Interaction of disopyramide enantiomers for sites on plasma protein

The binding of disopyramide enantiomers to donor plasma to which had been added human alpha-1 acid glycoprotein (AAG) was characterized alone and in the presence of the opposite enantiomer. At pre-dialysis concentrations of 10(-5) M, S(+)-disopyramide increased the percent of R(-)-disopyramide free (unbound) 2.6-fold from 11 to 30%. At similar pre-dialysis concentrations, R(-)-disopyramide increased the percent of S(+)-disopyramide free 2-fold from 4.1 to 9.0%. Differences in the binding of one enantiomer due to the presence of the other were due to apparent changes in association constant; no changes in capacity to bind the enantiomers were observed. It is concluded that the enantiomers of disopyramide compete with each other for one site on AAG.     

Determination of (R)- and (S)-Disophyramide in human plasma using a chiral α1-acid glycoprotein column

The direct resolution and quantitation of (R)- and (S)-disopyramide, isolated from human plasma, was accomplished using a chiral α₁-acid glycoprotein column. A LiChrosorb RP-2 column (50 × 3.0 mm I.D.) was used as a precolumn. Phosphate buffer, pH 6.20, containing 2-propanol and N,N-dimethyloctylamine was used as mobile phase, expressed as the relative standard deviation, was 1.8% and 3.3% for (R)- and (S)-disopyramide, respectively, at a drug level of 0.5 μg/ml. In two subjects who received a single capsule of racemic disopyramide (150 mg), the plasma levels of the (R) isomer were about half those of the (S) isomer. The half-lives of (R)- and (S)-disopyramide were similar.     

Measurement of non-specific binding by parallel or consecutive incubation procedures in receptor homogenates following tissue disintegrations by sonification

It was demonstrated with 125I-labeled insulin and homogenates of human placenta that the energy and duration of tissue disintegration by sonification markedly affected the extent and reproducibility of binding. If the non-specific binding was determined by consecutive incubation on identical samples, as was the total binding, the data obtained were more reproducible and closer to the affinity values and number of receptors that had been obtained on isolated trophoblast membranes when compared to experiments in which the non-specific binding was measured on different samples than for the total binding. On isolated membranes, the results were almost unaffected by the method of measuring non-specific binding. Therefore, we suggest that total and non-specific binding should be measured consecutively on identical samples if only small amounts of homogenized tissue are available.     

Stereoselective binding of zopiclone to human plasma proteins

The binding of racemic zopiclone (ZOP) and of its two enantiomers to plasma proteins, albumin and α1‐acid glycoprotein were compared. Our work shows that the binding of ZOP to human plasma proteins is stereoselective. The total plasma protein binding percentages were 79.3 ± 5.5%, 83.8 ± 5.2%, and 75.1 ± 2.1%, for racemic zopiclone, (−)zopiclone and (+)zopiclone, respectively. These results were confirmed by the analysis of samples obtained from healthy volunteers after the oral administration of ZOP. The anticoagulant used for sampling was also shown to have an influence on the percentage binding and on its stereoselectivity. Considering albumin and α1‐acid glycoprotein separately, stereoselectivity was also observed. Chirality 11:129–132, 1999. © 1999 Wiley‐Liss, Inc.     

Immobilization of Zinc and Cadmium in Polluted Soils by Polynuclear Al13 and Al-Montmorillonite

We investigated the suitability of two aluminum-based binding agents, polynuclear Al₁₃ and Al-coated montmorillonite (Al-mont-morillonite), for the immobilization of heavy metals in two contaminated agricultural soils: a loamy luvisol from an arable site in Rafz, Canton Zürich, Switzerland, and a sandy podsol from Szopienice, Upper Silesia, Poland. Both soils were polluted by lead, zinc, and cadmium: the soil from Szopienice by the emissions of a nearby zinc-lead smelter, and the soil from Rafz by sewage sludge applications. While the samples from Szopienice exhibited extremely high loads of these metals, the samples from Rafz were only moderately contaminated. The samples from both soils were slightly acidic. The Rafz soil contained 2.5% organic matter, that from Szopienice only 1.5%. Destruction of the organic matter in the Szopienice samples by H₂O₂ led to a significant release of Zn and Cd into solution. This indicated that organic matter is an important factor for the immobilization of heavy metals in this soil. The treatment of the Szopienice samples with 8?mmol Al₁₃ per kg dry soil resulted in a considerable mobilization of the two metals. As the pH of the samples did not decrease, this effect was presumably due to direct interactions between the applied aluminium and organic matter. After destruction of soil organic matter, the two binding agents exhibited an immobilizing effect on Zn, which, however, was weak compared with the binding of the metal by the organic matter prior to its destruction. In the case of the Rafz samples, metal mobilization was observed only for Al₁₃ if applied in high doses (4 and 8?mmol per kg soil), but not for Al-montmorillonite. In this soil, Al-montmorillonite as well as Al₁₃ at low doses (1.2?mmol per kg soil and less) decreased soluble zinc concentrations significantly. The mobilization of metals at high doses of the applied binding agents and the dependence of this effect on the type of soil show that care has to be taken with this remediation method and that the proper doses of applied binding agents can be crucial for the success of metal immobilization in polluted soils.     

Histochemical demonstration of estrogen and progesterone binding in endometriotic tissue and in uterine endometrium: A comparative study

Estrogen and progesterone binding to endometriotic and endometrial tissue was studied histochemically using estradiol and progesterone fluorochrome derivatives (E2-bovine serum albumin-fluorescein isothiocyanate and progesterone-bovine serum albumin-tetramethylrhodamine isothiocyanate). Thirty endometriotic samples from 21 women were studied, together with endometrial specimens obtained simultaneously from 14 of the women. In 77% of the endometriotic samples binding of the estrogen conjugate was indicated by specific fluorescence in more than half of the epithelial cell population, and in 20% in less than half. The corresponding figures for the progesterone conjugate binding were 75 and 18%, respectively. Blocking studies indicated a reasonable degree of ligand specificity. In endometrial tissue the corresponding figures were 64 and 29%, respectively, for binding of the estrogen conjugate and 54 and 38%, respectively, for binding of the progesterone conjugate. In 7 of 13 cases where evaluable samples of both tissues had been obtained, the relative proportion of fluorescent cells, with either reagent, was similar in the two tissue types. Our results suggest that the cytoplasm of epithelial cells in endometriotic tissue and in uterine endometrium contains specific binding sites for both estrogen and progesterone. The binding pattern of the two conjugates in endometriotic tissue was unrelated to the menstrual phase.     

Microarrays of tumor cell derived proteins uncover a distinct pattern of prostate cancer serum immunoreactivity

The broad characterization of the immune responses elicited by tumors has valuable applications in diagnostics and basic research. We present here the use of microarrays of tumor-derived proteins to profile the antibody repertoire in the sera of prostate cancer patients and controls. Two-dimensional liquid chromatography was used to separate proteins from the prostate cancer cell line LNCaP into 1760 fractions. These fractions were spotted in microarrays on coated microscope slides, and the microarrays were incubated individually with serum samples from 25 men with prostate cancer and 25 male controls. The amount of immunoglobulin bound to each fraction by each serum sample was quantified. Statistical analysis revealed that 38 of the fractions had significantly higher levels of immunoglobulin binding in the prostate cancer samples compared to the controls. Two fractions showed higher binding in the control samples. The significantly higher immunoglobulin reactivity from the prostate cancer samples may reflect a strong immune response to the tumors in the prostate cancer patients. We used multivariate analysis to classify the samples as either prostate cancer or control. In a cross-validation study, recursive partitioning classified the samples with 84% accuracy. A decision tree with two levels of partitioning classified the samples with 98% accuracy. Additional studies will allow further characterization of tumor antigens in prostate cancer and their significance for diagnosis. These results suggest that microarrays of fractionated proteins could be a powerful tool for tumor antigen discovery and cancer diagnosis.     

Injected TFF1 and TFF3 bind to TFF2-immunoreactive cells in the gastrointestinal tract in rats

Peptides of the trefoil factor family (TFF1, TFF2 and TFF3) are co-secreted with mucus in most organ systems and are believed to interact with mucins to produce high-viscosity, stable gel complexes. We have previously demonstrated that cells in the GI tract possess binding sites to TFF2 and that injected TFF2 ends up in the mucus layer. In the present study, tissue binding and metabolism of parenterally administered human TFF1 and TFF3 in rats were described and compared to the immunohistochemical localization of the TFF peptides. 125I-TFF1 monomer and 125I-TFF3 mono- and dimer were given intravenously to female Wistar rats. The tissue distribution was assessed by gamma counting of organ samples and by autoradiography of histological sections. The degradation of 125I-TFF3 was studied by means of trichloracetic acid (TCA) precipitation and the saturability of the binding by administration of excess unlabelled peptide. The TFF peptides were localized in histologic sections from the GI tract by immunohistochemistry. Injected TFF3 dimer (12%) was taken up by the GI tract. At autoradiography, grains were localized to the same cells that were immunoreactive to TFF2. The binding could be displaced by excess TFF3. Similar binding was observed for the TFF1 and TFF3 monomers apart from binding in the stomach, where the uptake was only 15% in comparison to the dimer. There was no specific binding outside the GI tract and no binding to TFF1 or TFF3 immunoreactive cells. In conclusion, the TFF2-binding cells in the gastrointestinal tract seem to have basolateral, receptor-like activity to all three TFF peptides. The mucous neck cells of the stomach predominantly take up TFFs with two trefoil domains, indicating a different receptor-like activity in the stomach compared to the rest of the GI tract.     

TDPAC studies of the metal-binding sites in serum transferrin: comparison between 181Hf-labeled human- and rat-serum transferrin

The binding of hafnium to human serum transferrin was studied using the time differential perturbed angular correlation (TDPAC-) technique. The samples were prepared in vitro by adding 181Hf-NTA solution to human serum. Two specific electric quadrupole interactions were observed, which correspond to two well-defined binding configurations. Their relative intensities depend on the pH, salt- and hafnium-concentrations, and on the incubation time. The present data may be compared with the results of a previous rat serum study, where the hafnium binding to transferrin behaved rather similarly. Small but significant differences, however, can be deduced from the TDPAC-parameters for these human and rat transferrin species. For either binding configuration, the electric field gradient (EFG) is slightly higher in the case of rat transferrin. The most characteristic difference, however, concerns the asymmetry parameter eta 2 of the second binding configuration, which is about 10% smaller for rat serum transferrin. The TDPAC-technique might be used as a sensitive and reliable analytical method to study the metal-binding sites of different transferrin species.     

Impact of ethanol on innate protection of gastric mucosal epithelial surfaces and the risk of injury.

Earlier investigations on the effect of ethanol on synthesis and posttranlational glycosylation of gastric mucus glycoprotein (mucin) revealed quantitative changes in the apoprotein assembly, glycosylation, and mucin retention on the mucosal surface (Slomiany et al.., Alcoholism: Clin. Exp. Res. 21, 417-423, 1998). To assess whether metabolic consequences of ethanol ingestion, documented in the in vitro system are also occurring in vivo the rats were subjected to 8 weeks of ethanol containing liquid diet. The retention of mucin on the surface of gastric mucosa was quantitated by measuring the binding of gastric mucin to Mucin Binding Protein (MBP) of gastric mucosa. The results were compared with those obtained with the rats subjected to pair-feeding the isocaloric-control diet. Before alcohol administration, and in two weeks' intervals thereafter, the gastric contents from the animals was collected and mucin purified. After 8 weeks of the respective diet, the animals were sacrificed and their gastric mucosa used for MBP preparation. The binding of mucin to MBP before ethanol, and after 2, 4, 6, and 8 weeks of ethanol diet was quantitated with Enzyme Linked Lectin Assay (ELLA). The study with standard mucin revealed that binding of mucin to MBP differs substantially between individual animals. The same variability in binding was observed with the individual mucin preparations collected at the onset of the experiment. However, with the progression of ethanol feeding, the mucin samples besides displaying the variable and animal-specific binding to MBP at the initiation of the experiment, also showed a dramatic decrease in binding. In five animals, after two weeks of ethanol diet, mucin binding to MBP decreased by 50%; in two animals, the drastic decrease in binding was observed in mucin collected after four weeks of alcohol feeding; and in one animal a 20% decrease in binding persisted for six weeks, and then decreased to 50% in the last collection. Also, in two animals, the mucin collected after 8 weeks of ethanol feeding retained only 6-9% of the initial binding capacity. In contrast, in pair-fed controls, the mucin binding to MBP remained the same or increased up to 20%. Results of the studies, performed on mucin of the individual animals and matching preparations of MBP, showed that each animal expresses different degree of mucin binding. Moreover, in chronic ethanol ingestion, the individual variations are accompanied by a decrease in mucin binding to MBP. Since the observed decrease in binding occurred in samples containing the same preparation of MBP, the component affected by alcohol resides on mucin. Thus, considering the in vitro impact of ethanol on generation of carbohydrate chains in Golgi, and the finding on mucin oligosaccharides-dependent mucin-MBP complex formation, we conclude that ethanol impairs the synthesis of mucin oligosaccharide structures required for binding with MBP, and the retention on gastric mucosal surfaces.     

Studies on 5alpha-androst-16-en-3-one binding to porcine serum, plasma and testicular cytosolic fraction and to human serum

The present study evaluated whether a specific androstenone-binding protein is present in porcine and human serum, and in the cytosolic fraction of porcine testis. The binding of [(3)H]-androstenone to serum and testicular cytosol was measured in the absence (total binding) and presence (non-specific binding) of unlabelled androstenone. The optimization of the assay is described. As a part of the assay validation, the binding of [(3)H]-dihydrotestosterone ([(3)H]-DHT) to porcine and human serum was also examined. As expected, specific binding of [(3)H]-DHT was detected in human serum, but not in porcine serum. No specific androstenone-binding protein was detected, either in porcine or human serum, or in the cytosolic fraction of porcine testis. The amount of non-specific binding of [(3)H]-androstenone was slightly lower in porcine serum compared to human serum. Between-animal variations in [(3)H]-androstenone binding were studied in plasma samples from 15 animals with androstenone concentrations ranging from 1.1 to 23.1 ng/mL. Mean values+/-standard deviations of binding in these samples were 15.2+/-0.9% for total binding and 15.9+/-0.8% for non-specific bindings. Low between-animal variations indicate that androstenone binding does not affect androstenone accumulation in fat.     

Relation between the mode of binding of nitrite and the energy distribution between the two photosystems

The reversibility of nitrite-induced inhibition in relation to energy distribution between the two photosystems was studied in spinach thylakoid membranes. Measurements of electron transfer rate catalyzed by photosystem I (PS I) and photosystem II (PS II), chlorophyll a (Chl a ) fluorescence induction kinetics, S₂ state multiline spectra, and room temperature electron paramagnetic resonance (EPR) signals indicated that nitrite anions bind PS II in two ways: dissociable (loose) and non-dissociable (tight). The inhibition caused by the dissociable binding was reversible in washed (nitrite-treated samples washed with nitrite-free medium) samples, while the inhibition caused by the non-dissociable binding was irreversible. At 77 K, an increase in absorption cross section of PS I (as inferred from the excitation spectra of Chl a fluorescence) and a decrease in absorption cross section of PS II in nitrite-treated sample when compared with sample washed with nitrite-free medium and control sample suggested that nitrite plays a role in regulating the distribution of absorbed excitation energy between the two photosystems. We propose, for the first time, that the removal of loosely bound nitrite leads to migration of light-harvesting complex II back to the PS II, and thus the mode of binding of nitrite regulates the extent of migration of antenna molecules between the two photosystems.     

Zero-field M?ssbauer studies of diferric human transferrin

Diferric transferrin samples labelled with 57Fe at the N- or the C-terminal binding sites are compared by M?ssbauer spectroscopy at 15 K and in zero magnetic field. The spectra of the samples are similar but the fitting of single Lorenzian lines to the data shows that some of the line positions differ in the two cases. According to this we can not exclude a difference between the chemical structures of the binding sites that can arise for example from the participation of different forms of the anion and/or water in the two lobes of transferrin. All other line parameters (line-width, intensity) are the same within the limits of errors.     

Serum prostacyclin binding and half-life in the umbilical circulation

Serum prostacyclin binding and half-life was measured in twenty pairs of maternal and umbilical venous samples and in twenty non-pregnant controls. When compared to non-pregnant values both umbilical and maternal samples demonstrated significantly lower albumin concentrations, percentage of prostacyclin binding and shorter prostacyclin half-life.     

Estradiol- and estriol-binding in human benign prostatic hyperplasia.

Binding of the natural estrogens, estradiol and estriol, was investigated, in 34 samples of human benign prostatic hypertrophy (BPH) tissue, using Scatchard analysis and agar gel electrophoresis. Saturation binding analysis using a wide range of concentrations of both ligands resulted in curvilinear Scatchard plots. This confirmed the presence of two binding forms for estradiol: a true estrogen receptor, and a protein with lower affinity and higher capacity. Both binding species were also demonstrated and quantified with estriol. The electrophoretic process, after incubation at low and high ligand concentrations also resulted in separation, for both estrogens, of two binding peaks. They are probably two distinct forms of the low affinity, high capacity binding measured by Scatchard. The procedure used in our laboratory was not able to provide accurate determination of the concentrations of these binding forms. Possible modifications to alleviate these drawbacks are discussed.     

Differential binding of folates by rat renal cortex brush border and basolateral membrane preparations

The binding of radioactive 5-methyltetrahydrofolate and folic acid was found to be greater in brush border than in basolateral membrane preparations of rat renal cortex. This appeared to be due to an increased amount of a specific folate binding protein in the brush border membrane preparations as compared to those of the basolateral membrane. The binding was saturable and inhibited by nonradioactive folic acid and, therefore, a specific, rather than nonspecific process. The Km's for folic acid binding in brush border and basolateral membrane preparations were similar and involved a single high-affinity binding site. In contrast, methotrexate was found to bind equally well to both brush border and basolateral membrane preparations. Moreover, folic acid binding was not inhibited by an equimolar amount of methotrexate. A folate binding protein could be extracted from either membrane preparation with 1% Triton X-100 and, to a lesser extent, with 0.6 M NaCl. These different extraction procedures resulted in different apparent molecular weights for folate binding protein (greater than 160,000 for Triton X-100-extracted samples and 40,000 for NaCl-extracted samples). The membrane preparation pellets remaining after NaCl extraction were able to rebind tritiated folic acid and also the 40,000-Da folate binding protein. On the other hand, membrane preparations extracted with Triton X-100 lost the ability to bind folic acid or the 40,000-Da folate binding protein. These differences in molecular weight and rebinding capacity may be explained by the existence of a receptor for folate binding protein which was extracted by Triton X-100, but not by NaCl. The greater concentration of folate binding protein in the renal tubule cell brush border membrane preparations as compared to those from basolateral membranes ascribes, for the first time, a functional role for folate binding protein in the renal reabsorption of folates which is required to prevent loss of folate in the urine and perhaps in the membrane transport of folates in general.     

Genetic polymorphism of the human sex hormone-binding globulin: evidence of an isoelectric focusing variant with normal androgen-binding affinities

Human sex hormone-binding globulin (hSHBG) is a plasma glycoprotein composed of two identical subunits. The protein, which has high affinity for testosterone and estradiol has been purified to homogeneity. In this study we have investigated, on neuraminidase-treated serum samples, the presence of genetic variations of hSHBG by polyacrylamide gel isoelectric focusing (IEF). Based on IEF analyses of 110 serum samples from adult Mexican individuals we have identified two distinct IEF-patterns. The most frequent phenotype (95.45%) was characterized by two IEF-bands with pIs of 6.50 and 6.63, respectively. In five serum samples, a different 4-band pattern with pIs of 6.50, 6.63, 6.70 and 6.76 was identified. Family studies showed that this pattern was genetically determined. The frequency of this variant was 4.55%, and the observed phenotypes were consistent with the expression of an autosomal genetic system. The estimated gene frequencies for both alleles were shown to be in genetic equilibrium. Affinity constants, binding kinetics and serum concentrations of hSHBG from individuals having a 4-band pattern were similar to those obtained in individuals with a 2-band pattern, thus suggesting that the mechanism responsible for the generation of polymorphic variants of hSHBG reported herein did not involve the steroid binding site of the molecule. These findings may be of broad interest, as other serum binding proteins express genetic variants, which may permit their further structural and functional subclassification.     

Akonni TruTip? and Qiagen? Methods for Extraction of Fetal Circulating DNA - Evaluation by Real-Time and Digital PCR

Due to the low percentage of fetal DNA present in maternal plasma (< 10%) during early gestation, efficient extraction processes are required for successful downstream detection applications in non-invasive prenatal diagnostic testing. In this study, two extraction methods using similar chemistries but different workflows were compared for isolation efficiency and percent fetal DNA recovery. The Akonni Biosystems TruTip technology uses a binding matrix embedded in a pipette tip; the Circulating Nucleic Acids Kit from Qiagen employs a spin column approach. The TruTip method adds an extra step to decrease the recovery of DNA fragments larger than 600 bp from the sample to yield an overall higher percentage of smaller molecular weight DNA, effectively enriching for fetal DNA. In this evaluation, three separate extraction comparison studies were performed - a dilution series of fragmented DNA in plasma, a set of clinical maternal samples, and a blood collection tube time point study of maternal samples. Both extraction methods were found to efficiently extract small fragment DNA from large volumes of plasma. In the amended samples, the TruTip extraction method was ~15% less efficient with overall DNA recovery, but yielded an 87% increase in % fetal DNA relative to the Qiagen method. The average percent increase of fetal DNA of TruTip extracted samples compared to the Qiagen method was 55% for all sets of blinded clinical samples. A study comparing extraction efficiencies from whole blood samples incubated up to 48 hours prior to processing into plasma resulted in more consistent % fetal DNA recoveries using TruTip. The extracted products were tested on two detection platforms, quantitative real-time PCR and droplet digital PCR, and yielded similar results for both extraction methods.     

Elevated epidermal growth factor receptor binding in plutonium-induced lung tumors from dogs

The objective of this study is to examine and characterize epidermal growth factor receptor (EGF-R) binding in inhaled plutonium-induced canine lung-tumor tissue and to compare it with that in normal canine lung tissue. Crude membrane preparations from normal and lung-tumor tissue from beagle dogs were examined in a radioreceptor assay, using 125I-labeled epidermal growth factor (EGF) as a ligand. Specific EGF receptor binding was determined in the presence of excess unlabeled EGF. We have examined EGF receptor binding in eight lung-tumor samples obtained from six dogs. Epidermal growth factor receptor binding was significantly greater in lung-tumor samples (31.38%) compared with that in normal lung tissue (3.76%). Scatchard plot analysis from the displacement assay revealed that there was no statistical difference in the binding affinity but significantly higher concentration of EGF-R sites in the lung-tumor tissue (619 fmol/mg) than in normal lung tissue (53 fmol/mg). The increase in EGF-R number in plutonium-induced dog lung tumors does not seem to correlate with increase in the initial lung burden exposure to plutonium. Our results demonstrate that there is a significant increase in EGF-R binding in inhaled plutonium-induced dog lung tumors.     

Acetylcholine Receptor Binding Properties at the Rat Neuromuscular Junction During Aging

Specific binding characteristics of acetylcholine receptors at the diaphragm neuromuscular junction of rats aged 10 (mature adult) and 28 (aged) months were assayed by measuring 125I-alpha-bungarotoxin binding. Maximal binding to intact tissue samples was greater in the older rats; this could be attributed to an age-related increase in terminal branching. The toxin concentration at which half-maximal binding occurred increased in the older rats. Binding kinetics were assayed in finely minced tissue samples, and the association rate constant was observed to decrease in the 28-month animals. Retardation of the initial rate of toxin binding by d-tubocurarine (dTC) in minced tissue was described by a two-component nonlinear Hofstee plot; IC50 values (7.1-7.2 microM and 39.0-46.5 nM) were about the same for both age groups, but there was a significant shift toward the low-affinity values in the aged rats. Rhodamine-conjugated alpha-bungarotoxin was used to visualize receptor localization. There were no major changes in receptor distribution, and nerve terminals were consistently associated with receptors and vice versa. The data indicate a shift toward lower binding affinity during aging, which may involve changes either in one of the two toxin-binding sites on individual receptors, in dTC blocking of the channel moiety, or in receptor types.