Teaching photosensitizers a new trick: red light-triggered G-quadruplex alkylation by ligand co-localization.

We propose a bimolecular approach for G-quadruplex alkylation, using a pro-reactive furan-containing ligand, activated by red-light irradiation of a proximate G4-binding photosensitizer. G4- over dsDNA alkylation can be achieved selectively and proves high-yielding at low ligand excess. HPLC and modelling studies allowed identifying potential residues involved in the alkylation.

Thermodynamic studies of substrate binding and spin transitions in human cytochrome P-450 3A4 expressed in yeast microsomes.

An approach to the quantitative spectral analysis of substrate binding and inactivation of cytochrome P-450 in microsomes is described. The method is based on the application of the principal component analysis technique on the Soret-region spectra measured at different temperatures at various concentrations of substrate. This approach allowed us to study the thermodynamic parameters of substrate binding and spin transitions in human cytochrome P-450 3A4 expressed in yeast (Saccharomyces cerevisiae) microsomes.

Multiphasic modelling of ligand/acceptor interactions. The hydrophobicity-dependent binding of relatively small amphiphilic substances to acceptor proteins and the nature and facedness of acceptor sites.

The modelling of multiphasic ligand/acceptor equilibrium binding systems proceeds at three logically distinct levels: (1) A suitable response quantity, e.g. the amount of acceptor-bound ligand nEL, is expressed as a function of the ligand concentrations [Li] (L = A,B,.

The substrate-depletion error on the multiple Michaelis-Menten equation with and without an added term linearly dependent on the substrate concentration.

Saturation curves for substrate interaction with an acceptor (enzyme, binding or carrier protein) are often analysed on the assumption that the amount of acceptor-bound substrate is negligible compared to its total amount. Analytical criteria permitting one to decide whether the assumption is justified or not for systems described by a single Michaelis-Menten equation have been derived previously. For more complicated systems, error formulae often cannot be obtained in closed form, and, if obtainable, are unwieldy.

Modelling of interaction of basic lipophilic ligands with cytochrome P-450 reconstituted in liposomes. Determination of membrane partition coefficients of S-(-)-nicotine and N,N-diethylaniline from spectral binding studies and fluorescence quenching.

The spectral interaction of N,N-diethylaniline and S-(-)-nicotine with cytochrome P450IIB4 reconstituted into large unilamellar vesicles could properly be described by a model for interaction of basic lipid-soluble ligands with membrane-bound acceptor sites in which linear partitioning of non-ionized ligand in the membrane is postulated. Apparent spectral dissociation constants Ksapp for type I binding of N,N-diethylaniline and for type II binding of S-(-)-nicotine increased linearly with increasing lipid volume fraction alpha L of the proteoliposomes. From plots of Ksapp vs.

Interaction of 7-n-alkoxycoumarins with cytochrome P-450(2) and their partitioning into liposomal membranes. Assessment of methods for determination of membrane partition coefficients.

A study was made of the binding of 7-ethoxy-, 7-n-propoxy- and 7-n-pentoxy-coumarin to cytochrome P-450(2) reconstituted into large unilamellar liposomes composed of a mixture of egg L-alpha-phosphatidylcholine, egg phosphatidylethanolamine and dipalmitoyl phosphatidic acid (2:1:0.06, by weight). The apparent spectral dissociation constants Ksapp.

Testing and characterizing enzymes and membrane-bound carrier proteins acting on amphipathic ligands in the presence of bilayer membrane material and soluble binding protein. Application to the uptake of oleate into isolated cells.

1. A multiphasic modelling approach [Heirwegh, Meuwissen, Vermeir & De Smedt (1988) Biochem. J.

Analysis of membrane-bound acceptors. A correction function for non-specific accumulation of poorly water-soluble hydrophobic or amphipathic ligands based on the ligand partition concept.

Non-specific ligand accumulation into membrane material, which may contribute considerably to the experimental signal obtained in binding studies with labelled amphipathic and hydrophobic ligands, may be accounted for by linear partition of the ligands into the membrane phase. For application to binding data obtained at a single membrane-lipid concentration, a fitting procedure is proposed which allows one to correct for non-specific ligand partition. If the assumption is met that the amount of acceptor-bound ligand is small compared to the total amount present in the system, one can validly interpret the data in terms of total ligand concentrations.

Synthesis, chromatographic purification, and analysis of isomers of biliverdin IX and bilirubin IX.

Neutral solvent systems were developed to isolate the alpha, beta, gamma, and delta isomers of biliverdin IX dimethyl ester by TLC. The individual free acids of biliverdin IX were obtained by saponification of the corresponding dimethyl esters. The bilirubin IX isomers were prepared by reducing the corresponding biliverdin IX isomers with NaBH3CN.

Generation and characterization of a neutralizing rat anti-rmTNF-alpha monoclonal antibody.

A rat anti-recombinant mouse tumour necrosis factor-alpha (rmTNF-alpha) monoclonal IgM antibody (1F3F3) with high specific binding activity for rmTNF-alpha was generated. The 1F3F3 monoclonal antibody (mAb) neutralizes the cytotoxic activity in vitro of rmTNF-alpha on L929 cells and inhibits the binding of radiolabelled rmTNF-alpha to its putative receptor on L929 cells. The 1F3F3 mAb binds to monomeric, dimeric and trimeric rmTNF-alpha and does not bind to reduced rmTNF-alpha, indicating that the recognized epitope is sensitive to denaturation.

Chromatographic analysis and structure determination of biliverdins and bilirubins.

Recent applications of thin-layer chromatographic (TLC) and high-performance liquid chromatographic (HPLC) procedures has revealed an unexpected wide variety of naturally occurring unconjugated and conjugated bilirubins. Biliverdins seems to occur only in unconjugated forms, mainly as the IX alpha isomer. Several synthetic biliverdins and bilirubins present interesting models for biochemical and metabolic studies.

Similarities in maximal biliary bilirubin output in the normal rat after administration of unconjugated bilirubin or bilirubin diglucuronide.

The rate-limiting step in the overall plasma-to-bile transport of a saturating load of bilirubin is still a matter of controversy. We reassessed the apparent maximal biliary bilirubin excretion following i.v.

Modelling of chemical reactions catalysed by membrane-bound enzymes. Determination and significance of the kinetic constants.

A model of multiphasic systems, based on the assumption of zero-order partition of substrates and products into the membranes, is applied to reversible mono-substrate and bi-substrate reactions catalysed by membrane-bound enzymes. Apart from replacement of single-phase kinetic constants by apparent kinetic constants, the derived kinetic expressions are formally identical with those for corresponding single-phase systems. The model confers to the apparent kinetic constants an experimentally verifiable meaning.

Thyroid hormones and the hepatic handling of bilirubin. I. Effects of hypothyroidism and hyperthyroidism on the hepatic transport of bilirubin mono- and diconjugates in the Wistar rat.

The effects of thyroidectomy and of thyroid hormone administration on the hepatic transport of endogenous bilirubin were investigated in the Wistar R/APfd rat. Hypothyroidism resulted in an enhanced hepatic bilirubin UDP-glucuronosyltransferase activity and in a decreased p-nitrophenol transferase activity. It caused a cholestatic condition with a 50% decrease in bile flow and bile salt excretion, and an increased proportion of conjugated bilirubin in serum.

Subcellular localization of UDP-glucuronyltransferase by differential centrifugation. Changes produced by pretreatment of rats with secretin, glucagon, vasoactive intestinal polypeptide and phenobarbitone.

Subcellular fractionation of liver homogenates from treated rats was carried out in order to study the mechanism of action of the gastrointestinal polypeptides on glucoronidation. Rats were treated for 90 min with an intravenous infusion of secretin (0.4 cU/h/100 g body weight), glucagon (100 micrograms/h/100 g body weight) and vasoactive intestinal polypeptide (VIP) (300 ng/h/100 g body weight); controls were sham-treated rats.

Liposomes as carriers of poorly water-soluble substrates: linear modelling of membrane systems with catalytic or binding sites of different facedness. Significance of experimental membrane partition coefficients and of kinetic and equilibrium parameters.

1. A multiphasic modelling approach to systems containing membrane-bound receptors or catalytic sites and a liposomal preparation as a substrate carrier is described. Kinetic expressions are derived for a single-substrate enzymic reaction operating at constant liposome concentration or at a fixed substrate/liposome concentration ratio.

Novel method for high-performance liquid chromatography of azo derivatives of conjugated and unconjugated bilirubin.

A method for the separation and quantitation of ethyl anthranilate or p-iodoaniline azo derivatives of bile pigments was developed using reversed-phase high-performance liquid chromatography. A convenient separation was achieved in 15 min, permitting the quantitation of the unconjugated azo-dipyrrole (alpha o) and its glucuronide (delta), xyloside (alpha 2) and glucoside (alpha 3) conjugates. The pathological beta- and gamma-azo pigments, derived from bilirubin glucuronide isomers that occur in cholestatic bile or plasma, are also detected in this system.

Transplantation of isolated hepatocytes into the pancreas.

In previous research into hepatocyte transplantation (HTX) the spleen was the preferred acceptor organ for isolated donor hepatocytes. In this study the pancreas was tested as an acceptor organ for HTX. HTX into the pancreas or spleen was performed by injection of 10(7) isolated hepatocytes into the parenchyma of these organs.

Principles of a competitive binding assay for the study of the interactions of poorly water-soluble ligands with their soluble binding partners. Application to bilirubin with the use of Sephadex G-10 as a competitive adsorbent.

1. A generally applicable method is described for obtaining experimental data on the interactions between a poorly water-soluble ligand and soluble binding factors, with the use of chemically inert solid adsorbent. The equilibrium distribution of the ligand between the liquid phase containing the soluble binders and the adsorbent must be measured and knowledge of the binding isotherm of the adsorbent is required.

Selective absorption and scattering of light by solutions of macromolecules and by particulate suspensions.

For particulate suspensions and for solutions that scatter light measurably the total absorbance A generally contains contributions due to specific absorption (Aa) and scattering of light (As). The quantity As is closely related to the turbidity tau. In general, spectrophotometry of such systems requires proper modification of the spectrophotometer used in order to permit accurate determination of the absorbance A and of the derived quantities Aa and As.

In vivo dynamic 99mTc-HIDA scintigraphy after hepatocyte transplantation: a new method for the monitoring of graft function.

In vivo dynamic 99mTc-HIDA scintigraphy was tested as a method for graft function monitoring after hepatocyte transplantation (HTX). Bilirubin uridyldiphosphate glucuronyl transferase-deficient rats received 10(7) viable isolated hepatocytes from congenic nondeficient donors by intrasplenic injection. The transplanted rats were divided into 2 groups.

A role for the interferon system in the pathogenesis of multiple sclerosis?

MS may be caused by an as yet unidentified virus whose life-long persistence in the body may deregulate the immune system so as to make it react against central nervous system tissue. Interferon are endogenous, hormone-like proteins with antiviral and immunomodulatory properties. This paper analyses current knowledge on the production and action of these proteins in the perspective of their possible involvement in the pathogenesis of MS.

The congenic normal R/APfd and jaundiced R/APfd-j/j rat strains: a new animal model of hereditary non-haemolytic unconjugated hyperbilirubinaemia due to defective bilirubin conjugation.

In this paper the production of the R/APfd-j/j strain which is congenic with the R/APfd strain is reported. The R/APfd-j/j completely lacks hepatic bilirubin UDP-glucuronyltransferase activity, as do our GUNNXR/Pfd-j/j rat strain and various other stocks of GUNN rats (j/j) described in the literature. Our recombinant inbred strain GUNNXR/Pfd-j/j was produced from non-inbred GUNN (j/j) rats.