Temperature-induced ultraviolet difference absorption spectrometry for determination of enthalpy changes Binding of 4-methylumbelliferyl glycosides to four lectins

AbstractRaising the temperature in a single mixture of a lectin and a chromophoric glycoside allows determination of the binding enthalpy. This is made possible by continuously monitoring the displacement of the complex from its equilibrium concentration with a sensitive difference absorption spectrophotometer. The method is illustrated with the following lectins: concanavalin A, soybean agglutinin, peanut agglutinin and Erythrina cristagalli agglutinin. The ligands are 4-methylumbelliferyl glycosides. The binding enthalpies found range from −60 kJ · mol−1 for the Galß1 → 3GalNAc-ßglycoside and peanut agglutinin to −30 kJ · mol−1 for a monosaccharide glycoside and the other lectins

TBX2 is a neuroblastoma core regulatory circuitry component enhancing MYCN/FOXM1 reactivation of DREAM targets

Chromosome 17q gains are almost invariably present in high-risk neuroblastoma cases. Here, we perform an integrative epigenomics search for dosage-sensitive transcription factors on 17q marked by H3K27ac defined super-enhancers and identify TBX2 as top candidate gene. We show that TBX2 is a constituent of the recently established core regulatory circuitry in neuroblastoma with features of a cell identity transcription factor, driving proliferation through activation of p21-DREAM repressed FOXM1 target genes. Combined MYCN/TBX2 knockdown enforces cell growth arrest suggesting that TBX2 enhances MYCN sustained activation of FOXM1 targets. Targeting transcriptional addiction by combined CDK7 and BET bromodomain inhibition shows synergistic effects on cell viability with strong repressive effects on CRC gene expression and p53 pathway response as well as several genes implicated in transcriptional regulation. In conclusion, we provide insight into the role of the TBX2 CRC gene in transcriptional dependency of neuroblastoma cells warranting clinical trials using BET and CDK7 inhibitors

RRM2 enhances MYCN-driven neuroblastoma formation and acts as a synergistic target with CHK1 inhibition

High-risk neuroblastoma, a pediatric tumor originating from the sympathetic nervous system, has a low mutation load but highly recurrent somatic DNA copy number variants. Previously, segmental gains and/or amplifications allowed identification of drivers for neuroblastoma development. Using this approach, combined with gene dosage impact on expression and survival, we identified ribonucleotide reductase subunit M2 (RRM2) as a candidate dependency factor further supported by growth inhibition upon in vitro knockdown and accelerated tumor formation in a neuroblastoma zebrafish model coexpressing human RRM2 with MYCN. Forced RRM2 induction alleviates excessive replicative stress induced by CHK1 inhibition, while high RRM2 expression in human neuroblastomas correlates with high CHK1 activity. MYCN-driven zebrafish tumors with RRM2 co-overexpression exhibit differentially expressed DNA repair genes in keeping with enhanced ATR-CHK1 signaling activity. In vitro, RRM2 inhibition enhances intrinsic replication stress checkpoint addiction. Last, combinatorial RRM2-CHK1 inhibition acts synergistic in high-risk neuroblastoma cell lines and patient-derived xenograft models, illustrating the therapeutic potential

Base-sequence specificity of Hoechst 33258 and DAPI binding to five (A/T)(4) DNA sites with kinetic evidence for more than one high-affinity Hoechst 33258-AATT complex.

A

Some physicochemical aspects of oligosaccharide binding to concanavalin A and wheat germ agglutinin

The binding of fluorescently labelled carbohydrates to concanavalin A and wheat germ agglutinin was studied at equilibrium and by the stopped-flow and temperature jump relaxation methods. Ligand were mainly die 4-methylumbelliferyl glycosides of alpha (1 → 2)-linked mannooligosaccharides and of beta (1 → 4)-linked chitooligosaccharides as limited homologous series. They offer distinct advantages, parti cularly for kinetic studies. Enthalpie and kinetic considerations suggest that concanavalin A specifically binds a single mannopyranosyl group in alpha (1 →2)-linked mannooligosaccharides. This occurs preferentially at the non-reducing end. Glycosylation of a carbohydrate with e.g. an aryl group does not afect die binding kinetics and for all carbohydrates the association rate is comparable but relatively slow, which indicates that a common process is involved in the binding of all carbohydrates to concanavalin A. The affinity of a carbohydrate for concanavalin A is determined by the dissociation-rate parameter, resulting in a longer residence time for a better ligand. Interaction of chitooligosaccharides with wheat germ agglutinin is complex. With the larger members of the 4-methylumbelliferyl chitooligosaccharides, binding studies were only possible at low fractional saturation to avoid formation of unsoluble complexes. The binding kinetics of wheat germ agglutinin are faster than with concanavalin A and are consistent with a wheat germ agglutinin binding region composed of two adjacent subsites. For binding of the monoside as well as the bioside, two consistent kinetic models apply. They have common that for each ligand there exist two complexes with comparable population

Kinetics of binding of hoechst dyes to DNA studied by stopped-flow fluorescence techniques.

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Binding of manno-oligosaccharides to concanavalin A: substitution titration with a fluorescent-indicator ligand

The association constants for binding of methyl alpha-D-mannopyranoside (I), mannobiose (II) and mannotriose (III) to concanavalin A were determined in the temperature range 285-313 °K by a substitution titration, using 4-methylumbelliferyl alpha-D-mannopyranoside as a carbohydrate-specific and fluorescent indicator. All binding equilibria are simple, but establish extremely slowly with II and III. At 298.3 °K, K increases moderately from I to III: (6.4 +/- 0.5) x 10(3), (1.2 +/- 0.1) x 10(4) and (1.10 +/- 0.05) x 10(5) M-1. For binding of I, II and III, the - delta H degree values are constant (36 +/- 2 kJ mol-1) and equal to the average value (36.1 +/- 0.6 kJ mol-1) obtained for the three corresponding 4-methylumbelliferyl alpha-D-mannooligosaccharides [Van Landschoot, A., Loontiens, F. G., and De Bruyne, C. K (1978) Eur. J. Biochem. 83, 277-285]. The data are interpreted as arising from specific binding to a single mannopyranosyl residue in (alpha 1 leads to 2)-linked mannooligosaccharides