Comparison of 3 assay systems using a common probe substrate, calcein AM, for studying P-gp using a selected set of compounds

The multidrug resistance protein 1 (MDR1) transporter is the most abundantly investigated adenosine triphosphate (ATP)-Binding Cassette (ABC) transporter protein. Multiple assay systems were developed to study MDR1-mediated transport and possible drug-drug interactions. Yet, as different probe substrates are used in these assays, it is difficult to directly compare the results. In this study, a common probe substrate was applied in 3 assay systems developed to study MDR1: the cellular dye efflux assay, the ATPase assay, and the vesicular transport assay. This probe substrate is calcein acetoxymethyl ester (calcein AM), the acetoxymethyl ester derivative of the fluorescent dye, calcein. Using a common probe allows the investigation of the effect of passive permeability on the result obtained by testing various compounds. In this study, 22 compounds with different logP values were tested in the above-mentioned 3 assay systems. The vesicular transport assay proved most sensitive, detecting 18 of 22 interactions with the protein. The ATPase assay detected 15 interactions, whereas the cellular dye efflux assay was the least sensitive with only 10 hits. A correlation was found between the hydrophobicity of the compound and the ratio of cellular and vesicular transport IC(50) values, indicating the effect of passive permeability on the result. Based on hydrophobicity, the current study provides guidelines on applying the most correct tool for studying MDR1 interactions.     

Quinidine as an ABCB1 probe for testing drug interactions at the blood-brain barrier: an in vitro in vivo correlation study

This study provides evidence that quinidine can be used as a probe substrate for ABCB1 in multiple experimental systems both in vitro and in vivo relevant to the blood-brain barrier (BBB). The combination of quinidine and PSC-833 (valspodar) is an effective tool to assess investigational drugs for interactions on ABCB1. Effects of quinidine and substrate-inhibitor interactions were tested in a membrane assay and in monolayer assays. The authors compared quinidine and digoxin as ABCB1 probes in the in vitro assays and found that quinidine was more potent and at least as specific as digoxin in ATPase and monolayer efflux assays employing MDCKII-MDR1 and the rat brain microcapillary endothelial cell system. Brain exposure to quinidine was tested in dual-/triple-probe microdialysis experiments in rats by assessing levels of quinidine in blood and brain. Comparing quinidine levels in dialysate samples from valspodar-treated and control animals, it is evident that systemic/local administration of the inhibitor diminishes the pumping function of ABCB1 at the BBB, resulting in an increased brain penetration of quinidine. In sum, quinidine is a good probe to study ABCB1 function at the BBB. Moreover, quinidine/PSC-833 is an ABCB1-specific substrate/inhibitor combination applicable to many assay systems both in vitro and in vivo.     

Effects of experimental calcium availability,egg parameters and laying order on Great Tit Parus major eggshell pigmentation patterns

Many bird species lay eggs speckled with protoporphyrin‐based spots, however, for most of them the function of eggshell spotting is unknown. A plausible hypothesis is that protoporphyrin might have a structural function in strengthening the eggshell and is therefore deposited when calcium is scarce. In this study, we experimentally provided Great Tit Parus major females with supplemental calcium to examine its effect on the protoporphyrin‐based maculation of their eggs. In addition, we studied variation in eggshell pigmentation patterns in relation to other egg parameters and laying order. Calcium‐supplemented females laid larger eggs but shell thickness was not significantly affected by the treatment. Calcium supplementation may reduce the time and energy females devote to searching for calcium‐rich material, so that they can collect more nutrients and so lay larger eggs. Furthermore, pigment darkness was associated with egg volume and shape, which suggests that female quality and environmental food availability may also influence the shell pigmentation pattern. Within clutches, later‐laid eggs had larger and darker spots that were distributed more unevenly on the shell surface. This within‐clutch pattern could be explained by the increase in egg volume and egg shape and a decline in shell thickness with egg‐laying order, which characteristics were all related to shell‐spotting pattern. Eggs with a coronal ring had thinner shells, but pigment intensity and spot size were not related to shell thickness. Thus, our results suggest that concentrated spotting distribution may have a mechanical function, supporting the structural‐function hypothesis.     

Adsorption and narcosis

Summary The adsorption of homologic alcohols by the sap ofCaulerpa prolifera was studied.It was found that the alcohols are adsorbed in an indirect relation to their respective capillary activities against air.Traube's rule is therefore not valid for this adsorption.It is supposed that the unadsorbed remainder is the cause of the narcotic effect and that the adsorption is a regulating device to remove the narcotizing substance from the more liquid phase of the cell interior.The validity ofTraube's rule for narcosis as a biological phenomenon can be understood on the basis of the fact, that the strongly capillary active substances are adsorbed at least and the little capillary active substances at most. Thus their narcotizing effect is indirectly proportional to their respective adsorbility.     

Über den Zusammenhang zwischen Wasserstoffionenkonzentration,Oberflächenspannung und Wachstumsgeschwindigkeit

Zusammenfassung Die Oberflächenspannung des Preßsaftes wird durch p_H in charakteristischer Weise verändert. Im p_H 4,7 wird ein Maximum der Oberflächenspannung beobachtet, so daß in dieser Wasserstoffionenkonzentration der isoelektrische Punkt des Pflanzenpreßsaftes liegen dürfte. Es wird der Zusammenhang zwischen p_H, Oberflächenspannung und Wachstum besprochen.Mit 3 Textabbildungen.     

Mutations in squirrel monkey glucocorticoid receptor impair nuclear translocation

To identify the determinants of impaired glucocorticoid receptor (GR) signaling in a model of glucocorticoid resistance, cloned GR from Guyanese squirrel monkeys (gsmGR) was tagged with enhanced green fluorescent protein, and nuclear translocation was examined in transfected COS1 cells. In keeping with evidence that gsmGR transactivational competence is impaired, we found that nuclear translocation is likewise diminished in gsmGR relative to human GR (hGR). Experiments with GR chimeras revealed that replacement of the gsmGR ligand binding domain (LBD) with that from hGR increased translocation. Truncated gsmGR constructs lacking the LDB after amino acid 552 also showed increased translocation even in the absence of cortisol. Three back-mutations of gsmGR to hGR (Thr551Ser, Ala616Ser, and Ser618Ala) in the LBD confirmed that these amino acids play a role in diminished translocation.     

Binding of cationic porphyrin to isolated DNA and nucleoprotein complex: quantitative analysis of binding forms under various experimental conditions

We studied the complex formation of tetrakis(4-N-methylpyridyl)porphyrin (TMPyP) with double stranded DNAs and T7 phage nucleoprotein complex. We analyzed the effect of base pair composition of DNA, the presence of capsid protein, and the composition of the microenvironment on the distribution of TMPyP between binding forms as determined by the decomposition of porphyrin absorption spectra. No difference was found in the amount of bound TMPyP between DNAs of various base compositions; however, the ratio of TMPyP binding forms depends on the AT/GC ratio. The presence of protein capsid opposes the binding of TMPyP to DNA. This behavior offers a possibility to investigate the protein capsid integrity due to the analysis of porphyrin binding. Increasing ionic strength of monovalent ions decreases the amount of bound porphyrin through the inhibition of intercalation, but does not influence the quantity of groove-binding forms when TMPyP interacts with isolated DNA. In the case of the nucleoprotein complex the groove-binding is also inhibited already at 140 mM ionic strength. The presence of 1 mM divalent cations (Mg(2+), Ca(2+), Cu(2+) and Ni(2+)) in a buffer solution of 70 mM ionic strength does not influence significantly the free to bound ration of TMPyP when it interacts with isolated DNA. The contribution of binding forms is remarkably different in Mg(2+)/Ca(2+) and Cu(2+)/Ni(2+) containing solutions. Transition metals significantly decrease the binding sites for intercalation in both DNA and nucleoprotein complex, but facilitate the groove-binding of TMPyP to isolated DNA.     

Induction of mutations inSerratia marcescens by a proteosynthesis block

The formation of colour mutations ofSerratia marcescens by the action of chloramphenicol was studied. Pink variants were the commonest; the proportion of white variants was much smaller. Almost 100% mutations were formed in a two-day culture containing 100 μg. chloramphenicol/ml. Comparative experiments showed that the change in pigment formation was hereditary, i.e. that actual mutation, and not selection of chloramphenicol-resistant mutants, occurred. Mutation occurred both in strain 151 and strain HY. The resultant mutations remained constant throughout ten passages on normal nutrient medium. The minimum chloramphenicol concentration which produced an increase in the mutation frequency was 5 μg./ml. The combined effect of X-ray irradiation and chloramphenicol treatment somewhat stimulated the increase in the frequency of mutation as compared with cells which were only irradiated. The increase in the frequency of mutation was much slower on solid medium containing chloramphenicol.     

Application of microarray technology in primate behavioral neuroscience research

Gene expression profiling of brain tissue samples applied to DNA microarrays promises to provide novel insights into the neurobiological bases of primate behavior. The strength of the microarray technology lies in the ability to simultaneously measure the expression levels of all genes in defined brain regions that are known to mediate behavior. The application of microarrays presents, however, various limitations and challenges for primate neuroscience research. Low RNA abundance, modest changes in gene expression, heterogeneous distribution of mRNA among cell subpopulations, and individual differences in behavior all mandate great care in the collection, processing, and analysis of brain tissue. A unique problem for nonhuman primate research is the limited availability of species-specific arrays. Arrays designed for humans are often used, but expression level differences are inevitably confounded by gene sequence differences in all cross-species array applications. Tools to deal with this problem are currently being developed. Here we review these methodological issues, and provide examples from our experiences using human arrays to examine brain tissue samples from squirrel monkeys. Until species-specific microarrays become more widely available, great caution must be taken in the assessment and interpretation of microarray data from nonhuman primates. Nevertheless, the application of human microarrays in nonhuman primate neuroscience research recovers useful information from thousands of genes, and represents an important new strategy for understanding the molecular complexity of behavior and mental health.