Factors influencing the choice of buffer in background electrolytes for indirect detection of fast anions by capillary electrophoresis

The suitability of relatively slow (low absolute value of mobility) coanionic buffers in background electrolytes (BGEs) for indirect photometric detection of anions by capillary electrophoresis was investigated. As a model system, 2-(cyclohexylamino)ethanesulfonic acid (CHES) was used to buffer the indirect detection electrolyte of sodium chromate. CHES (PKa 9.55) is a zwitterionic molecule carrying a net negative charge depending on the pH (effective charge -0.5 at pH = pKa). Within its useful pH buffering range CHES acted as a competing probe coanion. System peaks were induced which had deleterious effects on the detection sensitivity of slow to medium mobility anions. The mobility of the system peak was determined by the effective mobility of CHES, both of which increased with increasing pH. The peaks of analytes that migrated near or on the system peak were distorted and lost all quantitative properties. Analytes that migrated after the system peak either were not detected or reversed their responses. Analytes that migrated well before the system peak were unaffected. Consequently, the suitability of slow coanionic buffers is limited either to (i) fast anions or, (ii) a pH range much below the PKa, where the buffering capacity is not optimal.     

Components of the blood acid-base disturbance that accompanies urethane anaesthesia in rats during normothermia and hypothermia

1. We have studied the components of the metabolic acidosis that accompanies urethane anaesthesia in rats, both with and without the hypothermia that results from this anaesthesia. 2. Acid-base disturbances were analysed with an approach based on Stewart's analysis of acid-base chemistry. 3. The pH fall in the blood of normothermic anaesthetized rats (body temperature Tb) = 37 degrees C) was related to increases in plasma anions (lactate and [Cl-]), which decreased the strong ion difference ([SID]), as well as to increase the weak acid buffers due to increases in albumin. 4. A stronger metabolic acidosis was found in the blood of rats with hypothermia induced by urethane (Tb = 32 degrees C). Although plasma lactate was unchanged in hypothermic rats, [SID] decreased due to alterations in the plasma ionic balance. The metabolic acidosis found in hypothermia was also associated with increased weak acid buffers due to increases in albumin and inorganic phosphate. Further to hyperphosphataemia, signs of acute renal disfunction, such as increases in plasma [Mg2+] and blood urea nitrogen were found. Plasma retention of endogenous acids together with the retention of acid end-products of the metabolism of urethane because of acute renal failure may have contributed to strengthening the fall in pH and [HCO3-] found in urethane-induced hypothermic rats.     

Molecular cancer disposition diagnosis exemplified by colorectal carcinoma. What is the contribution of pathology?

The effect of low temperature on cytosolic pH regulation and buffering capacity was evaluated in the isolated rat liver. The pH changes were followed by phosphorus-31 nuclear magnetic resonance. Cooling from 37 to 4 degrees C, with Krebs-Heinseleit perfusion at an external pH of 7.35, induced an alkaline shift in cytosolic pH (pHcyt) of 0.13 or 0.75 pH units in the presence of bicarbonate, respectively (dpH cys/dT values were 0.004 and 0.022 unit/degrees C. With 4 degrees C perfusion, in the presence or absence of bicarbonate, acute changes of external pH (from 7.40 to 5.90) did not affect pHcyt. In contrast, intracellular loading with isobutyric acid or NH4Cl induced rapid pHcyt variations. The intrinsic buffering power value (10 to 50 slykes) measured in the absence of bicarbonate depended on pHcyt. The larger value was observed for pHcyt 7.30, a value near the pK value of the imidazole group of intracellular proteins at 4 degrees C. The presence of bicarbonate modified the amplitude of the pHcyt change by increasing the total buffering power. It was demonstrated that during hypothermia, ionic carriers are inactivated and the charged forms of molecules are unable to cross the cell membrane; thus, the pHcyt homeostasis depends essentially on intracellular buffering power.     

drp, a novel protein expressed at high cell density but not during growth arrest

In this study the disposition of 1,2-[14C]dibromoethane (1, 2-[14C]DBE) was investigated in male Wistar rats. 1,2-DBE is a cytotoxic and carcinogenic compound that has been used as an additive in leaded gasoline and as a fumigant. 1,2-[14C]DBE was administered orally or iv. Radioactivity was recovered (mostly within 48 hr after administration) in urine (75-82% of the dose), feces (3.2-4% of the dose), and expired air (0.53-7.2% of the dose). One hundred-sixty-eight hours after administration of 1,2-[14C]DBE, most of the radioactivity in tissues was found in the liver, lungs, and kidneys (<1% of the dose) and the red blood cells (0.3% of the dose). Identified urinary metabolites were S-(2-hydroxyethyl)mercapturic acid, thiodiacetic acid, and thiodiacetic acid sulfoxide, together accounting for, on average, 78% of the total amount of radioactivity in urine. In addition to S-(2-hydroxyethyl)mercapturic acid, thiodiacetic acid, and thiodiacetic acid sulfoxide, several compounds were anticipated as potential urinary metabolites of 1,2-DBE, i.e. S-(carboxymethyl)mercapturic acid, S-(2-hydroxyethyl)thioacetic acid, S-(2-hydroxyethyl)thiopyruvic acid, S-(carboxymethyl)thiopyruvic acid, S-(2-hydroxyethyl)thiolactic acid, and S-(carboxymethyl)thiolactic acid. All of the postulated urinary metabolites were synthesized and searched for in urine samples. None of these metabolites could be detected in urine, however. The data obtained in the present study might be useful for risk assessment and biomonitoring studies of 1,2-DBE and will also be used to further validate a physiologically based pharmacokinetic model for 1, 2-DBE in rats and humans that was recently developed.     

Generation of tryptic maps of alpha- and beta-globin chains by capillary electrophoresis in isoelectric buffers

A novel method for generating peptide maps, following tryptic digests of proteins, is reported here: capillary zone electrophoresis in the presence of isoelectric buffers as the sole buffering species. A typical buffer composition comprises 50 mM aspartic acid (pH = pI = 2.77), 0.5% hydroxyethyl cellulose (added as a dynamic coating agent for preventing peptide adsorption to weakly ionized silanols), 5% trifluoroethanol and 1% zwitterionic detergent (CHAPS). With this buffer composition, a high-voltage gradient can be applied (typically 600 V/cm in 75 microns I.D. and 900 V/cm in 50 microns I.D. capillaries), thus drastically reducing the analysis times. The method is applied to the generation of peptide maps of alpha- and beta-globin chains from human adult hemoglobin. In the case of beta-peptides, at an operative pH of 2.77, which represents a cross-over point in the titration curve of peptides T2 and T9, the two analytes merge into a single peak. However it is shown that it is possible to change the pH of the zwitterionic buffer by adjusting its concentration in solution. In 30 mM Asp (pH 3.0) or 20 mM Asp (pH 3.1) resolution of these two peptides is fully restored. Isoelectric, amphoteric buffers thus seem to represent a novel, powerful buffer system able to offer high resolution and high selectivity.     

Study of acid-base equilibria of fleroxacin

The acid-base equilibria of fleroxacin were studied by means of potentiometry and spectrophotometry. It was established that fleroxacin undergoes a complex acid-base equilibrium due to its zwitterionic nature and two proton-binding sites of similar acidity. The stoichiometric equilibrium constants were determined at 25 degrees C and constant ionic strength 0.1 M (NaCl). The acidity constants pK1 = 5.59 +/- 0.01 and pK2 = 8.08 +/- 0.04 were found by potentiometry, and pK1 = 5.61 +/- 0.03 and pK2 = 8.11 +/- 0.06 by spectrophotometry. The distribution diagram of the corresponding ionic species is given.     

Pyridyloxobutyl adduct O6-[4-oxo-4-(3-pyridyl)butyl]guanine is present in 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone-treated DNA and is a substrate for O6-alkylguanine-DNA alkyltransferase

The lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is activated to reactive metabolites that methylate or pyridyloxobutylate DNA. Previous studies demonstrated that pyridyloxobutylated DNA interferes with the repair of O6-methylguanine (O6-mG) by O6-alkylguanine-DNA alkyltransferase (AGT). The AGT reactivity of pyridyloxobutylated DNA was attributed to (pyridyloxobutyl)guanine adducts. One potential AGT substrate adduct, 2'-deoxy-O6-[4-oxo-4-(3-pyridyl)butyl]guanosine (O6-pobdG), was prepared. This adduct was stable at pH 7.0 for greater than 13 days and to neutral thermal hydrolysis conditions (pH 7.0, 100 degrees C, 30 min). Under mild acid hydrolysis conditions (0.1 N HCl, 80 degrees C), O6-pobdG was depurinated to yield O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-pobG). O6-pobdG was hydrolyzed to 4-hydroxy-1-(3-pyridyl)-1-butanone and guanine under strong acid hydrolysis conditions (0.8 N HCl, 80 degrees C). O6-pobG was detected in 0.1 N HCl hydrolysates of DNA alkylated with the model pyridyloxobutylating agent 4-(acetoxymethylnitrosamino)-1-(3-[5-3H]pyridyl)-1-butanone ([5-3H]NNKOAc). When [5-3H]NNKOAc-treated DNA was incubated with either rat liver or recombinant human AGT, O6-pobG was removed, presumably a result of transfer of the pyridyloxobutyl group from the O6-position of guanine to AGT's active site.     

Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis

We describe the extraction and enrichment of membrane proteins for separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) after differential solubilization of an Escherichia coli cell lysate. In a simple three-step sequential solubilization protocol applicable for whole cell lysates, membrane proteins are partitioned from other cellular proteins by their insolubility in solutions conventionally used for isoelectric focusing (IEF). As the first step, Tris-base was used to solubilize many cytosolic proteins. The resultant pellet was then subjected to conventional solubilizing solutions (urea, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, dithiothreitol, Tris, carrier ampholytes). Following the completion of this step, 89% of the initial E. coli sample mass was solubilized. Finally, the membrane protein rich pellet was partially solubilized using a combination of urea, thiourea, tributyl phosphine and multiple zwitterionic surfactants. Using N-terminal sequence tagging and peptide mass fingerprinting we have identified 11 membrane proteins from this pellet. Two of these outer membrane proteins (Omp), OmpW and OmpX, have previously been known only as an open reading frame in E. coli, while OmpC, OmpT and OmpTOLC have not previously been identified on a 2-D gel. The prefractionation of an entire cell lysate into multiple fractions, based on solubility, results in simplified protein patterns following 2-D PAGE using broad-range pH 3.5-10 immobilized pH gradients (IPGs). Additional advantages of sample prefractionation are that protein identification and gel matching, for database construction, is a more manageable task, the procedure requires no specialized apparatus, and the sequential extraction is conducted in a single centrifuge tube, minimizing protein loss.     

Capillary zone electrophoresis of oligonucleotides and peptides in isoelectric buffers: theory and methodology

The use of isoelectric buffers in capillary zone electrophoresis is reviewed. Such buffers allow application of extremely high voltage gradients (up to 1000 V/cm in relatively high bore capillary, e.g. 75 to 100 microm internal diameter), permitting separations of the order of a few minutes and thus favoring high resolution due to minimal, diffusion-driven peak spreading. The fundamental properties of ampholytes are first discussed, such as buffering power (beta) as a function of delta pK, i.e. of the distance between the pI value and neighboring protolytic groups. The highest possible relative beta value (= 2) is obtained for amphoteres possessing a delta pK = 0.6, a condition not met by existing amphoteric species. A novel parameter for ampholyte evaluation is then proposed, namely the beta/lambda ratio, i.e. the ratio between the beta power and conductivity at the pI value. It is additionally shown that the pI is not a constant value, but depends on ampholyte concentration in solution. In addition, at constant concentration, the theoretical pI can change as a function of delta pK. Isoelectric His and, to a lesser extent, Lys have been found to offer unique separations of oligonucleotides in sieving liquid polymers. In the absense of sieving media, isoelectric Asp, in presence of 7 M urea (apparent pH 3.77), permits unique separations of oligonucleotides having the same length but different nucleotide composition. Isoelectric Asp (pI 2.77 at 50 mM concentration) provides a medium of high resolving power for generating peptide maps. In difficult cases, of coincident titration curves, the pH can be moved up to higher values (e.g. pH 3.0 for 30 mM Asp) thus eliciting separation of unresolved peptides at pH 2.77. This was illustrated by running peptide maps of tryptic digests of human beta-globin chains. Also imino diacetic acid (pI 2.33 at 50 mM concentration) allows generation of high resolution peptide maps.     

Oleic acid binding and transport capacity of human red cell membrane

Resealed human red cell membranes, ghosts, bind oleate (OL) by a limited number of sites when equilibrated at 37 degrees C, pH 7.3 with OL bound to bovine serum albumin (BSA) in molar ratios below 1.5. The binding capacity is 34 +/- 2.2 nmol g-1 ghosts with a dissociation equilibrium constant (37 degrees C) Kdm 1.38 +/- 0.15 fold Kd of albumin binding Kdm is temperature independent and approximately 7-8 nM. Exchange efflux kinetics at 0 degrees C to buffers of various albumin concentrations ([BSAy]) is biexponential and is analysed in terms of a three-compartment model. Accordingly the ratio of inner to outer membrane leaflet binding sites is 0.450 +/- 0.018 and the rate constant of unidirectional flux from inside to outside is 0.067 +/- 0.01 s-1. The rate constant of flux from the extracellular side of the membrane to BSAy increases with the square root of [BSAy] as expected of an unstirred layer effect. This provides an estimate of the dissociation rate constant of OL-BSA complex at 0 degrees C of 0.0063 +/- 0.0003 s-1. Exchange efflux from ghosts containing four different [BSAi] obeys the expected kinetics of a three-compartment approximation of the theoretical model. Accounting for the effect of an unstirred fluid inside ghosts, the rate coefficients fit the values predicted by the parameters obtained by the studies of albumin-free ghosts. The results show that the OL transport across the membrane is mediated exclusively by the asymmetrically distributed binding sites. The differences between transport sites of three long-chain fatty acids suggest that they are protein determined microdomains of phospholipids.     

Mechanisms of adrenergic agonist induced allergy bioactivation and antigen formation

A density gradient electrophoresis apparatus made of Perspex (7 cm, O 2.2 cm) with a circular platinum anode and a palladium cathode was used for the separation of proteins in free liquid. Following a concept developed by M. Bier et al. (Electrophoresis 1993, 14, 1011-1018), mixtures of two suitable amphoteric buffers I and II provide for media with a fixed and electrophoretically stable pH or were used for the generation of preformed (electrophoretically stable) pH gradients covering about 1 pH unit. Amphoters I and II are considered suitable if there is overlap between (pK(1,1)-1-2) and the pK(2,II)+1+2) region. 3-(N-Morpholino)propanesulfonic acid (MOPS) and gamma-amino-n-butyric acid (GABA) were used as an example. Two approaches were followed: (i) rate-zonal separation of test proteins in a pH window, formed by a fixed ratio of MOPS/GABA. (ii) Isoelectric focusing in a shallow preformed pH gradient, made up of inverse reciprocal linear gradients of MOPS and GABA. At isopH, test proteins (bovine serum albumin, cytochrome c, ferritin, hemoglobin, lactoglobulin, myoglobin, and transferrin) were rate-zonally separated within a short time. Even the separation of the A and B forms of lactoglobulin was feasible at isopH. The glycoforms of transferrin were separated and enriched on a pH 5.2-6.1 pH gradient, indicating that pH differences of about 0.01 still permit resolution. Contrary to the ill-defined Ampholines, the cost of these well-defined amphoters is low.     

Continuous sucrose hydrolysis by yeast cells immobilized to wool

A novel immobilized biocatalyst with invertase activity was prepared by adhesion of yeast cells to wool using-glutaraldehyde. Yeast cells could be immobilized onto wool by treating either the yeast cells or wool or both with glutaraldehyde. Immobilized cells were not desorbed by washing with 1 M KCl or 0.1 M buffers. pH 3.5-7.5. The biocatalyst shows a maximum enzyme activity when immobilized at pH 4.2-4.6 and 7.5-8.0. The immobilized biocatalyst was tested in a tubular fixed-bed reactor to investigate its possible application for continuous full-scale sucrose hydrolysis. The influence of temperature, sugar concentration and flow rate on the productivity of the reactor and on the specific productivity of the biocatalyst was studied. The system demonstrates a very good productivity at a temperature of 70 degrees C and a sugar concentration of 2.0 M. The increase of the volume of the biocatalyst layer exponentially increases the productivity. The productivity of the immobilized biocatalyst decreases no more than 50% during 60 days of continuous work at 70 degrees C and 2.0 M sucrose, but during the first 30 days it remains constant. The cumulative biocatalyst productivity for 60 days was 4.8 x 10(3) kg inverted sucrose/kg biocatalyst. The biocatalyst was proved to be fully capable of continuous sucrose hydrolysis in fixed-bed reactors.     

Bronchial obstruction and exhaled nitric oxide response during exercise in cold air

This study examines whether exhausting exercise in cold air induces bronchial obstruction and changes in exhaled [NO] and in exhaled NO output (V'NO). Thus, eight well-trained males performed two incremental exercise tests until exhaustion, followed by 5 min of recovery in temperate (22 degrees C) and cold (-10 degrees C) environments, at random. At -10 degrees C, they were dressed in warm clothes. Ventilation (V'E), oxygen consumption (V'O2), carbon dioxide production, cardiac frequency (fC), and [NO] and V'NO were measured continuously. Before and after each test, the subjects' maximal expiratory flow-volume curves and peak expiratory flow, forced expiratory volume in one second (FEV1) and forced expiratory flow at 25 (FEF25), 50 (FEF50) and 75% (FEF75) of forced vital capacity were determined. At -10 degrees C, significant decreases in FEV1 and FEF75 were observed after exercise. At rest and at the same submaximal intensity, V'O2, V'E and fC did not differ significantly. At rest and up to approximately 50% peak V'O2, [NO] and V'NO values were lower at -10 degrees C than at 22 degrees C. Thereafter, and during recovery, the V'NO response became similar at both -10 and 22 degrees C. This study confirms that considerable hyperpnoea in cold air causes a detectable airway obstruction. This airway cooling also induces an initial decrease in the exhaled NO response. Since endogenous NO-production is involved in bronchial dilation, it cannot be excluded that this lack of production may favour the appearance of airway obstruction.     

Chain length specificity in the utilization of long chain alcohols for ether lipid biosynthesis in rat brain

A mixture of cis-9[1(-14)C] octadecenol and [1(-14)C] docosanol was injected into the brains of 19-day-old rats, and incorporation of radioactivity into brain lipids was determined after 3, 12, and 24 hr. Both alcohols were metabolized by the brain but at different rates; each was oxidized to the corresponding fatty acid, but oleic acid was more readily incorporated into polar lipids. Substantial amounts of radioactivity were incorporated into 18:1 alkyl and alk-1-enyl moieties of the ethanolamine phosphoglycerides and into 18:1 alkyl moieties of the choline phosphoglycerides. Even after the disappearance of the 18:1 alcohol from the substrate mixture (12 hr), the 22:0 alcohol was not used to any measurable extent for alkyl and alk-1-enylglycerol formation.     

Biotransformation of 17-alkylsteroids in the equine: gas chromatographic-mass spectral identification of ten intermediate metabolites of methyltestosterone

Parenterally administered domoic acid, a structural analog of the excitatory amino acids glutamic acid and kainic acid, has specific effects on brain histology in rats, as measured using different anatomic markers. Domoic acid-induced convulsions affects limbic structures such as hippocampus and entorhinal cortex, and different anatomic markers can detect these neurotoxic effects to varying degrees. Here we report effects of domoic acid administration on quantitative indicators of brain metabolism and gliosis. Domoic acid, 2.25 mg/kg i.p., caused stereotyped behavior and convulsions in approximately 60% of rats which received it. Six to eight days after domoic acid or vehicle administration, the animals were processed to measure regional brain incorporation of the long-chain fatty acids [1-(14)C]arachidonic acid ([14C]AA) and [9,10-(3)H]palmitic acid ([3H]PA), or regional cerebral glucose utilization (rCMRglc) using 2-[1-(14)C]deoxy-D-glucose, by quantitative autoradiography. Others rats were processed to measure brain glial fibrillary acidic protein (GFAP) by enzyme-linked immunosorbent assay. Domoic acid increased GFAP in the anterior portion of cerebral cortex, the caudate putamen and thalamus compared with vehicle. However, in rats that convulsed after domoic acid GFAP was significantly increased throughout the cerebral cortex, as well as in the hippocampus, septum, caudate putamen, and thalamus. Domoic acid, in the absence of convulsions, decreased relative [14C]AA incorporation in the claustrum and pyramidal cell layer of the hippocampus compared with vehicle-injected controls. In the presence of convulsions, relative [14C]AA incorporation was decreased in hippocampus regions CA1 and CA2. Uptake of [3H]PA into brain was unaffected. Relative rCMRglc decreased in entorhinal cortex following domoic acid administration with or without convulsions. These results suggest that acute domoic acid exposure affects discrete brain circuits by inducing convulsions, and that domoic acid-induced convulsions cause chronic effects on brain function that are reflected in altered fatty acid metabolism and gliosis.     

Further studies on the possible compartmentation of the precursor pool of cholesterol for the biosynthesis of cholic acid in the rat

In vivo and in vitro experiments with rats were carried out to investie precursor for the biosynthesis of cholic acid. When rats with a bile-fistula were given a mixture of [2-14C]mevalonate and [1,2-3H]cholesterol intravenously, the 14C:3H ratio in cholic acid in both whole homogenate and cytosol prepared from their lives was higher than that in free cholesterol in any subcellular fraction of the livers. When [2-14C] mevalonate was administered intravenously to bile-fistula rats, the specific radioactivity of free cholesterol in the hepatic microsomal fraction exceeded that in any other fraction, and the specific radioactivity of biliary cholic acid was remarkably high, exceeding that of microsomal free cholesterol. In similar experiments with [4-14C] cholesterol, the specific radioactivity of free cholesterol in the hepatic microsomal fraction exceeded that in any other subcellular fraction and the specific radioactivity of biliary cholic acid was lower than that of free cholesterol in any hepatic subcellular fraction. Tissue suspensions of rat livers in Krebs-Ringer bicarbonate (pH 7.4)-5.5 mM glucose were incubated with [2-14C]mevalonate in O2-CO2 (95:5, v/v) at 37 degrees. The specific radioactivity of free cholesterol in the microsomal fraction prepared from the incubated tissue exceeded the specific radioactivities of free cholesterol in the other subcellular fractions. The estimated specific radioactivity of taurocholate formed during the incubation was far higher than that of microsomal free cholesterol. These data indicate that hepatic microsomal free cholesterol which was newly synthesized in situ was preferentially incorporated into cholic acid.     

High-resolution density gradient electrophoresis of proteins and subcellular organelles

Following a concept developed by Bier et al. (Electrophoresis 1993, 14, 1011-1018), binary mixtures of amphoteric buffers with low conductivity and a good buffering capacity permit rapid rate zonal separation of proteins on a density gradient electrophoresis apparatus (7 cm, x 2.2 cm). At pH 8.66 and 250 V, beta-lactoglobulin (Mr 36600) was separated into the A and B isoforms within 44 min; human transferrin (Mr 76000-81000) was separated into its sialylated glycoforms and carbonic anhydrase (Mr 30000) separated into its isoenzymes. From these results we arrive at the term high-performance density gradient electrophoresis. Compartments belonging to the endosomal system were separated by density gradient electrophoresis. Early endosomes, recycling vesicles, intermediate endosomes, late endosomes and lysomes became well-separated after 80 min at 10 mA using [125I]transferrin and horseradish peroxidase as reporter molecules in pulse-chase regimes. Mixtures of Bier buffers and standard electrophoresis media permitted very short separation times (19 min at 10 mA) for the endosomal compartments. Concommittantly, endoplasmic reticulum and proteasomes were well resolved.     

Synthesis and structure-activity relationships of a new series of benzimidazoles as H1-antihistaminic agents

New 2-(4-(4-azolylbutyl)piperazinyl)-, 2-(4-(4-azolylbutyl) piperazinylmethyl)-, 2-(4-(-azolylbutyl)homopiperazinyl)- and 2-(4-(4-azolylbutyl)homopiperazinylmethyl)benzimidazoles were synthesized, characterized and tested for in vitro and in vivo H1-antihistaminic activity. Structure-activity relationships implied that the best antihistaminic activity required the simultaneous presence of a homopiperazinylbenzimidazole system (or a methylene linker between the benzimidazole and the piperazine rings) and an unsubstituted pyrazole ring. 1-(2-Ethoxyethyl)-2-?4-[4-(pyrazol-1-yl)butyl] homopiperazin-1-yl?benzimidazole (17), as its dimaleate salt, has been chosen for further development.     

Cis-Dichloro(diaminosuccinate diethyl ester)palladium (II) as Pd(II)/Pt(II) model compound for DNA-binding and antitumor properties: solution equilibria of their aqua-, hydroxo-, and/or chloro-species

Cis-Dichloro(diaminosuccinic acid)palladium(II), cis-[Pd(H2dasa)Cl2] (I), or cis-dichloro(diaminosuccinate diethyl ester)palladium(II), cis-[Pd(Et2dasa)Cl2] (II) reacts with two equivalents of AgClO4 to give insoluble Pd(dasa) or an aqueous solution of [Pd(Et2dasa) (H2O)2](ClO4)2, respectively. Three solutions of this salt were titrated with NaOH (I = NaClO4 (0.15M),37 degrees C), and 133 E(H+) data (3.5 < or = pH < or = 7) were treated by SUPERQUAD to fit log beta pqr of cis-aquahydroxo-(pqr = 10-1, -5.25(3)) and di-mu-hydroxo-species (20-2, -6.55(1)). At pH > 7 the ester hydrolysis prevents the calculation of log beta 10-2 for the cis-dihydroxo-complex. Another three solutions of such salt were titrated (I = 0.15M (NaClO4), 37 degrees C) with NaOH and NaCl simultaneously using two potentiometric systems (which measure H+ or Cl-). From 147 E(H+) and E(Cl-) data pairs and the above fixed log beta pqr, SUPERQUAD calculations yield log beta pqr for cis-chloro-aqua (pqr = 110, 3.65(1)), cis-chloro-hydroxo (11-1, -2.68 (4)), and cis-dichloro-species (120, 5.86(3)). Simulated and experimental titrations are in good agreement. Circular dichroism spectra of native DNA and drug:DNA complexes suggest that cis-Pd(H2dasa) and cis-Pd(Et2dasa) chelate moieties induce an opening and rotation of the stacked bases in the double helix. This finding is explained by the abundance of each one and of the total neutral and charged species of II in the tested CD solution.     

Purification and characterization of a feruloyl esterase from the fungus Penicillium expansum

An extracellular phenolic acid esterase produced by the fungus Penicillium expansum in solid state culture released ferulic and rho-coumaric acid from methyl esters of the acids, and from the phenolic-carbohydrate esters O-[5-O-(trans-feruloyl)-alpha-L-arabinofuranosyl]-(1-->3)-O-beta- D-xylopyranosyl-(1-->4)-D-xylopyranose (FAXX) and O-[5-O-((E)-rho-coumaroyl)-alpha-L-arabinofuranosyl]- (1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (PAXX). The esterase was purified 360-fold in successive steps involving ultrafiltration and column chromatography by gel filtration, anion exchange and hydrophobic interaction. These chromatographic methods separated the phenolic acid esterase from alpha-L-arabinofuranosidase, pectate and pectin lyase, polygalacturonase, xylanase and beta-D-xylosidase activities. The phenolic acid esterase had an apparent mass of 65 kDa under non-denaturing conditions and a mass of 57.5 kDa under denaturing conditions. Optimal pH and temperature were 5.6 and 37 degrees C, respectively and the metal ions Cu2+ and Fe3+ at concentrations of 5 mmol 1-1 inhibited feruloyl esterase activity by 95% and 44%, respectively, at the optimum pH and temperature. The apparent Km and Vmax of the purified feruloyl esterase for methyl ferulate at pH 5.6 and 37 degrees C were 2.6 mmol 1-1 and 27.1 mumol min-1 mg-1. The corresponding constants of rho-coumaroyl esterase for methyl coumarate were 2.9 mmol 1-1 and 18.6 mumol min-1 mg-1.