Self-association studies of two adenine derivatives by equilibrium ultracentrifugation

Among the purine derivatives, N-6-dimethyladenosine [6-(dimethylamino)-purine-ribonucleoside] and N-6,9-di-methyladenine [6-(methylamino)-9-methyl-purine] show an exceptionally high self-association tendency. The self-association of these two substances was studied by equilibrium ultiacentrifugation at several concentrations and temperatures. Thus, the thermo dynamic quantities ΔH⁰ and ΔS⁰ as well as the nonideality parameters could be evaluated. In both cases, the equilibrium constants at 25°C were found to he higher than the values reported in the literature. This may be due to the fact that in our work the influences of nonideality were taken into account.     

Affinities of methylphenidate derivatives for dopamine,norepinephrine and serotonin transporters

We have synthesized several derivatives of dl-threo-methylphenidate (Ritalin) bearing substituents on the phenyl ring. IC₅₀ values for binding of these compounds to rat brain monoamine transporters were assessed using [³H]WIN 35,428 (striatal membranes, dopamine transporters, DAT), [³H]nisoxetine (frontal cortex membranes, norepinephrine transporters, NET) and [³H]paroxetine (brain stem membranes, 5HT transporters, 5HTT). Affinities (1/Ki) decreased in the order: DAT > NET ⪢ 5HTT. Substitution at the para position of dl-threo-methylphenidate generally led to retained or increased affinity for the dopamine transporter (bromo > iodo > methoxy > hydroxy). Substitution at the meta position also increased affinity for the DAT (m-bromo > methylphenidate; m-iodo-p-hydroxy > p-hydroxy). Substitution at the ortho position with bromine considerably decreased affinity. Similar IC₅₀ values for binding of o-bromomethylphenidate to the dopamine transporter were measured at 0, 22 and 37 degrees. N-Methylation of the piperidine ring of methylphenidate also considerably reduced affinity. The dl-erythro isomer of obromomethylphenidate did not bind to the DAT (IC₅₀ > 50,000 nM). Affinities at the dopamine and norepinephrine transporters for substituted methylphenidate derivatives were well correlated (r² = 0.90). Abilities of several methylphenidate derivatives to inhibit [³H]dopamine uptake in striatal synaptosomes corresponded well with inhibition of [³H]WIN 35, 428 binding. None of the compounds examined exhibited significant affinity to dopamine D1 or D2 receptors (IC₅₀ > 500 or 5,000 nM, respectively), as assessed by inhibition of binding of [³H]SCH 23390 or [¹²³I]epidepride, respectively, to striatal membranes.     

Experimental and theoretical microdialysis studies of in situ metabolism

Microdialysis sampling was performed to monitor localized metabolism in vivo and in vitro. A mathematical model that accounts for analyte mass transport during microdialysis sampling was used to predict metabolite concentrations in the microdialysis probe during localized metabolism experiments. The model predicts that metabolite concentrations obtained in the microdialysis probe are a function of different experimental parameters including membrane length, perfusion fluid flow rate, and sample diffusive and kinetic properties. Different microdialysis experimental parameters including membrane length and perfusion fluid flow rate were varied to affect substrate extraction efficiency (E(d)), or loss to the sample matrix, in vivo and in vitro. Local hepatic metabolism was studied in vivo in male Sprague-Dawley rats by infusing acetaminophen through the microdialysis probe. Acetaminophen sulfate concentrations increased linearly with respect to acetaminophen E(d) in contrast to modeling predictions. Xanthine oxidase was used as an in vitro model of localized metabolism. In vitro experimental results partially matched modeling predictions for 10-mm probes. These results suggest that monitoring local metabolism using microdialysis sampling is feasible. It is important to consider system parameters such as dialysis flow rate, membrane length, and sample properties because these factors will affect analyte concentrations obtained during local metabolism experiments.     

Theory relating in vitro and in vivo microdialysis with one or two probes

In this paper, we further develop the general theory of microdialysis by extending the linear model of Bungay et al. to provide a theoretical basis for in vitro and in vivo microdialysis. Specifically, we considered the effect of active clearance processes on in vivo microdialysis, and thereby elaborated the theory of Benveniste et al. to endogenous compounds. We examined the use of steady state tissue diffusion resistance with negligible clearance processes to interpret microdialysis data. The influence of the tissue properties on the in vitro and in vivo recoveries in dual-probe microdialysis was analyzed and we simulated the effect of the operating parameters on dual probe microdialysis performance. We estimated that the minimum clearance rate constant detectable by microdialysis in a quasi-steady state is about 5.5 x 10(-5) s(-1). This minimum rate constant establishes a criterion, below which inhibition of the active clearance processes does not show detectable influences on the microdialysis extraction efficiency.     

Characterization of two Arabidopsis thaliana acyltransferases with preference for lysophosphatidylethanolamine

Background  

Two previously uncharacterized Arabidopsis genes that encode proteins with acyltransferase PlsC regions were selected for study based on their sequence similarity to a recently identified lung lysophosphatidylcholine acyltransferase (LPCAT). To identify their substrate specificity and biochemical properties, the two Arabidopsis acyltransferases, designated AtLPEAT1, (At1g80950), and AtLPEAT2 (At2g45670) were expressed in yeast knockout lines ale1 and slc1 that are deficient in microsomal lysophosphatidyl acyltransferase activities.     

Conformational studies of two isomeric ring-expanded purine nucleosides and their 5'-mono- and -diphosphate derivatives

The nucleosides Ia and IIa exist in syn and anti conformations, respectively, both in solid state and solution. Compound Ia undergoes significant conformational change, accompanied by increased population of the anti conformer, upon conversion to the corresponding 5'-mono- and- diphosphate derivatives, whereas conformation of IIa remains reasonably constant between nucleoside and nucleotides. While Ia possessed the C2'-endo-C3'-exo geometry, IIa had the opposite C2'-exo-C3'-endo conformation. The C5' of the two nucleosides bore axial and equatorial conformations, respectively.     

Interactions of two O-phosphorylresveratrol derivatives with model membranes

The hydrosoluble resveratrol derivative 3-O-phosphorylresveratrol was shown to be more cytotoxic against DU 145 prostate cancer cells than its analog 4'-O-phosphorylresveratrol. In an attempt to unveil the molecular determinants that lye at the root of their different biological effects, here we investigate the interactions of the two resveratrol derivatives with DMPC model membranes by using DSC, membrane permeation/poration assays and molecular dynamics. The results show that the 3-O-derivative interacts with DMPC membranes and diffuses across them. The 4'-O-derivative lies preferentially onto the surface of membrane. The MD simulations provide a molecular interpretation of the experiments and highlight that, in order to maximize the apolar interactions, the 3-O-derivative is embedded in the lipid hydrophobic region. This topographical position of the 3-O resveratrol analog perturbs the liquid-crystalline order of the lipid bilayer promoting membrane curvature and partial lipid loss from the vesicle. This finding reconciles with the lowering of the enthalpy of the lipid phase transition and the ability of the molecule to diffuse across membranes. The present data contribute to explain the different biological activity of the two molecules and evidence that membrane permeability is a key requirement for effective design of resveratrol derivatives to be used for therapeutic purposes.     

Interaction of two phenothiazine derivatives with phospholipid monolayers

This paper addresses the cooperative interaction of two phenothiazine drugs, viz. trifluoperazine (TFP) and chlorpromazine (CPZ), with phospholipid monolayers as the model membrane system. Surface pressure and surface potential isotherms were obtained for mixed Langmuir monolayers of either dipalmitoyl-phosphatidyl-choline (DPPC) or dipalmitoyl-phosphatidyl-glycerol (DPPG) co-spread with TFP or CPZ. The changes in monolayer behavior caused by incorporation of a few molar ratio of drug molecules were practically within the experimental dispersion for the zwitterionic DPPC, and therefore a more refined analysis will be required to probe the interactions in an unequivocal way. For the charged DPPG, on the other hand, the surface pressure and the dipole moment were significantly affected even for TFP or CPZ concentrations as low as 0.002 molar ratio. Overall, the effects from CPZ and TFP are similar, but small differences exist which are probably due to the different protonation properties of the two drugs. For both drugs, changes are more prominent at the liftoff of the surface pressure, i.e. at the gas-condensed phase transition, with the surface pressure and surface potential isotherms becoming more expanded with the drug incorporation. With DPPG/CPZ monolayers, in particular, an additional phase transition appears at higher CPZ concentrations, which resembles the effects from increasing the subphase temperature for a pure DPPG monolayer. The dipole moment for DPPG/CPZ and DPPG/TFP monolayers decreases with the drug concentration, which means that the effects from the charged drugs are not associated with changes in the double-layer potential. Otherwise, the effective dipole moment should increase with the drug concentration. The changes caused in surface pressure and dipole moment by small concentrations of TFP or CPZ can only be explained by some cooperative effect through which the contribution from DPPG molecules changes considerably, i.e. even DPPG molecules that are not neighbor to a CPZ or TFP molecule are also affected. Such changes may occur either through a significant reorientation of the DPPG molecules or to a change in their hydration state. We discuss the cooperativity semi-quantitatively by estimating the number of lipid molecules affected by the drug interaction. CPZ and TFP also affect the morphology of DPPG monolayers, which was confirmed with Brewster angle microscopy. The biological implications from the cooperative, non-specific interaction of CPZ and TFP with membranes are also commented upon.     

A linear sesterterpene,two squalene derivatives and two peptide derivatives from Croton hieronymi

Aerial parts of Croton hieronymi furnished in addition to a large number of plant sterols and triterpenes the C-25 analog of trans-phytol, the squalene derivatives all-trans-2,6,15,19,23-pentamethyltetracosa-2,6,10,(28),14,22,28-hexaene-11-ol and all-trans-10-methylene-2,6,10,14,18,22-pentamethyltetracosa-1,6,10,14,18,22-hexaen-3-ol, the sesquiterpenes epicubenol and T-cadinol, the acetophenone derivative xanthoxylin and the peptide derivatives aurentiamide acetate and N-benzoylphenylalanyl-N-benzoylphenylalaninate.     

Behavioural conflict in two strains of rat: Home cage preference versus dark preference

When confronted with a novel situation rats show a tendency either to stay in a dark place or to retreat to a familiar place, if available. To produce a conflict between these two tendencies the rat's home cage was connected by a runway to a dark but unfamiliar compartment. Three groups of rats were tested in this apparatus with different levels of illumination in the home cage. Tryon Maze Bright rats showed a strong preference for the home cage which was independent of the illumination level. This preference was also found in Wistar rats. With a very high level of illumination in the home cage, however, Wistar rats showed a preference for the home cage only in the first few trials or a series of repeated trials. In later trials they spent more time in the dark part of the apparatus. The results were regarded as an indication that in a novel situation rats primarily search for a familiar place. When such a place is available they tolerate aversive stimulation present in this place.     

Spectral luminescence studies of cells stained with acrichine derivatives

Excitation and fluorescence spectra are given of quinacrine derivative solutions, of buccal epithelium cell nuclei, of peripheral blood cells, and of isolated chromosomes treated with propyl-quinacrine mustard. It is confirmed that the differential cell treatment with quinacrine derivates may be observed in aqueous solutions only. Data obtained allow us to give some recommendations for employment of optimal filters and dichroic beam-splitters in the fluorescence microscopy of chromosomes treated with quinacrine derivatives.     

SAR studies of gymnasterkoreayne derivatives with cancer chemopreventive activities

We synthesized diyne triols based on gymnasterkoreayne and evaluated their cancer chemopreventive activities in terms of the chemopreventive index (CI) to reveal the structure-activity relationship, and discovered more active compounds than natural diynes.     

Electrochemical detection of free 3-nitrotyrosine: application to microdialysis studies

3-Nitrotyrosine (3-NT) is formed by the reaction of peroxynitrite with either free or protein-bound tyrosine residues and has been proposed as a biomarker of oxidative stress caused by reactive nitrogen species. This study describes the development of an HPLC electrochemical detection assay for free 3-NT capable of measuring this metabolite at the very low (nanomolar) levels encountered physiologically. We employed a dual-cell coulometric approach in which 3-NT is first reduced at an upstream cell to 3-aminotyrosine, which itself is then oxidized at the downstream cell. The method was shown to be linear over the range of 1-500 nM (r = 0.999), with a detection limit (signal/noise ratio of 3) of 0.5 nM (25 fmol on column). Ten consecutive injections of 2 and 20 nM 3-NT standards produced coefficients of variation of 5.88 and 1.87%, respectively. Validation of the identity of the 3-NT peak was confirmed by coelution with authentic standards and by the in vitro production of 3-NT by incubation of 3-morpholinylsydnoneimine (SIN-1, 100 microM), a molecule releasing nitric oxide and superoxide in solution at a pH of 7.0 or higher with tyrosine (10 microM). Using this method, 3-NT was detected in human liver microdialysate (levels up to 2.6 nM), although levels in rat spinal cord dialysate were below the limit of detection.     

Spectroscopic studies of stellacyanin derivatives

Two covalently modified derivatives of the apoprotein of the blue copper protein stellacyanin have been prepared. In one case, a dansyl group was linked to the cysteine at the copper binding site of apostellacyanin; in the other, a nitrophenol moiety has been attached to this same cysteine. Fluorescence yields and emission maxima of the dansylated protein and pK determinations of the nitrophenol group linked to the protein suggest that the solvent microenvironment at the copper binding site of apostellacyanin is quite similar to bulk water.