Exercise training reduces pulmonary ischaemia-reperfusion-induced inflammatory responses.

Physical exercise reduces the deleterious effects of cardiovascular and inflammatory disorders. The purpose of the present study was to evaluate the beneficial effects of physical training on the inflammatory responses following lung ischaemia-reperfusion (IR) in rats. Male Wistar rats were divided into sham-operated animals and sedentary and trained animals submitted to lung IR. The run training programme consisted of 5 sessions.week(-1), each lasting 60 min.day(-1), at 66% of maximal oxygen consumption for 8 weeks. The left pulmonary artery, bronchus and pulmonary vein were occluded for 90 min and reperfused for 2 h. Lung protein extravasation was measured as (125)I-human albumin accumulation, whereas lung neutrophil infiltration was measured as myeloperoxidase activity. Lung IR in sedentary rats resulted in marked increases in protein extravasation and neutrophil influx, and in significant elevations of serum tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta levels. Physical preconditioning attenuated the increased IR-induced protein leakage without affecting neutrophil influx. It also reduced serum TNF-alpha (and IL-1beta) levels, but had no effect on IL-10 levels. Plasma superoxide dismutase activity was significantly increased in trained IR rats. The present data show that physical preconditioning protects the rat lung from ischaemia-reperfusion injury by attenuating the pulmonary vascular permeability that may be a consequence of reduced levels of tumour necrosis factor-alpha and interleukin-1beta and elevated superoxide dismutase activity.

Conformational flexibility in T4 endonuclease VII revealed by crystallography: implications for substrate binding and cleavage.

The structure of the N62D mutant of the junction-resolving endonuclease VII (EndoVII) from phage T4 has been refined at 1.3 A, and a second wild-type crystal form solved and refined at 2.8 A resolution. Comparison of the mutant with the wild-type protein structure in two different crystal environments reveals considerable conformational flexibility at the dimer level affecting the substrate-binding cleft, the dimerization interface and the orientation of the C-terminal domains. The opening of the DNA-binding cleft, the orientation of the C-terminal domains relative to the central dimerization domain as well as the relative positioning of helices in the dimerization interface appear to be sensitive to the crystal packing environment. The highly unexpected rearrangement within the extended hydrophobic interface does change the contact surface area but keeps the number of hydrophobic contacts about the same and will therefore not require significant energy input. The conformational flexibility most likely is of functional significance for the broad substrate specificity of EndoVII. Binding of sulphate ions in the mutant structure and their positions relative to the active-site metal ions and residues known to be essential for catalysis allows us to propose a possible catalytic mechanism. A comparison with the active-site geometries of other magnesium-dependent nucleases, among them the homing endonuclease I-PpoI and Serratia endonuclease, shows common features, suggesting related catalytic mechanisms.

Single-chain repressors containing engineered DNA-binding domains of the phage 434 repressor recognize symmetric or asymmetric DNA operators.

Single-chain (sc) DNA-binding proteins containing covalently dimerized N-terminal domains of the bacteriophage 434 repressor cI have been constructed. The DNA-binding domains (amino acid residues 1 to 69) were connected in a head-to-tail arrangement with a part of the natural linker sequence that connects the N and C-terminal domains of the intact repressor. Compared to the isolated N-terminal DNA-binding domain, the sc molecule showed at least 100-fold higher binding affinity in vitro and a slightly stronger repression in vivo. The recognition of the symmetric O(R)1 operator sequence by this sc homodimer was indistinguishable from that of the naturally dimerized repressor in terms of binding affinity, DNase I protection pattern and in vivo repressor function. Using the new, sc framework, mutant proteins with altered DNA-binding specificity have also been constructed. Substitution of the DNA-contacting amino acid residues of the recognition helix in one of the domains with the corresponding residues of the Salmonella phage P22 repressor c2 resulted in a sc heterodimer of altered specificity. This new heterodimeric molecule recognized an asymmetric, artificial 434-P22 chimeric operator with high affinity. Similar substitutions in both 434 domains have led to a new sc homodimer which showed high affinity binding to a natural, symmetric P22 operator. These findings, supported by both in vitro and in vivo experiments, show that the sc architecture allows for the introduction of independent changes in the binding domains and suggest that this new protein framework could be used to generate new specificities in protein-DNA interaction.

Effect of the progestogen, allylestriol, on the bursal development of the chicken.

Fertile eggs were dipped on the fifth day of incubation into varying concentrations of allylestriol (AE), a synthetic progesterone, and testosterone propionate (TP). Concentrations of AE higher than 0.1% inhibited hatchability. The AE inhibited the differentiation of the bursal mesenchyme and follicular development and subsequently resulted in bursectomy. The AE caused bursectomy in 40-50 times lower concentrations than did TP. An unusual lymphocyte accumulation occurred around the postcapillary venules in the bursal mesenchyme of AE-treated birds. This observation suggested that inhibition of mesenchyme differentiation may lead to a modification in bursal function. The AE modification of bursal development was compared to those produced by TP. We demonstrated that AE caused immunosuppression of the B-cell system.

Structural changes in the mouse thymus and lymph node after emetine treatment.

The effect of emetine which is a potent immunosuppresant was studied on the thymus and lymph node. The subcapsular zone of the thymus was depleted and large number of adherent cells accumulated in this thymic region. The medulla enlarged but the cortico-medullary border remained distinct. In the paracortex (T dependent area) of the lymph node many non-lymphoid pyroninophil cells appeared which is followed by an increased cell proliferation 24 hours after emetine injection. 48-60 hours after administration many macrophage-like cells appeared in the medullary sinuses. This macrophage invasion precedes the adherent cell accumulation in the subcapsular zone of the thymus suggesting a possible non-lymphoid (adherent) cell migration from the lymph node's paracortex to the thymus.

Determination and characterization of diuretics in human urine by liquid chromatography coupled to pneumatically assisted electrospray ionization mass spectrometry.

The aim of this work was to develop a method for the characterization and determination of diuretics in human urine samples by liquid chromatography (LC) coupled to pneumatically assisted electrospray ionization (ES) mass spectrometry (MS). The diuretics studied were substances forbidden by the IOC such as trichlormethiazide, furosemide, canrenoic acid, benzthiazide, bendroflumethiazide, bumetanide, etacrynic acid and spironolactone. For this purpose, the operational parameters of electrospray, such as counter electrode voltage, capillary voltage, sample cone voltage and source temperature, were optimized in order to obtain the best signal stability and the highest sensitivity for the greatest number of diuretic agents. The optimized separation method was successfully coupled with the MS system to analyze the above-mentioned diuretics extracted from spiked urine samples by a liquid extraction and clean-up procedure at basic pH, using ethyl acetate as solvent and the salting-out effect (NaCl). The mass spectra obtained provide adequate information for identification purposes. Positive urine samples obtained from athletes were also analyzed. The presence of these substances in human urine was confirmed by this method, making LC/ES-MS an analytical tool to be considered in the area of antidoping control.

Beta-amyloid(Phe(SO3H)24)25-35 in rat nucleus basalis induces behavioral dysfunctions, impairs learning and memory and disrupts cortical cholinergic innervation.

Long-term behavioral effects, changes in learning and memory functions and aberrations of cholinergic fibers projecting to the parietal cortex were investigated after bilateral injections of beta-amyloid(Phe(SO3H)24)25-35 peptide in rat nucleus basalis magnocellularis (nbm). The beta-amyloid peptide used in these experiments contained the original beta-amyloid 25-35 sequence which was coupled to a phenylalanine-sulphonate group at position 24. This additional residue serves as a protective cap on the molecule without influencing its neurotoxic properties and results in water-solubility, stability and low rates of peptide metabolism. In this paper, home cage, locomotor and open-field activities, passive shock-avoidance and 'Morris' water maze learning abilities were assessed throughout a 35-day survival period. Subsequently, acetylcholinesterase (AChE) histochemistry was used to visualize alterations of parietal cortical cholinergic innervation. In response to the neurotoxic action of beta-amyloid(Phe(SO3H)24)25-35, a progressive hyperactivity developed in the rats in their home cages which were maintained throughout the 5-week post-injection period. This was accompanied by a significant hypoactivity in the novel environment of a locomotor arena. Beta-amyloid(Phe(SO3H)24)25-35-treated animals showed greatly impaired cortical memory functions in the step-through passive shock-avoidance paradigm, while spatial learning processes remained unaffected. Moreover, beta-amyloid(Phe(SO3H)24)25-35 injections in the nucleus basalis suppressed explorative behavior in rats and inhibited conditioned stress responses 28 days after surgery. Reductions of cortical cholinergic (AChE-positive) projections provided anatomical substrate for the behavioral changes. This indicated extensive, long-lasting neurodegenerative processes as a result of beta-amyloid(Phe(SO3H)24)25-35 infusion.

Separation of potentially therapeutic peptide hormones by liquid chromatography. Optimisation of the composition and pH of the mobile phase.

The aim of this work is to optimise the proportion of the organic modifier and the pH of the mobile phase, in order to separate a series of peptide hormones with therapeutic interest in the molecular mass range from 500 to 6000. The composition of the mobile phase was optimised by establishing relationships between retention parameters and either the scale of solvent polarity, or the Kamlet-Taft multiparameter solvent scale of the eluent, using linear solvation energy relationships. Likewise, linear correlations between the chromatographic retention and Reichardt's E(T)N parameter were obtained. These relationships allowed an important reduction of the experimental retention data needed for developing a given separation. In addition, a model describing the effect of the correctly measured pH of the mobile phase on retention in LC was established and tested for the series of selected peptides using an octadecylsilica column. The proposed equations permit the prediction of the optimum pH and also permit the determination of the acidity constants of the peptides in the hydro-organic mixtures using a minimum number of measurements.

Analysis of a peptide hormone mixture of therapeutic interest by liquid chromatography coupled to high-flow pneumatically assisted electrospray mass spectrometry.

High-flow pneumatically assisted electrospray ionization mass spectrometry (ESI-MS) has been extensively used for the characterization and determination of peptides and peptide hormones available for biomedical research and therapeutic applications. The aim of this study was to optimize a method of characterization and determination of a mixture of peptide hormones with therapeutic interest by liquid chromatography (LC) coupled to ESI-MS. In this work the linear solvation energy relationship methodology was used in order to optimize the mobile phase to be used in the LC separation of the peptide hormone series and the operational parameters of the source and analyzer of ESI were also optimized to obtain the best signal stability and the highest sensitivity. To validate the proposed method for peptide hormone analysis, quality parameters were determined and satisfactory results were obtained. Likewise, the method detection limit was picomole level for most of the peptides employing selected-ion monitoring of the [M+nH]n+ ions.

Prediction of retention behaviour and evaluation of pka values of peptides and quinolones in liquid chromatography.

The present paper examines the effect of the solute ionisation on the retention behaviour in liquid chromatography of a series of peptide and quinolone compounds of biological interest, using acetonitrile-water media as mobile phases and a polymeric-based stationary phase. Polymeric columns with polystyrene-divinylbenzene (PS-DVB) polymer show advantages over silica-based reversed-phase packings since the former are stable in a wide pH range. (s)(s)pKa values have been evaluated using chromatographic data in acetonitrile-water mixtures with acetonitrile percentages of 30, 35, 40 and 50% (v/v) for quinolones and 12.5 and 20% (v/v) for peptides. The quinolones show great retention on PS-DVB phase stationary. It was thus necessary to work with a higher acetonitrile content in the mobile phase than for the less retained peptides. The pH values were measured in the hydroorganic mixtures, used as mobile phases, instead of in water and account was taken of the effect of activity coefficients. The derived equations permit the chromatographic determination of (s)(s)pKa. values of the peptides and quinolones in acetonitrile-water mixtures by fitting it to the experimental data in a nonlinear least-square procedure and also permit the prediction of the effect of (s)(s)pH on their chromatographic behaviour. We have also compared the obtained (s)(s)pKa values with those previously obtained in acetonitrile-water mixtures from potentiometric measurements.

Solvatochromic parameter values and pH in acetonitrile-water mixtures. Optimization of mobile phase for the separation of peptides by high-performance liquid chromatography.

The proportion of the organic modifier and the pH of the mobile phase were optimized in order to separate a series of low-molecular-mass peptides by HPLC. The composition of the mobile phase was optimized by establishing relationships between retention parameters and Reichardt's ENT scale of solvent polarity, and between retention and the Kamlet-Taft multiparameter solvent scale of the eluent, using linear solvation energy relationships (LSER). The pH of the mobile phase was also successfully optimized by establishing relationships between the retention and the pH measured in the aqueous-organic mixture used as eluent.

Evaluation of chromatographic versus electrophoretic behaviour of a series of therapeutical peptide hormones.

In this work, models describing the effect of pH on chromatographic and electrophoretic behaviour for a series of polyprotic therapeutic peptide hormones were compared. taking into account the species in solution and the activity coefficients. The usefulness of the proposed equations is twofold, they permit the determination of the acidity constants in water and in the hydroorganic mobile phases used in liquid chromatography (LC) and capillary electrophoresis (CE) and can also be used for the selection of the optimum pH for the separation of mixtures of the modelled compounds. The proposed relationships allow an important reduction of the experimental data needed for the development of new separation methods. The accuracy of the proposed equations is verified by modelling the chromatographic and electrophoretic behaviour of a series of polyprotic therapeutic peptide hormones. By calculating the values of predicted resolutions, selection of the optimum pH to perform LC or CE separations of their mixtures becomes a rapid and simple process.

pKa values of peptides in aqueous and aqueous-organic media. Prediction of chromatographic and electrophoretic behaviour.

In the present work, models describing the effect of the pH on the chromatographic and electrophoretic behaviour for polyprotic peptides were compared. The proposed models can be simultaneously used for determination of dissociation constants and selection of the optimum pH for the separation of peptides, in water and acetonitrile-water mixtures widely used in liquid chromatography and in capillary electrophoresis. The models use the pH value measured in the acetonitrile-water mixture instead of the pH value in water and take into account the effect of the activity coefficients. They permit the determination of the acidity constants in the aqueous and hydro-organic mobile phase from chromatographic retention and electrophoretic migration measurements, respectively. The values obtained by both proposed techniques agree with the potentiometric values previously determined. The suitability of the proposed models for predicting chromatographic and electrophoretic behaviour of compounds studied from a limited number of experimental data was also compared. The separation between solutes by both techniques in a complex mixture can be easily predicted, making simple and rapid pH selection to achieve optimum separation.

Retention behaviour of peptides, quinolones, diuretics and peptide hormones in liquid chromatography. Influence of ionic strength and pH on chromatographic retention.

Peptides, quinolones, diuretics and peptide hormones are important substances of biomedical interest. In this work a model describing the effect of pH on retention in liquid chromatography (LC) is established and tested for these compounds using an octadecylsilica column. The suggested model uses the pH value measured in the hydro-organic mixture used as mobile phase instead of the pH value in water and takes into account the effect of the activity coefficients. The proposed equations permit the prediction of the pH optimum using a minimum number of measurements and also permit the determination of the acidity constants of the compounds considered in the medium used as mobile phase. Moreover, these equations can be combined with the previously derived equations, that relate the retention with the solvent composition of the mobile phase, to establish a general model that relates the elution behaviour of the solute with the significant mobile phase properties: composition, pH and ionic strength.

Electrophoretic behavior of peptides in capillary electrophoresis influence of ionic strength and pH in aqueous-organic media.

Through correct pH, pKa and activity coefficients values, a model describing the effect of pH on electrophoretic mobility of substances has been applied to a series of peptides in water and in acetonitrile-water mixtures. The derived equations permit prediction of the optimum pH for the electrophoretic separation from only a few experimental values and they also permit determination of pKa values of analytes in the aqueous-organic media employed. Furthermore, the electrophoretic resolution between pairs of substances can be predicted, in order to evaluate electrophoretic separations of the studied peptides.

Development and validation of a capillary electrophoresis method with ultraviolet detection for the determination of the related substances in a pharmaceutical compound.

A capillary electrophoresis (CE) method was successfully developed to quantify the impurity profile of a new substance of pharmacological interest: LAS 35917. CE method was developed in order to separate the chloromethylated, monomethylated and hydroxylated impurities (molecules with very similar chemical structures) having the three coelution in the reversed-phase LC method initially established. Taking into account the structure of the impurities of LAS 35917, separation by conventional liquid chromatography (LC) methods would be longer and tedious than separation by CE, which is an appropriate and versatile technique giving easier and quicker methods. Among the three potential impurities mentioned of LAS 35917, two are due to the synthesis route of this drug, and the third arises from degradation. These drug-related impurities were separated using a capillary of 56 cm of effective length and 50 microm I.D., a 60 mM tetraborate buffer, at pH 9.2, and a positive voltage of 20 kV. The optimised CE method was preliminary validated with regard to specificity, linearity, limits of detection and quantitation, repeatability and solution stability. The method allows the detection and quantitation of impurities above 0.04 and 0.08% level, respectively. All three related substances were separated, detected and quantified from their parent drug in the analysis of real samples of LAS 35917, stressed or not stressed, with this simple and fast CE method.

Characterization of human transferrin glycoforms by capillary electrophoresis and electrospray ionization mass spectrometry.

Carbohydrate Deficient Glycoprotein Syndrome (CDGS) is an inherited metabolic disease affecting all parts of the body. The biochemical diagnosis of this syndrome is based on the presence of a special marker in blood, Carbohydrate Deficient Transferrin (CDT), which is also a marker of chronic alcohol abuse. CDT is characterized by abnormal glycoforms of serum transferrin (Tf). In the present study, electrophoretic separation of human serum transferrin glycoforms was carried out using a bare fused-silica capillary and the glycoforms present in commercial Tf were baseline separated. The limit of detection (LOD) of human Tf was around the nmol concentration range. The LOD of the trisialo- and disialo-Tf, expressed as percentages of the tetrasialo-Tf peak area, were 0.5% for trisialo-Tf and 0.4% for disialo-Tf, and these values were appropriate for CDGS diagnosis. Moreover, Tf glycoforms were characterized using mass spectrometry (MS). The method was applied to the analysis of normal and pathological serum samples, after dilution. The results obtained suggest a way of making a rapid and simple CDGS diagnosis.

Determination of fluoroquinolones in human urine by liquid chromatography coupled to pneumatically assisted electrospray ionization mass spectrometry.

The fluoroquinolones are synthetic antimicrobial agents widely used in human and veterinary medicine. The aim of this work was to develop a method of characterization and determination of three widely used fluoroquinolones (norfloxacin, ciprofloxacin and ofloxacin) in human urine by liquid chromatography (LC) coupled to pneumatically assisted electrospray ionization (ESI) mass spectrometry (MS). For this purpose, the operational parameters of the electrospray interface were optimized in order to obtain the best signal stability and the highest sensitivity of the fluoroquinolones. The three fluoroquinolones studied and enrofloxacin, used as internal standard, were extracted from human urine samples by solid-phase extraction (SPE) and the previously established LC-UV method was successfully coupled with the MS system. The mass spectra obtained provide adequate information for identification purposes. Quality parameters were determined and satisfactory results were obtained. Likewise, the method detection limit was about 10 ng ml(-1) for the three fluoroquinolones studied employing selected-ion monitoring mode.

Protonation equilibria of quinolone antibacterials in acetonitrile-water mobile phases used in LC.

Ionization constants of nine quinolone antibacterials in acetonitrile-water mixtures containing 0, 5.5, 10, 16.3, 25, 30, 40, 50 and 70% (w/w) acetonitrile were obtained and assignment of these pK values to the several potentially ionizable functional groups was made. The variation of the pK values obtained over the whole composition range studied can be explained by consideration of the preferential solvation of electrolytes in acetonitrile-water mixtures. In order to obtain pK values in any of the unlimited number of possible binary solvent acetonitrile-water mixtures, relationships between pK values and different bulk properties (such as dielectric constant) were examined. The linear solvation energy relationships method, LSER, was applied to study the correlation of pK values with the solvatochromic parameters of acetonitrile-water mixtures. The equations obtained allow calculation of the pK values of the quinolone antimicrobials in any acetonitrile-water mixtures up to 70% (w/w) and thus permit the knowledge of the acid-base behaviour of these important antimicrobials in the widely used acetonitrile-water media.

Development and validation of a fully automated method for the chromatographic determination of content uniformity of drug tablets.

A fully automated method for the content uniformity analysis of LAS 34475 25mg tablets has been developed by using an automated procedure. This automated method has been validated within the requirements of ICH guidelines Q2A-Q2B. Standard and sample solutions are processed by an automated benchtop system. The operations automated include the phases of disintegration of the dosage form, filtration of the resultant homogenate and injection of the clear sample into the chromatographic system. Although a manual method validated according to ICH guidelines already existed for this compound, the benefits of applying appropriate automation should provide continuous operation, increased precision, an affordable electronic audit trail and significantly reduced time consumption as well as reducing the exposure of the analyst to the drug substance. The objective of this work was to adapt the manual method to an automated workstation. Considerable effort went into developing and validating an automated method. The results obtained in the validation of this automated method were equivalent to the manual method in terms of system precision, linearity, accuracy, robustness and sensitivity (limits of detection, LOD and limits of quantification, LOQ), and carry-over.