Biomedical subjects

A Perico

Publications and source records attributed to A Perico.

At least 19 recordsLinked to original sources

PubMed2004

Determination of N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC) in urine by high-performance liquid chromatography combined with mass spectrometry.

A simple, sensitive and specific method for the quantification of N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC) in urines of the general population and of exposed workers has been developed. Samples were first diluted with phosphate buffer followed by purification by solid-phase extraction (SPE) with SAX columns prior to analysis by liquid chromatography/mass spectrometry (LC/MS). An external standard was used for the quantification together with selected ion monitoring of m/z 219, [M-H]-. The linear calibration curve showed a good correlation (r2 = 0.98, p < 0.001) between 0.1-110 mg/L of AMCC, and the detection limit was calculated to be 2 microg/L. The within-day and between-day precision, calculated for exposed workers and general population AMCC levels in urine samples, were determined as 1.2 and 3.6%, respectively (as relative standard deviation (RSD)).

Acetylcysteine↗

PubMed2003

Modeling the dynamics of the solvated SL1 domain of HIV-1 genomic RNA.

A mode-coupling solution of the Smoluchowski diffusion equation (MCD theory), designed to describe the dynamics of wobbling macromolecules in water, is applied to a macromolecular bead model including water beads in the nearest layers. The necessary statistical averages are evaluated by time averaging along a molecular dynamics (MD) trajectory where both solute and water are introduced as atomistic models. The cross peaks in (1)H nuclear Overhauser effect spectroscopy (NOESY) NMR spectra that are routinely measured to determine biological structures are here calculated for the mutated 23 nucleotides stem-loop fragment of the SL1 domain in the HIV-1(Lai) genomic RNA. The calculations are in acceptable agreement with experiments without requiring any screening of the hydrodynamic interactions. The screening of hydrodynamics was necessary in previous MCD calculations obtained by using the same full atomistic MD trajectory, but a nonsolvated frictional model.

Base Sequence↗

PubMed2001

Backbone dynamics for the wild type and a double H52R/T56W mutant of the vnd/NK-2 homeodomain from Drosophila melanogaster.

The (15)N relaxation behavior and heteronuclear Overhauser effect data for the wild type and an H52R/T56W double mutant protein that encompasses the vnd/NK-2 homeodomain from Drosophila melanogaster were used to characterize and describe the protein backbone dynamics. This investigation, which includes a description of a model structure for the H52R/T56W double mutant vnd/NK-2 homeodomain, was carried out for the two proteins in both the free and DNA-bound states. The double residue replacement at positions 52 and 56 within the DNA recognition helix of vnd/NK-2 has been shown to lead to a significant secondary structural modification resulting in an increase in the length of the recognition helix for the unbound protein. These structural changes are accompanied by corresponding changes in the T(1) and T(1)(rho) relaxation times as well as in the heteronuclear Overhauser effect (XNOE) values that show that the structural stability of the protein is enhanced by the two residue replacements. The values of the rotational anisotropy, D(parallel)/D(perpendicular), derived from analysis of the (15)N T(1) and T(1)(rho) relaxation values are small (1.189 for the unbound homeodomain and 1.110 for the bound homeodomain; both analyzed as prolate ellipsoids of revolution). A comparison of the T(2) values of the wild type and double mutant homeodomain reveals the presence of a low-frequency exchange contribution for the wild type analogue. These relaxation studies show that the motional behavior of the protein primarily reflects the tertiary structure and stability of the homeodomain backbone as well as the respective changes induced upon site-directed residue replacement or DNA binding.

Animals↗

PubMed2001

Modeling the dynamics of a mutated stem-loop in the SL1 domain of HIV-1Lai genomic RNA by 1H-NOESY spectra.

The cross-peaks of 1H-NOESY spectra at different time delays are compared to a mode-coupling diffusion (MCD) calculation, including the evaluation of the full 1H relaxation matrix, in the case of a 23 nucleotide fragment of the stem-loop SL1 domain of HIV-1Lai genomic RNA mutated in a single position. The MCD theory gives significant agreement with 1H relaxation experiments enabling a thorough understanding of the differential local dynamics along the sequence and particularly of the dynamics of nucleotides in the stem and in the loop. The differential dynamics of this hairpin structure is important in directing the dimerization of the retroviral genome, a fundamental step in the infectious process. The demonstration of a reliable use of time dependent NOE cross-peaks, largely available from NMR solution structure determination, coupled to MCD analysis, to probe the local dynamics of biological macromolecules, is a result of general interest of this paper.

Computer Simulation↗

PubMed2000

Smoluchowski dynamics of the vnd/NK-2 homeodomain from Drosophila melanogaster: second-order maximum correlation approximation.

The mode coupling diffusion theory is applied to the derivation of local dynamics in proteins in solution. The rotational dynamics of the bonds along the protein sequence are calculated and compared to the experimentally measured nmr (15)N spin-lattice relaxation time T(1), at 36.5, 60.8, and 81.1 MHz of the vnd/NK-2 homeodomain from Drosophila melanogaster. The starting point for the calculations is the experimental three-dimensional solution structure of the homeodomain determined by multidimensional nmr spectroscopy. The higher order mode-coupling computations are compared also with the recently published first-order approximation calculations. The more accurate calculations improve substantially the first-order ORZLD calculations and show that the role of the strength of the hydrodynamic interactions becomes crucial to fix the order of magnitude of the rotational dynanics for these very compact molecules characterized by partial screening of the internal atoms to water. However, the relative mobility of the bonds along the sequence and the differential fluctuations depend only weakly on the hydrodynamic strength but strongly on the geometry of the three-dimensional structure and on the statistics incorporated into the theory. Both rigid and fluctuating dynamic models are examined, with fluctuations evaluated using molecular dynamics simulations. The comparison with nmr data shows that mode coupling diffusion accounts for the T(1) relaxation pattern at low frequency where the rotational tumbling dominates. An important contribution of internal motions in the nanosecond time scale is seen at high frequencies and is discussed in terms of diffusive concepts.

Animals↗

PubMed2000

Dynamics of a double stranded DNA oligomer: mode-coupling diffusion approach and reduced rigid fragment models.

The local dynamics of a double stranded DNA fragment [d(CpGpCpApApApTpTpTpGpCpG)]2 of twelve base pairs is obtained to second order in the mode-coupling expansion of the Smoluchowski diffusion theory. The DNA is considered a fluctuating three-dimensional (3D) structure undergoing rotational diffusion. The starting structure for the calculations is the B canonical structure of the fragment, while the fluctuations are evaluated using molecular dynamics simulations, with the ensemble averages approximated by time averages along a trajectory of length 1.5 ns. The rotational dynamics of the bonds along the double strands are calculated and compared to experimental NMR relaxation rates of different 13C along the sequence: R(Cz), R(Cxy) and R(Hz-->Cz). For a fluctuating 3D structure the mode-coupling diffusion theory is found to be in good agreement with several relative characteristics of the experimental relaxation parameters, while motivations are given for the few differences which are due mainly to poor statistics or to inaccuracies in the diffusion model. With a view to application to larger DNA fragments, discussion is dedicated to the validity of reducing the number of degrees of freedom in the double helix statistics by grouping the atoms in rigid fragments (e.g. the backbone atoms, the sugar atoms and the base atoms of each nucleotide). Consideration is given to the effect on local dynamics properties of reduced descriptions that include only three or four rigid bodies per nucleotide as well as five rigid bodies per base pair. It is found that in general these approximations almost uniformly produce slight increase in the correlation time pattern, which grows as the rigidity in the model increases. The relative effects on the dynamic pattern for the most accurate rigid body models are modest. The errors in C1' and C5' mobilities are more significant if C5' is included in the backbone rigid body. These results offer new tools to analyse NMR relaxation behaviour and new perspectives in studying the role of dynamics in biological macromolecules.

Algorithms↗

PubMed1999

Smoluchowski dynamics of the vnd/NK-2 homeodomain from Drosophila melanogaster: first-order mode-coupling approximation.

This work is the first in a series devoted to applying mode coupling diffusion theory to the derivation of local dynamics properties of proteins in solution. The first-order mode-coupling approximation, or optimized Rouse-Zimm local dynamics (ORZLD), is applied here to derive the rotational dynamics of the bonds and compare the calculated with the experimental nmr 15N spin-lattice relaxation time behavior of the vnd/NK-2 homeodomain from Drosophila melanogaster. The starting point for the calculations is the experimental three-dimensional structure of the homeodomain determined by multidimensional nmr spectroscopy. The results of the computations are compared with experimentally measured 15N spin-lattice relaxation times T1, at 34.5 and 60.8 MHz, to check the first-order approximation. To estimate the relative importance of internal and overall rotation, both rigid and fluctuating dynamic models are examined, with fluctuations evaluated using molecular dynamics (MD) simulations. The correlation times for the fundamental bond vector time correlation function and for the second-order bond orientational TCF are obtained as a function of the residue number for vnd/NK-2. The stability of the corresponding local dynamics pattern for the fluctuating structure as a function of the length of the MD trajectory is presented. Diffusive dynamics, which is essentially free of model parameters even at first order in the mode-coupling diffusion approach, confirm that local dynamics of proteins can be described in terms of rotational diffusion of a fluctuating quasi-rigid structure. The comparison with the nmr data shows that the first-order mode coupling diffusion approximation accounts for the correct order of magnitude of the results and of important qualitative aspects of the data sensitive to conformational changes. Indications are obtained from this study to efficiently extend the theory to higher order in the mode-coupling expansion. These results demonstrate the promise of the mode-coupling approach, where the local dynamics of proteins is described in terms of rotational diffusion of a fluctuating quasi-rigid structure, to analyze nmr spin-lattice relaxation behavior.

Animals↗

PubMed1999

Mode-coupling smoluchowski dynamics of a double-stranded DNA oligomer

The local dynamics of a double-stranded DNA d(TpCpGpCpG)(2) is obtained to second order in the mode-coupling expansion of the Smoluchowski diffusion theory. The time correlation functions of bond variables are derived and the (13)C-nmr spin-lattice relaxation times T(1) of different (13)C along the chains are calculated and compared to experimental data from the literature at three frequencies. The DNA is considered as a fluctuating three-dimensional structure undergoing rotational diffusion. The fluctuations are evaluated using molecular dynamics simulations, with the ensemble averages approximated by time averages along a trajectory of length 1 ns. Any technique for sampling the configurational space can be used as an alternative. For a fluctuating three-dimensional (3D) structure using the three first-order vector modes of lower rates, higher order basis sets of second-rank tensor are built to give the required mode coupling dynamics. Second- and even first-order theories are found to be in close agreement with the experimental results, especially at high frequency, where the differences in T(1) for (13)C in the base pairs, sugar, and backbone are well described. These atomistic calculations are of general application for studying, on a molecular basis, the local dynamics of fluctuating 3D structures such as double-helix DNA fragments, proteins, and protein-DNA complexes. Copyright 1999 John Wiley & Sons, Inc.

Journal Article↗

PubMed1999

Biological monitoring of occupational exposure to cyclohexane by urinary 1,2- and 1,4-cyclohexanediol determination.

OBJECTIVES: This article reports the results obtained with the biological and environmental monitoring of occupational exposure to cyclohexane using 1,2-cyclohexanediol (1,2-DIOL) and 1,4-DIOL in urine. The kinetic profile of 1,2-DIOL in urine suggested by a physiologically based pharmacokinetic (PBPK) model was compared with the results obtained in workers. METHODS: Individual exposure to cyclohexane was measured in 156 workers employed in shoe and leather factories. The biological monitoring of cyclohexane exposure was done by measurement of 1,2-DIOL and 1,4-DIOL in urine collected on different days of the working week. In all, 29 workers provided urine samples on Monday (before and after the work shift) and 47 workers provided biological samples on Thursday at the end of the shift and on Friday morning. Another 86 workers provided biological samples at the end of the work shift only on Monday or Thursday. RESULTS: Individual exposure to cyclohexane ranged from 7 to 617 mg/ m3 (geometric mean value 60 mg/m3). Urinary concentrations of 1,2-DIOL (geometric mean) were 3.1, 7.6, 13.2, and 6.3 mg/g creatinine on Monday (pre- and postshift), Thursday (postshift) and Friday (pre-shift), respectively. The corresponding values recorded for 1,4-DIOL were 2.8, 5.1, 7.8, and 3.7 mg/g creatinine. A fairly close, statistically significant correlation was found between environmental exposure to cyclohexane and postshift urinary 1,2-DIOL and 1,4-DIOL on Monday. Data collected on Thursday and Friday showed only a poor correlation to exposure with a wide scatter. Both metabolites have a urinary half-life of close to 18 h and accumulate during the working week. CONCLUSIONS: Comparison between data obtained from a PBPK model and those found in workers suggests that 1,2-DIOL and 1,4-DIOL are urinary metabolites suitable for the biological monitoring of industrial exposure to cyclohexane.

Cyclohexanes↗

PubMed1998

Multiple exposure to arsenic, antimony, and other elements in art glass manufacturing.

Art glass manufacturing is one of the most interesting examples of exposure to complex mixtures. Among the raw materials used are silica sand, borax, carbonates, nitrates of Ca, Na, K, and a great number of compounds that are mainly oxides of As, Sb, Al, Zn, Cr, Ni, Sn, Se, Cd, Mn, Cu, Co, Fe, Nd, Er, Eu, and La. In six art glass factories that use As or Sb as fining agents, the exposure to these elements was investigated in 32 workers by means of environmental and biological monitoring. Analysis was conducted by atomic absorption spectrometry (AAS) and inductively coupled plasma mass spectrometry (ICP-MS). The results confirmed that As, which is the main carcinogen in glass production, reaches high air concentrations and is generally above the American Conference of Governmental Industrial Hygienists (ACGIH) threshold limit value-time-weighted average (TLV-TWA) of 10 micrograms/m3. When partly substituted by antimonial compounds, As air concentrations dropped dramatically, while the air levels of Sb (which is considered less toxic but is classified as a class 2B carcinogen by IARC) were relatively low and below the ACGIH TLV-TWA of 500 micrograms/m3. Exposure to As and Sb also differed in the three types of jobs investigated: As was high in oven chargers, Sb was higher in batch mixers, and both elements were low in makers-formers. Eleven to 18 elements were detected: arsenic, Al, Ba, Sb, Pb, and Zn were the elements most frequently measured (in tens, and in some cases hundreds, of micrograms/m3), followed by B, Li, Mn, Se, Sn, Sr, Ce, La, and Nd (in micrograms or in some cases tens of micrograms/m3. The results of biological monitoring for As, Sb, and other elements were in agreement with environmental monitoring data. We concluded that multiple detection of elements is a useful tool (or the evaluation of exposure to complex mixtures such as those used in the art glass industry and that such detection also allows a more accurate evaluation of related epidemiological data.

Antimony↗

PubMed1998

Determination of urinary 2,5-hexanedione in the general Italian population.

OBJECTIVE: Determination of the urinary levels of 2.5-hexanedione (2,5-HD) was performed in subjects belonging to the Italian general population to define the reference value for this metabolite. MATERIALS AND METHODS: Urine samples were collected from 123 healthy Italian subjects who had not been occupationally exposed to n-hexane or methyl-n-butyl ketone (60 men and 63 women; 53 living in urban areas and 70 living in rural areas; 36 smokers and 87 nonsmokers; 65 aged above 35 years and 58 aged below 35 years). The determinations were performed by a gas chromatography method using a flame ionization detector (FID). A quality-control step was realized by analysis of 78 of these samples by high-performance liquid chromatography (HPLC) with UV detection. RESULTS AND DISCUSSION: The distribution of 2,5-HD concentration was log-normal and the corresponding centiles at the 95% confidence interval were as follows: the 50th centile, 0.270 mg/l for men and 0.191 mg/l for women; the 75th centile, 0.352 and 0.330 mg/l, respectively, for men and women; and the 95th centile, 0.762 and 0.582 mg/l, respectively, for men and women. The reference value, calculated as the upper unilateral 95% tolerance interval at 95% of confidence, was 0.795 mg/l for men and 0.627 for women.

Adult↗

PubMed1995

Biological monitoring of workers exposed to N, N-dimethylfomamide. I. Methods of analysis.

Some methods for analysing N,N-dimethylformamide and its metabolites [hydroxymethyl-N-methylformamide, hydroxymethylformamide and N-acetyl-S-(N-methylcarbamoyl)cysteine] in the urine of exposed workers are described. Unchanged dimethylformamide was measured after pretreatment of urine (2 ml) with silica gel cartridges and elution with methanol. The gas chromatographic analysis using a nitrogen phosphor detector made it possible to detect N,N-dimethylformamide in urine even when workers were exposed to low concentrations of the solvent (about 1 mg/m3). N-Hydroxymethyl-N-methylformamide and N-hydroxymethylformamide were analysed as N-methylformamide and formamide respectively after direct injection of urine into the gas chromatograph. The injection port temperature played an important role in the gas chromatographic determination of these products. Reliable results were obtained when direct or split injections were performed at 250 degrees C. The splitless injection gave the same reliable results at 150 degrees C. In urine samples from occupationally non-exposed persons, N-methylformamide could not be detected. In contrast, formamide (or its precursor, hydroxymethylformamide) was present in every urine sample. Our results in respect of 19 urine samples analysed with the injection port of the gas chromatograph at 250 degrees C gave a mean of 8.6 mg/l of formamide. N-Acetyl-S-(N-methylcarbamoyl)cysteine was determined using a modified method for analysing organic acid in urine samples. The metabolite was extracted with ethyl ether in an acid environment, treated with a silylating reagent and measured by gas chromatography/mass spectrometry.

Acetylcysteine↗

PubMed1994

Synthesis, characterization, and analytical studies of adosupine, a potential new drug for urinary incontinence.

The synthesis and the spectroscopic characterization of a new potential drug for urinary incontinence, adosupine, is described. Adosupine and its potential synthesis impurities were analyzed by a new HPLC method that was developed with a C18 reversed-phase column. The analysis was made under isocratic conditions, with a mobile phase of acetonitrile:water (15:85, v/v). Resolution of all synthesis impurities was allowed. The method was also applied to stability studies of adosupine in solid state and in solution under different conditions. With the conditions used, only one degradation product was shown by HPLC analysis; it was isolated, characterized, and identified as the hydrolysis product of the lactam ring present in the adosupine structure.

Chromatography, High Pressure Liquid↗

PubMed1993

Rapid simultaneous determination of tryptophan and tyrosine in synthetic peptides by derivative spectroscopy.

A method for the simultaneous quantitative determination of tryptophan and tyrosine in synthetic peptides by second-order derivative diode-array spectroscopy is reported. The method does not require hydrolysis of the peptides or a derivatization reaction; the sample is dissolved in 0.1 N NaOH and directly scanned between 262 and 264 nm to detect tyrosine and between 304 and 306 nm to detect tryptophan. From these results the peptide content of the synthetic sample can be easily calculated in a very simple and fast way. The results obtained by the described method with several peptides are compared with those obtained by classical high-performance liquid chromatographic analysis of amino acids after peptide hydrolysis. A very good correlation was found both for the tryptophan/tyrosine molar ratio and the peptide content.

Amino Acid Sequence↗

PubMed1993

High-performance liquid chromatographic method for assay of otilonium bromide, diazepam, and related compounds in finished pharmaceutical forms.

A rapid, simple, stability-indicating assay procedure for otilonium bromide, a smooth muscle relaxant agent, and diazepam in composite tablet analysis was developed with high-performance liquid chromatography. The tablet matrix was dissolved with water, and drugs were extracted with acetonitrile containing an internal standard. An aliquot was centrifuged and chromatographed on a 5-microns, reversed-phase column with 0.5 M sodium acetate trihydrate buffer containing 5 mM 1-heptanesulfonic acid monohydrate sodium salt:methanol (30:70; v/v; adjusted to pH 6.0 with glacial acetic acid) as the mobile phase. The selectivity of the chromatographic system was demonstrated by resolving both compounds from various potential degradation products of each compound. The method is linear, quantitative, and reproducible.

Chromatography, High Pressure Liquid↗

PubMed1992

High-performance liquid chromatographic determination of N-methylformamide and N-methyl-N-(hydroxymethyl)-formamide in human urine.

A reliable high-performance liquid chromatographic (HPLC) method which allows the determination in human urine of two important metabolites of N,N-dimethylformamide (DMF), namely N-methylformamide (MMF) and N-methyl-N-(hydroxymethyl)formamide (DMFOH), is reported. A single-step rapid purification of urine was performed on a C18 solid-phase extraction column and the eluate was injected directly on to the HPLC column. HPLC was carried out isocratically on Aminex Ion Exclusion HPX-87H column using 7.5.10(-4) M sulphuric acid as the mobile phase with ultraviolet detection at 196 nm. The method is specific, accurate, precise and sufficiently sensitive to be applied to the biological monitoring of MMF and DMFOH in workers exposed to DMF.

Chromatography, High Pressure Liquid↗

PubMed1992

High-performance liquid chromatographic method for determination of ronactolol in urine and plasma: evaluation of pharmacokinetic parameters in healthy humans.

Ronactolol [(+/-)-4'-[2-hydroxy-3-(isopropylamino)propoxy]-p-anisanilide], a new aminopropanol derivative showing beta-adrenoreceptor blocking activity, was administered orally as capsules to healthy humans at three single doses (30, 60, and 120 mg). Two HPLC methods were developed separately for determination of drug levels in urine and plasma. For plasma samples, after addition of internal standard (IS), a single-step extraction of alkalinized plasma was performed with methylene chloride. The organic layer was evaporated to dryness under reduced pressure, and the residue was taken up and chromatographed on a microbore silica column. Ronactolol and IS were detected by a UV detector at a wavelength of 278 nm. Excellent linearity was observed between the peak height ratios (ronactolol:IS) and concentrations in plasma. The lowest limit of detection (signal:noise, 3:1) was 1.5 ng/mL of plasma. Urine samples were directly injected and chromatographed on a microbore C18 column with an ion-pairing mobile phase. Excellent linearity was observed between the peak areas and concentrations in urine. The lowest limit of detection (signal:noise, 3:1) was 75 ng/mL of urine. The assay was used to determine the main pharmacokinetic parameters in healthy humans.

Adrenergic beta-Antagonists↗

PubMed1992

Simultaneous determination of otilonium bromide and diazepam by first-derivative spectroscopy.

A rapid, simple assay procedure was developed for simultaneous analysis of otilonium bromide, a smooth-muscle relaxant, and diazepam in tablets containing 20 mg of otilonium bromide and 2 mg of diazepam (20:2 tablets) or 40 mg of otilonium bromide and 2 mg of diazepam (40:2 tablets) by "zero-crossing" first-derivative spectroscopy. The tablets were dissolved in 0.01 N HCl, mixtures were centrifuged at 3500 rpm (2472 x g) for 5 min, and first-derivative spectra were recorded. The absolute values of the derivative were measured at 264 nm for determination of otilonium bromide and between 406 and 408 nm (380 nm for analysis of 40:2 tablets) for determination of diazepam. The method is linear, quantitative, and reproducible and can also be used for the tablet dissolution test. Ten tablets of the same batch were analyzed by the described method and by a high-performance liquid chromatographic method, and the results were in good agreement.

Calibration↗