a significant concentration of a protein that binds oestradiol with high affinity. To demonstrate the activity, certain alterations in the conventional method of preparation of cytosol must be made. Homogenization in sucrose-containing buffer at pH 8.4 in the presence of proteinase inhibitors and rapid fractionation of the cytosol with (NH4)2SO4 enables demonstration of a single class of oestradiol-binding sites with a Kd of about 1 nM and specificity only for oestrogens. The concentration is about 300 sites per cell in liver from 2-week-old cockerels. Oestradiol treatment in vivo decreases the number of exchangeable cytosol oestradiol-binding sites by about 80% for 1--4h, after which time it is gradually restored. Gel filtration of the cytosol preparation in the presence of high salt concentrations reveals that most of the oestradiol-binding activity is in high-molecular-weight aggregates, but a mild trypsin treatment generates a specific binding protein with an approximate mol.wt. of 40 000. This protein may be an oestrogen receptor.