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H J Gabius

Publications and source records attributed to H J Gabius.

At least 325 records · Page 18Linked to original sources
PubMed1984

Biochemical characterization of endogenous carbohydrate-binding proteins from spontaneous murine rhabdomyosarcoma, mammary adenocarcinoma, and ovarian teratoma.

Three entirely different tumor types were investigated biochemically for the presence and characteristics of endogenous carbohydrate-binding proteins in an inbred Brown Norway rat, an outbred Sprague-Dawley rat, and an outbred Han:NMRI mouse. The patterns under investigation included specificities for alpha- and beta-galactosyl, alpha-mannosyl, and alpha-fucosyl moieties, respectively, and specificities for heparin, analyzed by affinity chromatography on resins with immobilized sugars or glycoproteins and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The patterns were divided into categories according to dependence of the binding activity on the presence of Ca2+ and dependence on extraction conditions. Rhabdomyosarcoma revealed only Ca2+-independent activities, i.e., activities with specificity for beta-galactosides at a molecular weight of 12,000, with specificity for alpha-galactosides at molecular weights of 29,000, 43,000, and 45,000, with specificity for heparin at molecular weights of 13,000 and 16,000, and with specificities for mannose and fucose at molecular weights ranging from 62,000 to 70,000. For the spontaneous mammary adenocarcinoma the pattern was entirely different and more diverse, including species with the Ca2+ requirement. Extracts with the use of 0.2 M NaCl (salt) and 2% Triton X-100 (detergent) from teratoma contained at least nine different carbohydrate-binding proteins. The only similarities between the pattern of endogenous carbohydrate-binding proteins from teratoma and from mammary adenocarcinoma were beta-galactoside-binding proteins, one with a Ca2+ requirement and one without a Ca2+ requirement, and the heparin-binding proteins. These heparin-binding proteins were the only types of carbohydrate-binding proteins common to all three tumor types. The analysis indicates that certain bands represented newly identified proteins capable of binding to galactose-, mannose- or fucose-containing glycoconjugates, respectively. When assayed with rabbit erythrocytes, the different fractions showed agglutination activity. They can thus be termed "endogenous lectins." The use of endogenous lectin patterns as potential diagnostic markers in addition to the corresponding changes in the glycoconjugate composition is proposed.

Adenocarcinoma↗
PubMed1983

Evolutionary aspects of accuracy of phenylalanyl-tRNA synthetase. Accuracy of fungal and animal mitochondrial enzymes and their relationship to their cytoplasmic counterparts and a prokaryotic enzyme.

Phenylalanyl-tRNA synthetases from mitochondria of yeast and hen liver resemble their corresponding cytoplasmic counterparts. Whereas slight intraspecies differences at the amino acid binding site, reflecting variations in the structures of these distinct enzymes, are exploitable by phenylalanine analogues, no intraspecies difference can be noted for the strategies to achieve the high fidelity of protein synthesis. While the yeast mitochondrial enzyme follows the pathway of posttransfer proofreading, the hen liver mitochondrial enzyme uses a tRNA-dependent pretransfer proofreading in the case of the natural amino acids. The accuracy of mitochondrial phenylalanyl-tRNA synthetases appears to be even better than the accuracy of the corresponding cytoplasmic enzymes. Interspecies rather than intraspecies differences for the functional role of certain amino acid residues of the enzymes further indicate the close relationship of the intracellular heterotopic isoenzymes. By use of a highly sensitive immunospotting procedure, common antigenic determinants are detected only within the enzymes from the two intracellular compartments of the same organism. The results suggest the origin of the cytoplasm-mitochondrion isoenzyme pair by independent gene duplication of the ancestral nuclear gene. A similarity of mitochondrial enzymes to the phenylalanyl-tRNA synthetase from Escherichia coli is not observed.

Adenosine Monophosphate↗
PubMed1983

Age-related changes in different steps of protein synthesis of liver and kidney of rats.

Protein synthesis in cell-free systems of rat liver and kidney decreases markedly with age. Examination of activity changes of the different steps revealed for both types of organs that reduced binding of aminoacyl-tRNA to ribosomes and reduced peptidyl transfer might be of major importance for the decrease in overall protein synthesis whereas ageing has only little effect on translocation as well as on initiation and termination.

Aging↗
PubMed1983

An RNA ligase from wheat germ which participates in transfer RNA splicing in vitro.

Transfer RNA half-molecules are intermediates in the splicing of tRNA precursors containing intervening sequences. We have utilized yeast tRNA half-molecules to identify and partially purify an ATP-dependent RNA ligase activity from extracts of wheat germ. This activity can complement a yeast tRNA endonuclease in vitro to efficiently splice 10 different yeast tRNA precursors. The products of in vitro splicing are a covalently joined tRNA and a circular intervening sequence RNA. The internucleotide bond formed at the splice junction is a 2'-phosphomonoester, 3',5'-phosphodiester structure. The 2'-phosphate originates from the 2',3'-cyclic phosphate at the 3' terminus of the 5' half-tRNA. The phosphodiester phosphate is derived from the gamma-phosphate of ATP.

Phosphorus Radioisotopes↗
PubMed1983

Evolutionary aspects of accuracy of phenylalanyl-tRNA synthetase. A comparative study with enzymes from Escherichia coli, Saccharomyces cerevisiae, Neurospora crassa, and turkey liver using phenylalanine analogues.

The phenylalanyl-tRNA synthetases from Escherichia coli, Saccharomyces cerevisiae, Neurospora crassa, and turkey liver activate a number of phenylalanine analogues (tyrosine, leucine, methionine, p-fluorophenylalanine, beta-phenylserine, beta-thien-2-ylalanine, 2-amino-4-methylhex-4-enoic acid, mimosine, N-benzyl-L- or N-benzyl-D-phenylalanine, and ochratoxin A), as demonstrated by Km and kcat of the ATP/PPi pyrophosphate exchange. Upon complexation with tRNA, the enzyme-tRNAPhe complexes show a significantly increased initial discrimination of these amino acid analogues expressed in higher Km and lower kcat values, as determined by amino-acylation of tRNAPhe-C-C-A(3'NH2). The overall accuracy is further enhanced by a second discrimination, a proofreading step. The strategies employed by the enzymes with respect to accuracy differ. Better initial discrimination in the aminoacylation and less elaborated proofreading for the E. coli enzyme can be compared to a more efficient proofreading by other synthetases. In this way the comparatively poor initial amino acid recognition in the case of the S. cerevisiae and N. crassa enzymes is balanced. The extent of initial discrimination is therefore inversely coupled to the hydrolytic capacity of the proofreading. A striking difference can be noted for the proofreading mechanisms. Whereas the enzymes from E. coli, S. cerevisiae, and N. crassa follow the pathway of posttransfer proofreading, namely, enzymatic hydrolysis of the misaminoacylated tRNA, the turkey liver enzyme uses tRNA-dependent pretransfer proofreading in the case of natural amino acids. In spite of the same subunit structure and similar molecular weight, the phenylalanyl-tRNA synthetases from a prokaryotic and lower and higher eukaryotic organisms show obvious mechanistic differences in their strategy to achieve the necessary fidelity.

Amino Acyl-tRNA Synthetases↗
PubMed1983

Phenylalanyl-tRNA synthetases from yeast cytoplasm and mitochondria. The presence of a carbohydrate moiety in the mitochondrial enzyme and immunological evidence for structural relationship.

Homogeneous yeast cytoplasmic and mitochondrial phenylalanyl-tRNA synthetases (L-phenylalanine:tRNAPhe ligase (AMP-forming), EC 6.1.1.20) are analysed for structural differences. Only the large subunit of the mitochondrial enzyme is a glycoprotein with nearly 3% carbohydrate by weight. The carbohydrates present are: glucose, N-acetylglucosamine, mannose, galactose and N-acetylneuraminic acid. Removal of the sugar moieties yields an activity increase, but no significant change of sensitivity to proteolytic degradation. Antibodies to both homogeneous enzymes demonstrate a structural similarity for both types of subunit using the highly sensitive immunoblotting technique.

Amino Acyl-tRNA Synthetases↗
PubMed1983

Activity patterns of aminoacyl-tRNA synthetases, tRNA methylases, arginyltransferase and tubulin: tyrosine ligase during development and ageing of Caenorhabditis elegans.

As a step in the characterization of development and ageing in the nematode Caenorhabditis elegans, the activities of different groups of enzymes that supposedly exert modulating functions in and after protein synthesis have been determined. From embryonic (E), the four juvenile larval stages (L1-L4) and the gravid adult (A,A+), the selection of defined developmental stages extends to two different preparations of aged nematodes (S10, S12). Some aminoacyl-tRNA synthetase activities remain nearly unchanged in all stages up to the adult, some increase continuously during the larval stages and the remaining activities show stage-specific alterations. Upon ageing all activities except the one for tryptophan decrease sharply, tRNA methylase activities increase from E to L4, decrease from L4 to adult and to aged nematodes with only qualitative alterations in substrate specificity. The activity of tubulin: tyrosine ligase exhibits a parallel pattern, while arginyltransferase activity has a plateau between L2 and L4. The results are consistent with the idea of a modulation of protein synthesis and other cellular processes by quantitative activity changes during development and ageing.

Acyltransferases↗
PubMed1982

Organ pattern of age-related changes in the aminoacyl-tRNA synthetase activities of the mouse.

The specific activities of 17 aminoacyl-tRNA synthetases from liver, lung, heart, spleen, kidney, small intestine and skeletal muscle of young (2 months) and aged (39 months) female Han:NMRI mice were determined under standard conditions of sample preparation and assay. The average reduction of total activity during ageing is 70% for liver, 50% for lung and spleen, nearly 40% for heart and kidney and nearly 20% for intestine and skeletal muscle. Detailed comparison reveals no general, but an organ-specific pattern. Aminoacyl-tRNA synthetases were, furthermore, found to be ribosome-associated in higher proportions in liver tissue from aged mice.

Aging↗
PubMed1982

Phenylalanyl-tRNA synthetases from hen liver cytoplasm and mitochondria, yeast cytoplasm and mitochondria, and from Escherichia coli: substrate specificity relationship with regard to ATP analogs.

Twelve structural analogs of ATP have been tested in the aminoacylation reaction of phenylalanyl-tRNA synthetases from hen liver cytoplasm and mitochondria, yeast cytoplasm and mitochondria and E. coli. Three compounds are substrates for all five phenylalanyl-tRNA synthetase, three are completely inactive, while the other ATP analogs show differing properties with the different enzymes. Their Km, Ki and V values have been determined. The importance of the amino group in Position 6, the nitrogen in Position 7 and an unsubstituted Position 8 of the purine moiety as well as the supposed anti-conformation of the glycosidic bond and coordination of the magnesium cation to N-7 appear to be conserved through evolution. Bulky substituents on the 2' and 3' of the ribose moiety are generally not tolerated. Graduation of substrate properties of some analogs are similar for the intracellular heterotopic isoenzymes from yeast and hen liver.

Adenosine Triphosphate↗
PubMed1982

Phenylalanyl-tRNA synthetases from cytoplasm and mitochondria of yeast and hen liver: comparison of their structural and catalytic properties.

Amino acid compositions and tryptic maps of the cytoplasmic and mitochondrial phenylalanyl-tRNA synthetases from yeast and hen liver, respectively, demonstrate the similarity of these enzymes, although they are clearly not identical. Moreover, similarity is noted for catalytic properties like stoichiometries of complex formation with tRNAPhe and negative cooperativity of tRNAPhe binding, triggered by substrates. Analysis of the kinetics at saturating and subsaturating substrate concentrations indicates the contribution of the transfer of phenylalanine from the adenylate to tRNAPhe to the rate-determining step in aminoacylation and subunit interactions in the tetrameric enzymes. Furthermore, the locations of substrate-binding sites appear rather constant within species and in interspecies comparison. Subtle differences at certain sites, although homology exists, are exemplified by a special regulatory effect on activity by other amino acids only in the case of the cytoplasmic enzyme from hen liver. The results support the idea of a common ancestry by gene duplication for the cytoplasmic and mitochondrial phenylalanyl-tRNA synthetases in fungi and animals, respectively.

Amino Acids↗
PubMedDate not supplied

Further refinement of the description of the ligand-binding characteristics for the galactoside-binding mistletoe lectin, a plant agglutinin with immunomodulatory potency.

The galactoside-binding lectin from mistletoe (Viscum album L.) is a biological response modifier, eliciting e.g. enhanced secretion of cytokines. This immunological activity warrants the further analysis of its ligand-binding properties with special attention paid to blood group epitopes. To avoid the microheterogeneity and complexity of naturally occurring glycoproteins, chemically strictly defined neoglycoconjugates and a panel of synthetic oligosaccharides were employed in solid-phase assays for direct binding and assessment of the relative inhibitory capacity. Since label incorporation into the lectin, although performed under protective conditions, or surface immobilization by adsorption to plastic may affect its affinity characteristics, the extent of neoglycoconjugate binding in the absence of any interfering substance and in the presence of oligosaccharides was determined comparatively with labeled and with immobilized lectin. In principle, these two factors could be excluded to markedly alter binding features. In addition to lactose, the blood group determinants H and B were strongly reactive. A fucose residue can thus especially be accommodated to the binding site when linked to the non-reducing unit. N-Acetyllactosamine was nearly as potent as an inhibitor as lactose. Lec and the A determinant were notably inferior to the other ABH blood group epitopes. Le(a) and Le(x) and their sialylated derivatives displayed only very weak binding capacity. Among the two natural isomers of sialyllactose, the alpha 2,6-form displayed a higher level of inhibitory capacity than the alpha 2,3-derivative. Isomeric variants of the Thomsen-Friedenreich antigen, too, reduced lectin binding to the lactose-carrying polymer. Their capacities were surpassed by those of the H and the B determinants and a related form of the latter, the P1 epitope. An overlap of specificity with the immunomodulatory human galectin-3 is thus measurable for H/B-like structures. The documented differential reactivity of the mistletoe lectin to blood group oligosaccharides may have a bearing on the responsiveness of blood group-positive cell populations.

Adjuvants, Immunologic↗
PubMedDate not supplied

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.

Adenocarcinoma↗
PubMedDate not supplied

Galectins-1 and -3 and their ligands in tumor biology. Non-uniform properties in cell-surface presentation and modulation of adhesion to matrix glycoproteins for various tumor cell lines, in biodistribution of free and liposome-bound galectins and in their expression by breast and colorectal carcinomas with/without metastatic propensity.

Protein (lectin)-carbohydrate (cellular glycoconjugate) recognition is operative in biochemical information transfer. Galectins constitute a family of endogenous galactoside-binding lectins with conserved features in the binding site. The members of this lectin category are assumed to be involved in cell adhesion and growth regulation. To assess to what extent the different modes of binding-site presentation and/or carbohydrate fine-specificities will affect aspects of galectin behavior, homodimeric cross-linking galectin-1 and monomeric chimeric galectin-3, with its collagenase-sensitive stalk linked to the carbohydrate-recognition domain, were investigated. Cell-surface expression of the two galectins and accessible galectin-binding sites on various tumor cell lines was ascertained by FACScan analysis. In particular, ligand accessibility for the two galectins differed for the tested cell line types. Binding of tumor cells to laminin and plasma or placental fibronectin was generally reduced by treatment of cells or matrix with galectins. Galectin-3 was more efficient than galectin 1 at impairing laminin's potency as matrix. Cell binding of galectin-1, on the other hand, proved on average more effective for blocking cell association to fibronectins after its preincubation with cell suspensions. Differences were also apparent in the biodistribution of the galectins, where an avian homolog of galectin- served as the control to distinguish effects of spatial and sugar-binding features. Histopathological analysis of lymph-node-negative and -positive breast and colorectal carcinomas (n = 180 including 60 metastatic lesions) indicated a correlation of either increased galectin-1 binding and reduced galectin-3 expression or reduced binding of both galectins with the occurrence of lymph node lesions. Together with data on the heparin-binding lectin, revealing reduced expression to be associated with a positive lymph-node status in the breast cancer group, these results can be interpreted to reflect cell-type-dependent requirements of galectin ligand presentation during the metastatic cascade. By introducing mammalian lectins to lectin-histochemical studies, the detection of quantitative differences in glycosylation brings an understanding of its cell biological significance one step closer.

Adenocarcinoma↗
PubMedDate not supplied

Conjugation of p-aminophenyl glycosides with squaric acid diester to a carrier protein and the use of neoglycoprotein in the histochemical detection of lectins.

The coupling of p-aminophenyl 2-acetamido-2-deoxy-3-O-beta-D- galactopyranosyl-beta-D-galactopyranoside (gal-beta 1,3-galNAc) to bovine serum albumin (BSA) was achieved by using 1,2-diethoxycyclobutene-3,4-dione (squaric acid diester) as a new coupling reagent. Two selective consequential steps afforded the desired neoglycoprotein: reaction of the p-aminophenyl group of gal-beta 1,3-galNAc with squaric acid diester gave the corresponding squaric acid amide ester, which was transformed into the BSA conjugate by coupling with the lysyl epsilon-amino group of BSA through formation of a squaric acid 1,2-bisamide. The experimental conditions for the reactions and the optimization of average were performed by using p-anisidine as model substance, the methyl group substituting for the carbohydrate part of a p-aminophenylglycoside. Neoglycoproteins have proven to be valuable tools for lectin detection. To evaluate the properties of this type of probe, the obtained neoglycoprotein with the histochemically crucial T-antigen structure was used for glycocytological and glycohistochemical studies. Three cultured human tumor cell lines and tissue sections from human breast carcinomas were chosen. Its efficiency was similar in comparison to measurements with a probe, derived by diazotization with the p-aminophenyl glycosides of gal-beta 1,3-galNAc and already shown to be a reliable marker for lectin localization in tissue sections and cultured cells.

Antigens, Tumor-Associated, Carbohydrate↗
PubMedDate not supplied

Neoglycoproteins with the synthetic complex biantennary nonasaccharide or its alpha 2,3/alpha 2,6-sialylated derivatives: their preparation, assessment of their ligand properties for purified lectins, for tumor cells in vitro, and in tissue sections, and their biodistribution in tumor-bearing mice.

Neoglycoproteins were prepared with chemoenzymatically synthesized complex biantennary N-glycan derivatives the nonreducing ends of which bear typical sequences found in glycoproteins. A chemically obtained biantennary heptasaccharide-azide was reduced and acylated with a 6-aminohexanoyl spacer. Elongation of the deprotected heptasaccharide using glycosyltransferases yielded a biantennary nonasaccharide with terminal galactose residues and two undecasaccharides terminating with alpha 2,6- or alpha 2,3-linked sialic acid. The free amino group of the spacer of these oligosaccharides was converted into an isothiocyanate. Its subsequent coupling to bovine serum albumin gave neoglycoproteins with a yield of 2.4-3.6 glycan chains per carrier molecule. This versatile synthetic pathway allows employment of a wide variety of complex-type glycans, which can be introduced to various test systems in vitro and in vivo to evaluate potential biomedical applications. Solid-phase assays with biotinylated sugar receptors revealed discriminatory binding properties of the three neoglycoproteins, especially for the mistletoe lectin. This direct assay system is preferable to the measurement of inhibitory capacities with respect to model ligands. Ligand type- and cell type-dependent quantitative differences in the binding properties of the probes were detected by FACScan analyses with a panel of tumor cell lines and by monitoring of staining in tissue sections for small cell and non-small-cell lung cancer and mesotheliomas. Biodistribution of iodinated neoglycoproteins in mice gave a prolonged presence of the sialylated probes in serum. Relative to the nonasaccharide, the uptake, especially of the iodinated neoglycoprotein with alpha 2,3-sialylated ligand chains, was clearly elevated in mice for kidneys and Ehrlich tumors. On the basis of the documented feasibility of these applications, it is concluded that the further elaboration of glycan chain variants by the described synthetic approach in combination with the given test panel is warranted to evaluate the potential of complex glycan chain-carrying neoglycoproteins for diagnostic and therapeutic purposes.

Animals↗
PubMedDate not supplied

Mapping of receptors for carbohydrate constituents of glycoconjugates in well-differentiated and malignant ependymomas: a glycohistochemical study.

Recognitive interactions between the carbohydrate part of cellular glycoconjugates and endogenous receptors supposedly govern important biological processes. Consequently, their elucidation can be of considerable value in tumour diagnosis. The histochemical patterns of expression of endogenous sugar receptors (e.g. endogenous lectin-like proteins) of 10 cases of well-differentiated ependymoma and 10 cases of malignant ependymoma were analysed, using a panel of 18 biotinylated (neo)glycoproteins and a standardized staining protocol. Within this panel, differences in the extent of staining for intracellular sugar receptors in well-differentiated and malignant ependymomas were histochemically detectable. In comparison to the well-differentiated ependymomas, the anaplastic form of the tumour exhibited a generally higher capacity to specifically bind labelled (neo)glycoproteins, containing alpha- or beta-glucosides and a disaccharide, characteristic for one type of beta-galactoside-terminated chain structure of glycoproteins. A significantly reduced binding was seen for tumours of the anaplastic type with labelled markers, carrying histochemically indispensable glucuronic acid residues. These findings suggest that labelled neoglycoproteins are a valuable tool for assessing the endogenous sugar-binding capacity in diagnostic histopathology. Our descriptive analysis of endogenous sugar receptors may also be a rational basis for studies on the functional significance of changes in the expression of their endogenous ligands and the cellular glycoconjugates. Further investigations are also possible on the correlation between the degree of differentiation and expression of both parts of a recognitive system, based on protein (receptor)-carbohydrate (ligand) interactions, in tumours of the central nervous system, especially ependymomas.

Brain Neoplasms↗