Biomedical subjects

F Cramer

Publications and source records attributed to F Cramer.

At least 37 records · Page 2Linked to original sources

PubMed1986

Carbohydrate-binding proteins of tumor lines with different growth properties. II. Changes in their pattern in clones of transformed rat fibroblasts of differing metastatic potential.

A tumor model system of clones of myeloproliferative sarcoma virus (MPV)-transformed rat fibroblasts (NRK) with different growth properties and metastatic potential was studied. The relationship between metastatic behavior and composition of carbohydrate-binding proteins (lectins) was analyzed by affinity chromatography. The metastatic variant differs qualitatively from its parental clone in the presence of galactoside-binding proteins at apparent molecular weight of 42 kDa. The alpha-glucosyl-binding proteins at apparent molecular weights of 67 kDa and 53 kDa and a galactoside-binding protein of apparent molecular weight of 34 kDa, however, are not detectable in the metastatic variant in comparison to its parental clone. In this respect the parental clone shows closer resemblance to the clone 5-8#1 with different growth properties and low metastatic potential than to its own metastatic variant. Furthermore, only the parental clone has a melibiose- and a mannan-binding protein of an apparent molecular weight of 64 kDa and 14 kDa, respectively. Rosette formation as model system for intercellular interaction reveals differences in the inhibition pattern with sugar between the two clones 5-8#1 and 5-20#20, whereas the metastatic variant 5-20#20 (s) exhibits drastically reduced capability to form rosettes. Initial experiments demonstrate the feasibility of drug targeting to transformed fibroblasts via carbohydrate-binding proteins.

Animals↗

PubMed1986

Localization of endogenous lectins in normal human breast, benign breast lesions and mammary carcinomas.

Specific antisera against three mammalian beta-galactoside-specific lectins of apparent molecular weights 14.5 kDa, 18 kDa and 29 kDa have been used to localize these lectins in normal breast, and in benign and malignant mammary lesions. In normal breast tissue discrete localization of two lectins (Mrs 14.5 kDa and 18 kDa) was demonstrated in fibroblasts, smooth muscle cells, myoepithelial cells and capillary endothelium. Extracellular localization of one lectin (Mr 14.5 kDa) in collagen was apparent. The third lectin (Mr 29 kDa) labelled preferentially luminal cells and their secretory product. Two benign tumours (an analyzed fibroadenoma and a papilloma) revealed strong staining with two lectins (Mrs 18 kDa and 29 kDa). Of the 24 mammary carcinomas examined, the lectin (Mr 14.5 kDa) was expressed by only occasional tumour cells, the lectin (Mr 18 kDa) occurred in many tumour cells and the lectin (Mr 29 kDa) labelled tumour cells in nearly all cases. The expression of these beta-galactoside-specific endogenous lectins therefore appears to be regulated differently in normal breast compared with mammary tumours.

Breast↗

PubMed1986

Phenylalanyl-tRNA synthetases as an example for comparative and evolutionary aspects of aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases are indispensable components of protein synthesis in all three lines of evolutionary descent, eubacteria, archaebacteria and eukaryotes. Furthermore they are also present in the translational apparatus of the semi-autonomous organelles, mitochondria and chloroplasts, of the eukaryotic cell. Therefore aminoacyl-tRNA synthetases are appropriate objects for comparative molecular biology in order to obtain a comprehensive picture of the evolution of the translational process. The analysis of the phenylalanyl-tRNA synthetase in a large variety of organisms and organelles in this respect is the most advanced. In addition to comparison of quaternary structure, analysis includes functional aspects of accuracy mechanisms (proofreading) and comparison of structural features by means of substrate analogs. Evolutionary relationships are furthermore elucidated using the immunological approach and heterologous aminoacylation.

Amino Acyl-tRNA Synthetases↗

PubMed1986

Pattern of endogenous lectins of a human sarcoma (Ewing's sarcoma) reveals differences to human normal tissues and tumors of epithelial and germ cell origin.

A human sarcoma, Ewing's sarcoma, contains activities of endogenous lectins. Fractionation of salt and detergent extracts by affinity chromatography on columns with immobilized sugars or glycoproteins results in the pattern of endogenous lectins for alpha- and beta-galactosides, alpha-mannosyl- and alpha-fucosyl-moieties. Whereas some lectins are known from normal, non-malignant human tissues or from a human epithelial tumor or a human germ cell tumor, a Ca2+-independent alpha- and beta-galactoside-binding protein at apparent molecular weight of 58 kilodaltons has so far not been characterized from any human source. The patterns for the tumors and human normal tissues reveal various differences in comparison between each other. These differences, documented for the first human sarcoma, human tumors of different histogenetic lineage and normal tissues are a first step to a lectin-based diagnosis and therapy of certain human cancer types.

Apudoma↗

PubMed1986

Isoleucyl-tRNA synthetase from baker's yeast. Influence of substrate concentrations on aminoacylation pathways, discrimination between tRNAIle and tRNAVal, and between isoleucine and valine.

The influence of substrate concentrations on aminoacylation pathways and substrate specificities was investigated in the acylation reaction catalyzed by isoleucyl-tRNA synthetase from yeast. For the cognate substrates isoleucine and tRNAIle two Km values each differing by a factor about five were determined; the higher values were observed at concentrations higher than 1 microM, the lower values below 1 microM isoleucine or tRNAIle, respectively. At substrate concentrations below 1 microM also kcat values of the isoleucylation reaction are lowered. With the noncognate substrates valine and tRNAVal such differences could not be detected. The substrate ATP did not show any change of its Km value as far as the reaction was measurable. Under six different new assay conditions orders of substrate addition and product release followed sixtimes a sequential ordered ter-ter steady-state mechanism with ATP as the first substrate to be added, isoleucine as the second, and tRNAIle as the third one; pyrophosphate is the first product to be released, isoleucyl-tRNA the second, and AMP the third one. In one case this mechanism was modified by a rapid equilibrium segment for addition of ATP and isoleucine. From kcat and Km values and from AMP formation rates discrimination factors for discrimination between tRNAIleII and tRNAValI as well as between isoleucine and valine were determined. In the first case discrimination factors can vary up to a factor of thirty by changes of tRNA or amino-acid concentrations, in the second case discrimination factors are practically invariant. The two different Km values are hypothetically explained by assumption of anticooperativity in a flip-flop mechanism. Two hypothetical catalytic cycles are postulated.

Adenosine Monophosphate↗

PubMed1986

Comparison of endogenous lectins in human embryonic carcinoma and yolk sac carcinoma.

Salt and detergent extracts of xenografted human embryonic carcinoma and a human yolk sac carcinoma were fractionated on different sets of Sepharose columns covalently derivatized with lactose, asialofetuin, melibiose, mannan and fucose. Successive elution by chelating reagent and specific sugar resulted in isolation of endogenous Ca2+-dependent and Ca2+-independent carbohydrate-binding proteins, as analyzed by gel electrophoresis. The salt extracts of the embryonic carcinoma contained a Ca2+-dependent carbohydrate-binding protein at Mr66,000, that was undetectable in yolk sac tumor extracts. Also, a Ca2+-independent fucose-binding protein at Mr62,000 could be purified. In general, the pattern of carbohydrate-binding proteins showed quantitative and qualitative differences as compared to normal tissue. The carbohydrate-binding proteins were assayable as agglutinin and showed no enzymatic activity. They can thus be defined as lectins. Their presence within certain stages of differentiation and developmental regulation may contribute to improvement of the classification, diagnosis and therapy of this tumor class. These new lectins may also be functionally related to the stage-specific embryonic antigens like SSEA-1.

Adult↗

PubMed1986

Differences in the pattern of endogenous lectins from spontaneous rat mammary tumors.

Three spontaneously occurring rat mammary tumors, a fibroadenoma and two tubulopapillary adenocarcinomas, differing in their degree of histologic differentiation and malignancy, were investigated. They revealed significant differences in the pattern of carbohydrate-binding proteins with specificities for alpha- and beta-galactosyl, alpha-mannosyl, and alpha-fucosyl moieties, respectively. Analysis was performed by affinity chromatography on resins with immobilized sugars or glycoproteins and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The patterns were divided into categories according to the dependence of binding activity on the presence of Ca2+ and to dependence on extraction conditions. In comparison with both adenocarcinomas, the fibroadenoma contained alpha- and beta-galactoside-binding proteins at apparent molecular weights of 74,000 and 130,000. These proteins were not found in either adenocarcinoma. Comparison between the two adenocarcinomas revealed qualitative differences for all classes of carbohydrate-binding proteins under investigation. These differences between tumors of the same tumor class (mammary tumors) and tumor type (tubulopapillary adenocarcinoma) allow for the proposal that the pattern of endogenous carbohydrate-binding proteins may be potentially useful as a diagnostic marker.

Adenocarcinoma↗

PubMed1985

Isoleucyl-tRNA synthetase from bakers' yeast: multistep proofreading in discrimination between isoleucine and valine with modulated accuracy, a scheme for molecular recognition by energy dissipation.

For discrimination between isoleucine and valine by isoleucyl-tRNA synthetase from yeast, a multistep sequence is established. The initial discrimination of the substrates is followed by a pretransfer and a posttransfer hydrolytic proofreading process. The overall discrimination factor D was determined from kcat and Km values observed in aminoacylation of tRNAIle-C-C-A with isoleucine and valine. From aminoacylation of the modified tRNA species tRNAIle-C-C-3'dA and tRNAIle-C-C-A (3'NH2), the initial discrimination factor I (valid for the reversible substrate binding) and the proofreading factor P1 (valid for the aminoacyl adenylate formation) could be determined. Factor I was computed from ATP consumption and D1, the overall discrimination factor for this partial reaction which can be obtained from kinetic constants, and P1 was calculated from AMP formation rates. Proofreading factor P2 (valid for aminoacyl transfer reaction) was determined from AMP formation rates observed in aminoacylation of tRNAIle-C-C-A and tRNAIle-C-C-3'dA. From the initial discrimination factor I and the AMP formation rates, discrimination factor DAMP in aminoacylation of tRNAIle-C-C-A can be calculated. These values deviate by a factor II from factor D obtained by kinetics which may be due to the fact that for acylation of tRNAIle-C-C-A an initial discrimination factor I' = III is valid. The observed overall discrimination varies up to a factor of 16 according to conditions. Under optimal conditions, 38 000 correct aminoacyl-tRNAs are produced per 1 error while the energy of 5.5 ATPs is dissipated. With the determined energetic and molecular flows for the various steps of the enzymatic reaction, a coherent picture of this new type of "far away from equilibrium enzyme" emerges.

Amino Acyl-tRNA Synthetases↗

PubMed1985

Evolutionary aspects of accuracy of phenylalanyl-tRNA synthetase. Accuracy of the cytoplasmic and chloroplastic enzymes of a higher plant (Phaseolus vulgaris).

The phenylalanyl-tRNA synthetases from cytoplasm and chloroplasts of bean (Phaseolus vulgaris) leaves employ different strategies with respect to accuracy. The chloroplastic enzyme that is coded for by the nuclear genome follows the pathway of posttransfer proofreading, also characteristic for enzymes from eubacteria and cytoplasm and mitochondria of lower eukaryotic organisms. In contrast, the cytoplasmic enzyme uses pretransfer proofreading in the case of noncognate natural amino acids, characteristic for higher eukaryotic organisms and archaebacteria. Dependent on the nature of the noncognate amino acid, pretransfer proofreading in this case occurs without tRNA stimulation or with tRNA stimulated with no or little effect of the nonaccepting 3'-OH group of the terminal adenosine. The fundamental mechanistic difference in proofreading between the heterotopic intracellular isoenzymes of the plant cell supports the idea of the origin of the chloroplastic gene by gene transfer from a eubacterial endosymbiont to the nucleus. Origin by duplication of the nuclear gene, as indicated for mitochondrial phenylalanyl-tRNA synthetases [Gabius, H.-J., Engelhardt, R., Schroeder, F.R., & Cramer, F. (1983) Biochemistry 22, 5306-5315], appears unlikely. Further analyses of the ATP/PPi pyrophosphate exchange and aminoacylation of tRNAPhe-C-C-A(3'NH2), using 11 phenylalanine analogues, reveal intraspecies and interspecies variability of the architecture of the amino acid binding part within the active site.

Amino Acyl-tRNA Synthetases↗

PubMed1985

Archaebacterial phenylalanyl-tRNA synthetase. Accuracy of the phenylalanyl-tRNA synthetase from the archaebacterium Methanosarcina barkeri, Zn(II)-dependent synthesis of diadenosine 5',5'''-P1,P4-tetraphosphate, and immunological relationship of OFFnylalanyl-tRNA synthetases from different urkingdoms.

Phenylalanyl-tRNA synthetase from the archaebacterium Methanosarcina barkeri activates a number of phenylalanine analogues (methionine, p-fluorophenylalanine, beta-phenylserine, beta-thien-2-ylalanine, 2-amino-4-methylhex-4-enoic acid and ochratoxin A) in the absence of tRNA, as demonstrated by Km and kcat of the ATP/PPi exchange reaction. Upon complexation with tRNA, AMP formation from the enzyme X tRNA complex in the presence of ATP, one of the above analogues or tyrosine, leucine, mimosine, N-benzyl-L- or N-benzyl-D-phenylalanine indicates activation of the analogues under conditions of aminoacylation. Natural noncognate amino acids are not transferred to tRNAPhe-C-C-A or tRNAPhe-C-C-A-(3'-NH2). This pretransfer proofreading mechanism, together with the comparatively low ratio of synthetic to successive hydrolytic steps, resembles the mechanism of liver enzymes of vertebrates. In contrast, eubacterial phenylalanyl-tRNA synthetases achieve the necessary fidelity by post-transfer proofreading, a corrective hydrolytic event after transfer to tRNAPhe. Diadenosine 5',5'''-P1,P4-tetraphosphate synthesis is shown to be a common feature for phenylalanyl-tRNA synthetases from all three lineages of descent. The immunological approach demonstrates that aminoacyl-tRNA synthetases do not belong to the group of enzymes in gene expression with high structural conservation.

Adenine Nucleotides↗

PubMed1985

Carbohydrate-binding proteins of tumor lines with different growth properties. I. Differences in their pattern for three clones of rat fibroblasts transformed with a myeloproliferative sarcoma virus.

Three clones of myeloproliferative virus (MPV)-transformed rat fibroblasts (NRK) with different growth properties and morphology were transplanted to athymic nude mice. Presence of carbohydrate-binding proteins was inferred by fluorescence microscopy using fluorescent, glycosylated markers. Salt and detergent extracts of tumors from this model system were fractionated under identical conditions on different sets of Sepharose columns, to which lactose, asialofetuin, melibiose, mannan and fucose had been covalently linked. Successive elution by chelating reagent and specific sugar resulted in isolation of the different Ca2+ -dependent and Ca2+ -independent endogenous carbohydrate-binding proteins that were assayable as agglutinins. In comparison, the different tumors displayed a pattern with qualitative and quantitative alterations. Since protein-carbohydrate interaction mediated by carbohydrate-binding proteins (lectins) is of importance for cognitive processes, it is remarkable that the pattern of membrane glycoproteins, isolated by affinity chromatography on resins with immobilized plant lectins, had also been found to reveal certain individual properties for receptors specific for peanut agglutinin (PNA) and Ulex europaeus agglutinin (UEA). These demonstrated differences within the system of protein-carbohydrate interaction suggest that endogenous lectins and their ligands have potential significance as markers defining a certain phenotype within this tumor model system.

Animals↗

PubMed1985

Endogenous tumor lectins: a new class of tumor markers and targets for therapy?

Endogenous lectins of normal tissues can play a functional role in recognition processes and cell adhesion. These functions are areas of particular relevance to tumor growth and metastasis. Our initial results on endogenous lectins of different tumors lead to the working hypothesis that the pattern of endogenous lectins is qualitatively and quantitatively different between different types of tumors and between tumors and normal, nonmalignant tissues. The endogenous lectins may thus prove to be potentially important in establishing a new concept for a rational lectin-based type of diagnosis and therapy of various tumors.

Animals↗

PubMed1985

Pattern of endogenous lectins in a human epithelial tumor.

Salt and detergent extracts of a malignant epithelial tumor, obtained by extraction of acetone powder, were fractionated on different sets of Sepharose columns covalently derivatized with lactose, asialofetuin, melibiose, mannan, fucose, and heparin. Successive elution by chelating reagent and specific sugar resulted in isolation of different Ca2+-dependent and Ca2+-independent endogenous carbohydrate-binding proteins, as analyzed by gel electrophoresis. It appears from the analysis that certain bands represent newly identified proteins capable of binding to lactose (at Mr 64,000), melibiose (at Mr 28,000), and fucose (at Mr 62,000 and 70,000). Other carbohydrate-binding proteins isolated from this human tumor have been identified in normal, especially embryonic, tissues of different nonhuman vertebrates. The carbohydrate-binding proteins are assayable as agglutinin with rabbit erythrocytes and show no detectable enzymatic activity. They can thus be defined as lectins. The presence of a complex pattern of endogenous lectins and their biochemical characteristics may contribute to an understanding of intercellular interaction during the complex process of metastatic spread and may furthermore allow a new tool for diagnosis and a lectin-based therapy.

Apudoma↗

PubMed1985

Cell surface lectins of transplantable human teratocarcinoma cells: purification of a new mannan-specific endogenous lectin.

Fractionation of detergent extracts of transplanted tumors of human teratocarcinoma cells by affinity chromatography yields one predominant protein with apparent molecular weight of 14,000 and further, for less abundant protein with apparent molecular weight of 35,000 from lactose-sepharose and one protein with apparent molecular weight of 68,000 from mannan-sepharose. No further carbohydrate-binding protein can be isolated on columns derivatized with asialofetuin, melibiose and L-fucose, to which the extract is applied successively. Both proteins agglutinate trypsinized, glutaraldehyde-fixed rabbit erythrocytes in the absence of Ca2+ and can thus be defined as endogenous human teratocarcinoma lectins. Inhibition of heterotypic and homotypic aggregation of human teratocarcinoma cells by D-mannose, D-galactose and glycoproteins rich in one of these sugars is consistent with a functional role of these Ca2+-independent lectins in cell aggregation. Visualization of these activities by fluorescent mannosylated and lactosylated markers on the cell surface further supports the cell surface localization of these detergent extractable lectins. The mannan-specific lectin, in particular, has so far not been detected in any mammalian tissue or tumor and is of potential value for a lectin-based diagnosis and therapy of embryonal carcinomas.

Carbohydrates↗

PubMed1984

Synthesis and reactions of a nucleoside derivative of phosphoric sulfonic anhydride. Studies related to the mechanisms of coupling reactions in the chemical synthesis of oligodeoxyribonucleotides by phosphotriester procedures.

The synthesis of a model compound, diphenylphosphoric toluene-p-sulfonic anhydride, an arylsubstituted phosphoric sulfonic mixed anhydride, is described. Using the same procedure a thymidyl substituted derivative was prepared. The phosphoric sulfonic anhydride is the presumed intermediate in oligonucleotide coupling reactions involving phosphodiester activation by arenesulfonyl derivatives. This mixed anhydride reacts with a variety of nucleophiles. It can be converted to phophotriester derivatives in the presence of simple alcohols. Phosphotriester formation using the 5'-hydroxyl of a thymidine derivative requires additionally a catalyst such as N-methylimidazole. The reactive intermediate produced upon the addition of N-methylimidazole to the phosphoric sulfonic anhydride has been observed spectroscopically using 31P-NMR.

Chemical Phenomena↗

PubMed1984

Preparation of oligonucleotides corresponding to the acceptor stem of yeast tRNAPhe and their interaction with yeast ATP(CTP):tRNA nucleotidyltransferase.

Seven oligonucleotides corresponding to the 3' and 5' sequences of the acceptor stem of yeast tRNAPhe have been prepared by chemical synthesis, chemical-enzymatic synthesis or by isolation from tRNA hydrolysates. The oligonucleotides have been examined as substrates for phosphodiester bond synthesis in the presence of ATP as catalysed by yeast ATP (CTP): tRNA nucleotidyltransferase. Oligonucleotides which correspond to the sequence of the 3'-strand of the tRNA acceptor stem and possess no secondary structure exhibit little or no activity with the enzyme. The ability of the enzyme to catalyse the synthesis of a phosphodiester linkage using ATP and an oligonucleotide corresponding to the 3'-strand of the acceptor stem is in general dramatically increased when an oligonucleotide corresponding to the sequence of the 5'-strand of tRNA acceptor stem is present. In cases where significant activity was observed kinetic parameters have been determined.

Adenosine Monophosphate↗

PubMed1984

Phenylalanyl-tRNA synthetase from the archaebacterium Methanosarcina barkeri.

Phenylalanyl-tRNA synthetase from the archaebacterium Methanosarcina barkeri was purified 1620-fold with 24% overall yield. It appears to be a tetrameric enzyme with a molecular mass of 270 kDa, as determined by gel filtration, with a subunit structure of alpha 2 beta 2 (alpha = 63 kDa, beta = 70 kDa), as determined by sodium dodecyl sulfate gel electrophoresis. No conservation of common antigenic determinants is noted with polyclonal antibodies raised against the enzymes of Escherichia coli, yeast, and hen liver. Heterologous aminoacylation of tRNA with high selectivity for archaebacterial tRNA and substrate properties of ATP analogues reveals a unique pattern, reflecting the supposed genealogical difference between the urkingdoms of archaebacteria, eubacteria, and eukaryotes.

Adenosine Triphosphate↗

PubMed1984

Purification and properties of phenylalanyl-tRNA synthetase from a higher plant (Phaseolus vulgaris).

Phenylalanyl-tRNA synthetase from beans (Phaseolus vulgaris) was purified 2 800-fold to homogeneity with a 16% overall yield by salting-out chromatography, salting-out affinity chromatography, gel filtration and chromatography on DEAE-cellulose and hydroxylapatite. This combination minimizes potentially harmful effects of proteinases and products of the secondary metabolism of a green plant during the early steps. The molecular mass is 260 000 Da with a subunit structure of alpha 2 beta 2 (alpha = 59 000, beta = 70 000 Da). Enzymatic activity was optimal with 20mM Mg2+ and 10mM KCl at pH 6.5 and pH 8.5, depending on the buffer substance. Kinetic measurements at low temperature and steady-state kinetics indicate that the esterification of tRNA or a step preceding it, but not the activation, are rate-determining at pH 7.65. The cognate tRNAPhe is exclusively aminoacylated at the 2'-OH group. tRNAs from Escherichia coli and bean chloroplasts are not aminoacylated. No immunological relationship of the plant enzyme to other phenylalanyl-tRNA synthetases was revealed by immuno-diffusion and immunotitration with polyclonal antibodies raised against the enzymes from E. coli, yeast and hen liver. ATP analogs revealed a unique pattern of substrate properties with indication of conservation of ATP binding in the form of an ATP-Mg2+ complex in the anti-conformation with a coordination of the cation to the nitrogen in position 7 of the purine moiety.

Amino Acyl-tRNA Synthetases↗