Structural characterization of glycoprotein carbohydrate chains by using diagoxigenin-labeled lectins on blots

The carbohydrate structures of blotted glycoproteins can be analyzed by probing them with lectins. Here we describe a method where lectins conjugated with digoxigenin are used in combination with an anti-digoxigenin antibody AP conjugate as a very sensitive detection system for this type of analysis. The specificity of the lectins used, and the sensitivity of the detection system, provide valuable conclusions on the glycan structures. Only small amounts of glycoproteins are required for the analysis. The binding specificity of a set of lectins is demonstrated with various glycoproteins of defined carbohydrate structure. The application of these labeled lectins in combination with specific glycosidases for the characterization of the carbohydrate chains of recombinant tissue plasminogen activator and erythropoietin is presented.     

Characterization of cloned murine cytolytic T cell lines

Murine cytolytic T lymphocytes can be kept in continuous culture apparently indefinitely by repeated passage in a concanavalin A-induced growth promoting medium. Some of these long-term cell lines maintain their cytolytic activity. Starting from three such populations, several cloned cytolytic T cell lines were derived and subsequently subcloned one or more times. Considerable variation in the levels of cytolytic activity was observed between different subclones; some initially active subclones lost activity with prolonged culture. In addition, one of the clones appeared to progressively lose the relative specificity demonstrated during the earlier passages of the parent cell line.     

Ultrastructure of reticulum cells in the bone marrow

In this study the attempt was made to classify the reticulum cells of the bone marrow on the basis of electron-microscopic findings. The basis of the differentiation was the ability of the cells to phagocytize substances or not. For two cell types the intracytoplasmic filaments were used as distinctive marks. The following classification resulted: (a) phagocytic reticulum cells, (b) undifferentiated reticulum cells, (c) fibrous reticulum cells of type I, which contain filaments of 4-8 nm diameter and are located near the blood sinus of the bone marrow, (d) fibrous reticulum cells of type II, which contain intracytoplasmic filaments of 10 nm diameter; since these cells contain neutral fat bodies, the possibility of a reversible conversion to fat cells has to be assumed and (e) fibroblasts, cells which synthesize the substance of the extracellular space. A connection of reticulum cells to haematopoietic functions or to stem cell functions could be found.     

Metabolism of sulfated glycosaminoglycans in cultivated bovine arterial cells. II. Quantitative studies on the uptake of 35SO4-labeled proteoglycans.

Cultured arterial fibroblasts were used for a quantitative study on adsorption, uptake and degradation of [35S]proteoglycans derived from secretions of cultured arterial or skin fibroblasts. The following results were obtained: 1) Proteoglycans added to the culture medium are integrated into the pool of cell membrane-associated (trypsin-removable) glycosaminoglycans by a saturable process, which depends on time and temperature. 2) Up to 17% of the added proteoglycans are taken up by the cells within 24 h. The uptake exhibits saturation kinetics, characteristic for adsorptive pinocytosis. Proteoglycan concentrations required for half-maximum uptake are higher than for half-maximum saturation of the glycosaminoglycan pool associated with the cell membrane. 3) After a lag phase, inorganic 35SO4 appears in the culture medium as a degradation product of the internalized proteoglycans. Pinocytosed proteoglycans are catabolized more rapidly than proteoglycans which remain inside the cell after their biosynthesis. 4) Pinocytosis exhibits specificity, the individual proteoglycans being internalized at different rates. The highest rate of uptake was measured for a dermatan-sulfate-rich proteoglycan. No competition of uptake between a dermatan-sulfate-rich and a heparan-sulfate-rich proteoglycan was observed. 5) Optimum pinocytosis requires an intact protein moiety and, presumably, undegraded carbohydrate chains of the proteoglycans.     

Reaction of a nucleoside 2, 4-dinitrophenyl phosphate with fluoride; a convenient method for the preparation of the nucleoside phosphorfluoridate.

Examination of the reaction of 2, 4-dinitrofluorobenzene with thymidine-5' phosphate in detail reveals that the initial product is the 2, 4-dinitrophenyl ester. This reacts with fluoride to produce thymidine-5' phosphorfluoridate. This second reaction provides the basis for the conversion of preformed thymidine-5' 2, 4-dinitrophenyl phosphate to thymidine-5' phosphorofluoridate.     

G4-quadruplex-binding proteins: review and insights into selectivity

There are over 700,000 putative G4-quadruplexes (G4Qs) in the human genome, found largely in promoter regions, telomeres, and other regions of high regulation. Growing evidence links their presence to functionality in various cellular processes, where cellular proteins interact with them, either stabilizing and/or anchoring upon them, or unwinding them to allow a process to proceed. Interest in understanding and manipulating the plethora of processes regulated by these G4Qs has spawned a new area of small-molecule binder development, with attempts to mimic and block the associated G4-binding protein (G4BP). Despite the growing interest and focus on these G4Qs, there is limited data (in particular, high-resolution structural information), on the nature of these G4Q-G4BP interactions and what makes a G4BP selective to certain G4Qs, if in fact they are at all. This review summarizes the current literature on G4BPs with regards to their interactions with G4Qs, providing groupings for binding mode, drawing conclusions around commonalities and highlighting information on specific interactions where available.     

Mitochondrial targeting sequences may form amphiphilic helices.

Twenty three mitochondrial targeting sequences have been analysed with regard to their potential for forming amphiphilic helices. It is shown that most if not all of these sequences can be expected to form helices with high hydrophobic moments in a suitable environment. In the few cases studied so far, the segments of maximal hydrophobic moment coincide closely with 'critical' regions defined by deletions and point mutations.     

Sequence differences between glycosylated and non-glycosylated Asn-X-Thr/Ser acceptor sites: implications for protein engineering

In N-glycosylated glycoproteins, carbohydrate is attached to Asn in the sequence Asn-X-Ser/Thr, where X denotes any amino acid. However, the presence of this consensus peptide does not always lead to glycosylation. We have compiled an extensive collection of glycosylated and non-glycosylated Asn-X-Thr/Ser sites and present a statistical study based on this data set. Our results indicate that non-glycosylated sites tend to be found more frequently towards the C termini of glycoproteins, and that proline residues in positions X and Y in the consensus Asn-X-Thr/Ser-Y strongly reduce the likelihood of N-linked glycosylation. Beyond this, there are no obvious local sequence features that seem to correlate with the absence or presence of N-linked glycosylation. These findings are discussed in terms of the prediction and engineering of glycosylation sites in secretory proteins.