Polyelectrolyte effects on 9-aminoacridine-DNA binding

The 9-aminoacridine-DNA binding curve is analyzed in two ways: with polyelectrolyte effects neglected and with polyelectrolyte effects included. It is found that the analysis which includes polyelectrolyte effects is consistent with the violation of neighbor exclusion displayed by diacridine complexes as observed by Atwell et al. and by Zimmerman and coworkers. However the analysis which neglects polyelectrolyte effects is inconsistent with the diacridine results. This comparison supports the necessity of including polyelectrolyte effects in the analysis of drug-DNA binding curves.     

Binding of CDK9 to TRAF2

CDK9 has been recently shown to have increased kinase activity in differentiated cells in culture and a differentiated tissue-specific expression in the developing mouse. In order to identify factors that contribute to CDK9's differentiation-specific function, we screened a mouse embryonic library in the yeast two-hybrid system and found a tumor necrosis factor signal transducer, TRAF2, to be an interacting protein. CDK9 interacts with a conserved domain in the TRAF-C region of TRAF2, a motif that is known to bind other kinases involved in TRAF-mediated signaling. Endogenous interaction between the two proteins appears to be specific to differentiated tissue. TRAF2-mediated signaling may incorporate additional kinases to signal cell survival in myotubes, a cell type that is severely affected in TRAF2 knockout mice. J. Cell. Biochem. 71:467–478, 1998. © 1998 Wiley-Liss, Inc.     

Effects of Radioiodination on Hormone Binding Ability

Abstract

This paper presents the effects of ¹²⁵I labelling on human chorionic gonadotropin binding ability. The results obtained showed a marked difference in the half-life of the specific radioactivity of the radiolabelled hormone comparing the one obtained by “self-displacement” analysis and that calculated considering the half-life of the radioactive isotope (60 days for ¹²⁵I). The decrease in the hormone half-life (t1/2= 3.10 days) was demonstrated by binding experiments performed at various days after hormone labelling. The affinity constant (Ka) and the number of binding sites obtained after Scatchard analysis using rat testes as receptor source, remained constant when the “self-displacement” specific radioactivity was used while they varied significantly if the 60 days half-life was considered for the data processing. The importance of the labelled hormone degradation as a possible cause of the decrease in the binding ability is also described.     

Mapping of the Complement C9 Binding Region on Clonorchis sinensis Paramyosin

Heliminthic paramyosin is a multifunctional protein that not only acts as a structural protein in muscle layers but as an immune-modulatory molecule interacting with the host immune system. Previously, we found that paramyosin from Clonorchis sinensis (CsPmy) is bound to human complement C9 protein (C9). To analyze the C9 binding region on CsPmy, overlapping recombinant fragments of CsPmy were produced and their binding activity to human C9 was investigated. The fragmental expression of CsPmy and C9 binding assays revealed that the C9 binding region was located at the C-terminus of CsPmy. Further analysis of the C-terminus of CsPmy to narrow the C9 binding region on CsPmy indicated that the region flanking ⁷³¹Leu–⁷⁸⁰Leu was a potent C9 binding region. The CsPmy fragments corresponding to the region effectively inhibited human C9 polymerization. These results provide a precise molecular basis for CsPmy as a potent immunomodulator to evade host immune defenses by inhibiting complement attack.     

Ultrastructural Effects of Lactoferrin Binding on Giardia lamblia Trophozoites

ABSTRACT. Lactoferrin and its derived N-terminal peptide may be important host defenses against Giardia lamblia . We showed earlier that lactoferrin and the derived peptides have potent giardicidal activity in vitro. Using indirect immunofluorescence, we now demonstrate binding of lactoferrin and the peptides to the live trophozoite surface. Iron strongly inhibited binding of lactoferrin, and decreased binding of the peptides, while certain divalent metal ions decreased binding of all forms by about half. Lactoferrin and the peptedes caused striking and complex morphologic changes in the trophozoite plasmalemma, endomembranes and cytoskeleton, and increases the electron density of the lysosome-like peripheral vacuoles.     

Effects of N-Acetylimidazole on Oxytocin Binding in Bovine Mammary Tissue

Abstract

The effects of N-acetylimidazole on specific binding of oxytocin to microsomal fractions of bovine mammary gland were studied. N-acetylimidazole suppressed oxytocin binding, with time and concentration dependence. Decreased oxytocin binding activity appeared to be due to decreased affinity of the hormone for its receptor. Acetylation of oxytocin, rather than of oxytocin receptors, seemed to be responsible for the decreased binding.     

Effects of Chilling Temperatures on Ethylene Binding by Banana Fruit

Banana fruit are highly susceptible to chilling injury during low temperature storage. Experiments were conducted to compare ethylene binding during storage at chilling (3 and 8 °C) versus optimum (13 °C) temperatures. The skins of fruit stored at 3 and 8 °C gradually darkened as storage duration increased. This chilling effect was reflected in increasing membrane permeability as shown by increased relative electrolyte leakage from skin tissue. In contrast, banana fruit stored for 8 days at 13 °C showed no chilling injury symptoms. Exposure of banana fruit to the ethylene binding inhibitor 1-methylcyclopropene (1 l l^-1 1-MCP) prevented ripening. However, this treatment also enhanced the chilling injury accelerated the occurrence of chilling injury-associated increased membrane permeability. ¹⁴C-ethylene release assay showed that ethylene binding by banana fruit stored at low temperature decreased with reduced storage temperature and/or prolonged storage time. Fruit exposed to 1-MCP for 12 h and then stored at 3 or 8 °C exhibited lower ethylene binding than those stored at 13 °C. Thus, chilling injury of banana fruit stored at low temperature is associated with a decrease in ethylene binding. The ability of tissue to respond to ethylene is evidently reduced, thereby resulting in failure to ripen.     

Intramolecular Binding of the Rad9 C Terminus in the Checkpoint Clamp Rad9-Hus1-Rad1 Is Closely Linked with Its DNA Binding

The human checkpoint clamp Rad9-Hus1-Rad1 (9-1-1) is loaded onto chromatin by its loader complex, Rad17-RFC, following DNA damage. The 120-amino acid (aa) stretch of the Rad9 C terminus (C-tail) is unstructured and projects from the core ring structure (CRS). Recent studies showed that 9-1-1 and CRS bind DNA independently of Rad17-RFC. The DNA-binding affinity of mutant 9^ΔC-1-1, which lacked the Rad9 C-tail, was much higher than that of wild-type 9-1-1, suggesting that 9-1-1 has intrinsic DNA binding activity that manifests in the absence of the C-tail. C-tail added in trans interacted with CRS and prevented it from binding to DNA. We narrowed down the amino acid sequence in the C-tail necessary for CRS binding to a 15-aa stretch harboring two conserved consecutive phenylalanine residues. We prepared 9-1-1 mutants containing the variant C-tail deficient for CRS binding, and we demonstrated that the mutant form restored DNA binding as efficiently as 9^ΔC-1-1. Furthermore, we mapped the sequence necessary for TopBP1 binding within the same 15-aa stretch, demonstrating that TopBP1 and CRS share the same binding region in the C-tail. Indeed, we observed their competitive binding to the C-tail with purified proteins. The importance of interaction between 9-1-1 and TopBP1 for DNA damage signaling suggests that the competitive interactions of TopBP1 and CRS with the C-tail will be crucial for the activation mechanism.     

Polyelectrolyte and rheological studies on the polysaccharide welan

This paper investigates the polyelectrolyte behaviour of welan in aqueous solution. From conductivity and activity measurements it is demonstrated that a double-helical conformation is adopted irrespective of pH and ionic strength. The intrinsic pK of its carboxylic groups is found to be abnormally low (pK₀ = 2.2) and no conformational transition was observed during neutralization. The viscometric behaviour was studied as a function of degree of neutralization, polymer concentration, shear rate and ionic strength. The linear relation [η]=f(C_s^−1/2) allows the determination of B, the stiffness parameter from Smidsrod, whose value characterizes a very stiff molecule.     

Binding affinities of CRBPI and CRBPII for 9-cis-retinoids

Background

Cellular retinol binding-protein I (CRBPI) and cellular retinol binding-protein II (CRBPII) serve as intracellular retinoid chaperones that bind retinol and retinal with high affinity and facilitate substrate delivery to select enzymes that catalyze retinoic acid (RA) and retinyl ester biosynthesis. Recently, 9-cis-RA has been identified in vivo in the pancreas, where it contributes to regulating glucose-stimulated insulin secretion. In vitro, 9-cis-RA activates RXR (retinoid × receptors), which serve as therapeutic targets for treating cancer and metabolic diseases. Binding affinities and structure–function relationships have been well characterized for CRBPI and CRBPII with all-trans-retinoids, but not for 9-cis-retinoids. This study extended current knowledge by establishing binding affinities for CRBPI and CRBPII with 9-cis-retinoids.

Methods

We have determined apparent dissociation constants, K′_d, through monitoring binding of 9-cis-retinol, 9-cis-retinal, and 9-cis-RA with CRBPI and CRBPII by fluorescence spectroscopy, and analyzing the data with non-linear regression. We compared these data to the data we obtained for all-trans- and 13-cis-retinoids under identical conditions.

Results

CRBPI and CRBPII, respectively, bind 9-cis-retinol (K′_d, 11 nM and 68 nM) and 9-cis-retinal (K′_d, 8 nM and 5 nM) with high affinity. No significant 9-cis-RA binding was observed with CRBPI or CRBPII.

Conclusions

CRBPI and CRBPII bind 9-cis-retinol and 9-cis-retinal with high affinities, albeit with affinities somewhat lower than for all-trans-retinol and all-trans-retinal.

General significance

These data provide further insight into structure–binding relationships of cellular retinol binding-proteins and are consistent with a model of 9-cis-RA biosynthesis that involves chaperoned delivery of 9-cis-retinoids to enzymes that recognize retinoid binding-proteins.     

Effects of Chronic Nicotine Infusion on Kinetics of High-Affinity Nicotine Binding

Abstract: It is well established that chronic nicotine treatment produces a dose-dependent increase in high-affinity l -[³H]nicotine binding. This increase may be due to chronic desensitization of the receptor. Sophisticated kinetic analyses of high-affinity nicotine binding to rat brain have demonstrated that the association rate is biphasic; the fast phase may represent binding to a high-affinity predesensitized state and the slow phase may represent binding to a lower affinity ground state that then isomerizes to form the high-affinity binding site. This isomerization presumably leads to receptor desensitization. The studies reported here assessed whether binding to mouse brain nicotinic receptors shows these same properties and whether chronic intravenous infusion of nicotine results in changes in these kinetic properties. The results obtained indicate that mouse brain nicotine binding also shows biphasic association kinetics and uniphasic dissociation kinetics, which supports the assertion that the receptor exists in two interconvertible states. However, unlike other results obtained with rat brain, the rate of the slow association process did not change with ligand concentration. Chronic infusion resulted in a dose-dependent increase in l -[³H]-nicotine binding, but the ratio of fast/slow phases of binding was not changed by these treatments. These results suggest that chronic infusion does not alter measurably the kinetics of nicotinic receptor binding when measured in vitro.     

Effects of Enucleation on High-Affinity Binding Sites in Chick Optic Tecta

Abstract: The effect of unilateral eye extirpation on the development of the chick optic tectum has been studied in both the embryo and the newly hatched chick. Although the prevention of normal afferentation of the embryonic tectum retarded its growth, there appeared to be a significant increase of muscarinic acetylcholine binding site in the noninnervated tectum. This phenomenon was repeated also in the posthatch denervated system wherein the functioning optic nerve is severed. A significant increase in the number of binding sites as well as reduced dissociation constant of the interactions of this receptor with [³H]quinuclindinyl benzilate was found in the deafferented optic tectum. This may suggest the presence of a denervation-supersensitivity-like modulation. Similar increases were not detected with other binding sites studied in either the noninnervated embryonic or deafferented posthatch optic lobes. The possibility that acetylcholine is a primary neurotransmitter of the optic system is discussed.     

Predicting RNA-Metal Ion Binding with Ion Dehydration Effects

Metal ions play essential roles in nucleic acids folding and stability. The interaction between metal ions and nucleic acids can be highly complicated because of the interplay between various effects such as ion correlation, fluctuation, and dehydration. These effects may be particularly important for multivalent ions such as Mg²⁺ ions. Previous efforts to model ion correlation and fluctuation effects led to the development of the Monte Carlo tightly bound ion model. Here, by incorporating ion hydration/dehydration effects into the Monte Carlo tightly bound ion model, we develop a, to our knowledge, new approach to predict ion binding. The new model enables predictions for not only the number of bound ions but also the three-dimensional spatial distribution of the bound ions. Furthermore, the new model reveals several intriguing features for the bound ions such as the mutual enhancement/inhibition in ion binding between the fully hydrated (diffuse) ions, the outer-shell dehydrated ions, and the inner-shell dehydrated ions and novel features for the monovalent-divalent ion interplay due to the hydration effect.     

Polyelectrolyte Nature of Bacterial Teichoic Acids

Several physicochemical properties of the teichoic acid of Bacillus subtilis 168 have been determined. The teichoic acid partial specific volume was found to be 0.57 ml/g. The apparent weight-average molecular weight of the polymer was 24,800. Sedimentation was strongly dependent on solvent. The sedimentation coefficient of the teichoic acid was found to have a value of s(20.w) (0) = 1.90S. In dilute buffers and distilled water, the teichoic acid possessed a rigid rod or extended conformation. Salts induced a loss of secondary structure in the polymer, resulting in a random coil configuration. Salt-induced structural changes in the teichoic acid were determined by viscosities, ultraviolet difference spectra, and inhibition of precipitation with concanavalin A. Divalent cations such as Mg(2+) had little effect on the teichoic acid structure. The salt-induced structural changes were reversible, as evidenced by return of the original properties upon dialysis of the teichoic acid against water. Sodium chloride inhibited the adsorption of bacteriophage ?25 to B. subtilis cell walls. Teichoic acid conformation may have a significant influence on the physiology of bacteria.     

Multiscale Simulations Examining Glycan Shield Effects on Drug Binding to Influenza Neuraminidase

Influenza neuraminidase is an important drug target. Glycans are present on neuraminidase and are generally considered to inhibit antibody binding via their glycan shield. In this work, we studied the effect of glycans on the binding kinetics of antiviral drugs to the influenza neuraminidase. We created all-atom in silico systems of influenza neuraminidase with experimentally derived glycoprofiles consisting of four systems with different glycan conformations and one system without glycans. Using Brownian dynamics simulations, we observe a two- to eightfold decrease in the rate of ligand binding to the primary binding site of neuraminidase due to the presence of glycans. These glycans are capable of covering much of the surface area of neuraminidase, and the ligand binding inhibition is derived from glycans sterically occluding the primary binding site on a neighboring monomer. Our work also indicates that drugs preferentially bind to the primary binding site (i.e., the active site) over the secondary binding site, and we propose a binding mechanism illustrating this. These results help illuminate the complex interplay between glycans and ligand binding on the influenza membrane protein neuraminidase.