Mouse Oct-1 contains a composite homeodomain of human Oct-1 and Oct-2.

Members of the Oct family of transcription factors specifically interact with the octamer motif, ATGC-AAAT, a regulatory element important for tissue- and cell-specific transcription as well as for the expression of housekeeping genes. Except for Oct-1, all Oct factors are expressed in a temporally and spatially restricted mode during murine development and their number varies in a given cell type. Despite its ubiquitous expression pattern Oct-1 may play a role in murine development. As a first step towards elucidating the role of Oct-1 we report the complementary DNA cloning of the mouse Oct-1 gene. Two large transcripts of 5 and 14 kb are derived from a single gene. The expression patterns of three splicing products of Oct-1 are similar in a number of cells and tissues. In the POU region murine Oct-1 differs in four amino acids from the human homologue and these differences are restricted to helices 1 and 2. Interestingly, two of the four variant amino acids are identical to those in human and mouse Oct-2 and thus the murine Oct-1 homeodomain is intermediary in sequence between human Oct-1 and Oct-2. These two amino acids together with a third one have been shown to be relevant for the interaction between human Oct-1 and herpes simplex virus transactivator VP16. Nevertheless, VP16 interacts albeit weakly with murine Oct-1. We speculate that the differences in the human and mouse Oct-1 homeodomains reflect host-specific differences in protein-protein interactions.

A nexus between Oct-4 and E1A: implications for gene regulation in embryonic stem cells.

Oct-4 is a transcription factor expressed in the pluripotent progenitor cells of the early mouse embryo. Additional factors are required for the distal activation of genes in differentiated cells containing ectopically expressed Oct-4. Here we show that Oct-4 and E1A are sufficient for distance-independent activation of the basal transcription machinery. The ratio of Oct-4 to E1A is critical for transcriptional activation, because higher levels of either factor are less efficient. Activation depends on a transactivation domain linked to the POU domain of Oct-4 and also on the conserved domain 3 of the 289RE1A protein. This domain is required for binding to the C-terminal part of Oct-4 including the POU domain. Our results indicate that E1A can serve as a bridging factor between Oct-4 and the basal initiation complex, and we postulate that an E1A-like factor acts as a cellular bridging factor of Oct-4 in pluripotent cells.

Glycopeptide-albumin derivative: it preparation and histochemical ligand properties.

Carrier-immobilized mono- or disaccharides and other carbohydrate structures, derived by custom-made chemical synthesis, have already proven to be valuable ligands for localizing carbohydrate-binding proteins in tissue sections. Defined purified glycopeptides, as components of neoglycoproteins, offer the possibility of increasing their structural complexity and, thereby, their receptor selectivity. To test the feasibility of this approach, the glycopeptide man6-glcNAc2-asparagine derived from ovalbumin was purified after pronase digestion. It was coupled to bovine serum albumin as carrier protein with the homobifunctional linking agent bis-(sulphosuccinimidyl)suberate to yield the diglycosylated concanavalin A-reactive product. Following biotinylation, it was used to detect mannose-specific binding sites in fixed cells of seven human leukemia or lymphoma lines and in fixed, paraffin-embedded sections of human breast cancer. In comparison to chemically mannosylated bovine serum albumin with ten sites of glycosylation or to ovalbumin, this derivative produced a similar pattern of reaction with a quantitatively lower extent of staining in most cases. Remarkably, the presence of potential endogenous ligands for the detected receptor sites was ascertained using the plant lectin concanavalin A. Thus, the conjugation of a purified, deliberately selected glycopeptide to a suitable carrier produces a histochemical tool for detecting glycopeptide-specific binding sites.

Carrier-immobilized derivatized lysoganglioside GM1 is a ligand for specific binding sites in various human tumor cell types and peripheral blood lymphocytes and monocytes.

Biotinylation of ganglioside-protein conjugates, derived from selective N-acylation of the sphingoid amino group of deacylated ganglioside GM1 or a ganglioside mixture, yielded probes to detect specific binding sites in fixed specimens. GM1-containing neoligandoprotein significantly bound to tumor cells in sections of 15 out of 16 cases of human lung cancer, while the probe, derived from the mixture, was ineffective under these conditions. The same graduation of staining was under identical conditions observed with these two probes on fixed human tumor cells and on peripheral blood lymphocytes and monocytes. Attempts of biochemical isolation of proteins, responsible for this binding capacity, from tumor cell extracts in the presence of the abundant endogenous ligands led to protein bands with apparent molecular weights of 44,000, 68,000 and 72,000 with yields of 0.1-0.24 micrograms/mg protein, after the detergent extracts had been passed over a resin, exposing gangliosides of the markedly less efficient mixture, to exclude binding by non-specific ionic or hydrophobic interactions.

Detection of metastasis-associated differences for receptors of glycoconjugates (lectins) in histomorphologically unchanged xenotransplants from primary and metastatic lesions of human colon adenocarcinomas.

Clinically applicable markers for tumor progression may be uncovered by selective analysis of biochemical parameters, supposedly participating in this complex process. Owing to the importance of specific protein-carbohydrate interactions in diverse biological processes, the pattern of the receptor part in this glycobiochemical recognition system, the sugar receptors (lectins), conceivably reflects biological properties of tumor cells in glycobiochemical terms. Therefore, we established and characterized xenografts from surgically removed specimens of a human primary colon adenocarcinoma and its metastatic lesions to liver of the same patient and of a histomorphologically similar primary colon adenocarcinoma of another patient in nude mice. Xenotransplantation and subsequent glycobiochemical analysis of material from early passages with a standardized procedure had been preferred to cell culture in monolayer on account of maintenance of a higher degree of organized histotypic assembly. Despite histomorphological similarities, the sugar receptor profile revealed significant differences in tumor-tumor and tumor-metastasis comparison, especially for alpha- and beta-galactoside-binding proteins. Tumor-metastasis differences were substantiated by a second successfully xenotransplanted pair of specimens. Comprehensive expansion of these initial data may eventually lead to desirable functional correlations with the different biological properties of histomorphologically similar primary colon adenocarcinomas and of the metastatic phenotype and to a rational development of therapeutic modalities to restrict tumor growth and spread.

Lineage- and differentiation-dependent alterations in the expression of receptors for glycoconjugates (lectins) in different human hematopoietic cell lines and low grade lymphomas.

Important biological functions and cellular recognition phenomena are supposedly governed by specific sugar-protein interactions. Human hematopoietic cell lines offer an excellent model for the study of the expression of endogenous receptors for the carbohydrate part of glycoconjugates with respect to cell lineage and modulation by differentiation. Initially, a panel of fluorescent (neo)glycoproteins was successfully employed to demonstrate cytologically the actual presence of such receptors on different cell lines: the B lymphoblast line, Daudi; the T cell lymphoblastic leukemia line, P12; the multipotent leukemic line, K562 and the promyelocytic line, HL060. Biochemical analyses were performed using affinity chromatography on supports with immobilized lactose and asialofetuin (simple or complex beta-galactosides), melibiose (alpha-galactoside), fucose, N-acetyl-D-galactosamine, maltose (alpha-glucoside), the mannose-rich yeast glycoprotein, mannan, glycopeptides containing sialic acid residues and heparin. Subsequently, sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis was used to detect cell lineage-dependent changes in theses parameters. Differentiation-dependent changes in the expression of receptors with specificity to galactose, N-acetylgalactosamine, maltose and heparin were similarly uncovered upon dimethyl sulfoxide-induced differentiation of HL60 cells. Differences in this type of cellular characteristic were also apparent for lymphoma cells from patients with various histological subtypes of lowgrade lymphomas. This initial description of lineage- and differentiation-dependent differences in various human hematopoietic cell lines and in cells from patients with lowgrade lymphomas suggests that advances in the knowledge of the composition of endogenous sugar receptors (lectins) may aid in understanding aspects of the biological behavior of hematopoietic cells and their related malignancies via participation of sugar-protein (lectin) interactions.

Endogenous sugar receptor (lectin) profiles of human retinoblastoma and retinoblast cell lines analyzed by cytological markers, affinity chromatography and neoglycoprotein-targeted photolysis.

Plant lectins have previously been employed to map the composition of cellular glycoconjugates on retinoblastoma and other tumor cells. To characterize the cellular receptors in protein-carbohydrate interactions, we have applied cytological markers (fluorescent neoglycoproteins) containing common carbohydrate building-blocks to investigate the presence of endogenous carbohydrate-binding proteins in six human retinoblastoma cell lines and one established human retinoblast cell line. The staining patterns showed similar expression of endogenous sugar receptors in all cell lines, with few qualitative differences. However, application of affinity chromatography using resins with immobilized carbohydrates as affinity ligands to isolate sugar receptors (lectins) with binding specificities for beta-D-galactosides, alpha-D-mannosides, alpha-L-fucosides alpha-D-glucosides and to N-acetyl-D-glucosamine and N-acetyl-D-galactosamine, respectively, revealed significant differences between the cell lines, emphasizing the value of complementary biochemical analysis. To demonstrate the practical use of this type of glycobiochemical profiling, selective photodestruction of retinoblastoma cells in vitro was accomplished following incubation with synthetic neoglycoprotein-hematoporphyrin conjugates and subsequent exposure to light. This phototherapeutical approach thus combined the inherent specificity of a neoglycoprotein for a particular cellular phenotype with targeted drug activation.

Histopathologic evaluation of application of labeled neoglycoproteins in primary bronchus carcinoma.

Neoglycoproteins are readily available conjugates of a histochemically inert carrier protein and histochemically crucial carbohydrate moieties which are covalently attached to the carrier protein by chemical synthesis. Biotinylation renders these conjugates detectable in formalin-fixed, paraffin-embedded tissue sections of human lung cancer by standard staining protocols, thereby localizing endogenous receptors for carbohydrate moieties. Examination of 30 cases of main types of human lung cancer revealed the presence of alpha-fucosyl-, alpha-mannosyl-, and alpha-glucosyl-specific receptors in adenocarcinomas or epidermoid carcinomas with high positivity rates. The extent of the expression of receptors for alpha- and beta-galactosides appeared to be comparatively lower. Within the standard protocol, using a concentration of the biotinylated probes of 10 micrograms/mL, this panel of probes consistently failed to detect endogenous sugar receptors in ten cases of small cell anaplastic carcinoma of the lung. Whereas none of the sections from the tumor cases bound the sulfated fucan fucoidan, the accompanying inflammatory cells, especially the granulocytes, expressed receptors for the sulfated fucan. Pronounced labeling for macrophages was observed for the alpha-galactoside-specific probe, whereas no binding to inflammatory cells and pneumocytes was detectable for the beta-galactoside-specific probe. The results indicate that expression of endogenous receptors for neoglycoproteins may be useful in discriminating between small cell and non-small cell lung carcinoma and carcinomatous cells from accompanying inflammatory cells.

Xenografts from a human colon carcinoma and its metastases: establishment, characterization and differences in the pattern of carbohydrate-binding proteins.

Investigation of the pathogenesis of human colorectal carcinoma metastasis can be rendered experimentally possible by suitable human cell biological model systems. The purpose of these studies was to establish xenografts in nude mice from human colon carcinoma and from its metastasis in the same patient as an appropriate model. Surgically removed biopsy specimens from a colon adenocarcinoma (grade 3) and its local relapse two years later with metastases in the small intestine were established as xenotransplants and their growth characteristics examined. Both tissue types shared common characteristics with respect to marker expression (carcinoembryonic antigen, neuron-specific enolase, cytokeratin). The primary tumor showed remarkable development of necrotic effusion with cytotoxic activity that ceased after several passages. The profile of endogenous carbohydrate-binding proteins (lectins), the receptors for cellular glycoconjugates in a recognitive protein-carbohydrate interplay with potential relevance to metastases formation, revealed differences between these two human tumor samples of identical origin, especially with respect to beta-galactoside-specific receptors. This glycobiochemical analysis employed standardized procedures. Prolonged passaging was also shown to result in profile alterations, as was similarly noted in comparison to another species. These studies may encourage the application of systems of primary tumor and its metastases in the same patient in attempts to correlate the expression of cellular characteristics with the biological and clinical behavior of human colonic tumor cells.

Urdamycins, new angucycline antibiotics from Streptomyces fradiae. V. Derivatives of urdamycin A.

Derivatives of the angucycline urdamycin A (1) were prepared in order to study structure-activity relationships in this group of antitumor antibiotics. Derivatives of 1 formed by methanolysis, O-acylation, hydrogenation and treatment with diazomethane were isolated and characterized by their spectroscopic data. Urdamycin G (20) was isolated from Streptomyces fradiae by shortening the fermentation time. The different glycosidation pattern of the aglycone 14 did not lead to significant differences in the biological activity. O-Acylation was shown to enhance the in vitro activity of 1 against stem cells of murine L1210 leukemia depending on the lipophilicity of the molecules. The importance of the 5,6-double bond of 1 with regard to the antitumor activities is discussed.

An 8- to 10-fold enhancement in sensitivity for quantitation of proteins by modified application of colloidal gold.

We have modified the highly sensitive protein assay of C. M. Stoscheck (1987, Anal. Biochem. 160, 301-305), resulting in a further 8- to 10-fold enhancement of sensitivity. This assay, responding to protein quantities with a detection limit of 1 ng, involves the single step of addition of colloidal gold solution, as now commonly used in histochemistry and protein blotting, to the protein sample, followed by simple measurement of the change in absorbance at 590 nm within minutes. By increasing the concentration of the colloidal gold, by using gold sol that has been stabilized with 0.01% polyethylene glycol and adjusted to pH 3.8, and by adapting the assay to microtiter plates, this type of assay can be applied to reliably determine proteins in the complete nanogram range. This assay therefore compares favorably to other assay procedures in terms of rapidity, sensitivity, expense, and lack of interference by many laboratory reagents, although like the others it suffers from the drawback of differences in response of different proteins, which is inherent in dye-binding assays.