N-acetyl functions and acetate detected by nuclear magnetic resonance spectroscopy of urine to detect renal dysfunction following aminoglycoside and/or glycopeptide antibiotic therapy.

BACKGROUND/AIMS: N-acetylneuraminidine (NeuNAc), N-acetylglutamine (GIcNAc) and acetate are metabolites present in normal urine. In patients treated with aminoglycosides and/or glycopeptides, elevation of these metabolites in urine suggests renal tubular injury. NeuNAc, GIcNAc and acetate are easily detected by magnetic resonance spectroscopy (MRS), in contrast to other bioanalytical methods. In the present study, these urinary metabolites were detected using MRS and compared with standard biochemical markers of renal injury in intensive care unit patients treated with aminoglycosides and/or glycopeptides. METHODS: 16 patients with clinical and biochemical signs of renal dysfunction were included in the study. Proton magnetic resonance spectra were obtained from 134 urine samples. The resonance intensity of NeuNAc, GIcNAc and acetate were reported relative to the resonance intensity of creatinine (ct). These ratios were compared with classical parameters of renal dysfunction, such as plasma creatinine and urea concentration, and 24-hour urine volume, by logistic regression and general linear models. RESULTS: Statistical analysis showed that changes in plasma creatinine and urea concentration were reliably reflected in changes in the NeuNAc/ct ratio, and that plasma urea concentration changes also correlated with the acetate/ct ratio; however, the GIcNAc/ct ratio was not related to these measures of overall renal function. CONCLUSIONS: NeuNAc/ct may be a useful marker of renal dysfunction in patients treated with aminoglycosides and glycopeptides; by MRS it can be both straightforward and informative to follow the renal function of patients treated with these antibiotics.

A hydroxyethylated cholesterol-based cationic lipid for DNA delivery: effect of conditioning.

We have synthesised a novel cholesterol-based cationic lipid to promote DNA transfer in cells. This lipid, dimethyl hydroxyethyl aminopropane carbamoyl cholesterol iodide (DMHAPC-Chol) contains a biodegradable carbamoyl linker and a hydroxyethyl group in the polar amino head moiety and is characterised by NMR. Liposomes prepared from this lipid and dioleoyl phosphatidyl ethanolamine (DOPE) in equimolar proportion showed a weak cytotoxicity as revealed by MTT assays and are efficient to deliver plasmids DNA evaluated by the expression of reporter genes in vitro and in vivo. In this paper, we present an original method to determine the lipid concentration based on the colorimetric detection of the colipid DOPE and the measure of the molar ratio DOPE/cationic lipid in the liposome by FTIR spectroscopy. The liposomes and lipid/DNA complexes structures were characterized by transmission electron microscopy (TEM) and by quasi-elastic light scattering (QLS). TEM indicated that the complexes correspond to aggregates containing globular substructures with liposomes size. The method of immuno-gold labelling was used to detect plasmid in the complex and reveals the presence of DNA inside the aggregates. Transfection results showed efficient DNA transfer depending on the charge ratio and liposomes conditioning. Gel retardation results indicated that at a molar charge ratio between X = 1.5 and X = 2.5 (depending on the liposome conditioning), all DNA was taken by liposomes. We showed that conditioning by freeze-drying (lyophilization) facilitates storage and improves transfection efficiency. When the liposomes were lyophilized prior to DNA addition or when the complexes were subjected to freeze-thawing cycles, the obtained complexes showed a transfection with levels enhanced up to four and five-fold respectively for the lyophilized liposomes and freeze-thawed complexes. NMR was used to characterize the modifications under freezing which showed an effect on 31P spectra.

Solution conformation of an RNA--DNA hybrid duplex containing a pyrimidine RNA strand and a purine DNA strand.

RNA--DNA hybrid duplexes are involved in transcription, replication and reverse transcription of nucleic acids. Information on such duplexes may shed some light on the mechanism of these processes. For this purpose, the influence of base composition on the structure of a polypyrimidine--polypurine RNA--DNA duplex r(cucuccuucucuu). d(GAGAGGAAGAGAA) has been studied using 1H, 31P and 13C NMR experiments, molecular modeling (JUMNA program) and NOE back-calculation methods. The resulting structure of the 13-mer hybrid duplex shows that the RNA strand is in the expected A-type conformation while the DNA strand is in a very flexible conformation. In the DNA strand, the desoxyribose sugars retain the C2'-endo B-type conformation. The duplex helical parameters (such as inclination, twist and displacement of the bases) are close to the A-type conformation. No bending was observed for the global axis curvature. The major groove width is close to the B-form value and the minor groove width is intermediate between standard values for A and B-forms. These results are in favour of the independence of minor groove size (where RNase H interacts) and the base composition of the hybrid duplexes.

Magnetic resonance spectroscopy of cellular lipid extracts from sensitive, resistant and reverting K562 cells and flow cytometry for investigating the P-glycoprotein function in resistance reversion.

The proton NMR spectra of K562 cells contain resonances of lipids. When these cells acquire multidrug resistance phenotype, the NMR lipid signals are modified and partially recovered when the resistance is reversed. The goals of the present study are to elucidate the mechanism of the resistance phenotype reversion and to investigate the possible origin of lipid signals detected in whole cells with proton NMR spectroscopy. Therefore, the K562 drug-sensitive cell line, its adriamycin resistant counterpart and two reverting derivates, obtained by verapamil treatment and long term culture in drug-free medium, were used in this study. The P-glycoprotein (P-gp) pump function was measured by flow cytometry and lipids were extracted to be analysed by proton and phosphorus spectroscopy. The phenotype reversion is due to the decrease of the P-gp function and an increased entrance of anthracycline drug when compared with the resistant cells. The spectra obtained on extracts showed no modification of the fatty acid composition and of the ratio of total cholesterol to fatty acid content. A different phospholipid composition in sensitive and resistant cells was found, but the reversion of resistance did not produce a recovery of these lipids. Thus, the lipid NMR spectra of extracts could not explain the spectral modifications observed on whole cells, in relation to acquiring and reverting drug resistance. These results are in favour of a different lipid organization or of localization within the cell.

Current Awareness.

In order to keep subscribers up-to-date with the latest developments in their field, John Wiley & Sons are providing a current awareness service in each issue of the journal. The bibliography contains newly published material in the field of NMR in biomedicine. Each bibliography is divided into 9 sections: 1 Books, Reviews ' Symposia; 2 General; 3 Technology; 4 Brain and Nerves; 5 Neuropathology; 6 Cancer; 7 Cardiac, Vascular and Respiratory Systems; 8 Liver, Kidney and Other Organs; 9 Muscle and Orthopaedic. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted.

Magnetic resonance spectroscopy and fluorescence microscopy to investigate mobile lipids in sensitive, resistant and reverting K562 cells and their membranes.

The erythroleukaemic K562 cell line and its adriamycin resistant counterpart were used to study resistance, its reversion and their consequences on the levels and localisation of lipids detected in proton nuclear magnetic resonance (NMR) spectra. On whole cells, the mobile lipids giving rise to a NMR signal were significantly decreased in the resistant cells when compared to the sensitive ones; these lipids recovered partially in the reverting cells. According to the spinlattice relaxation times (T1), the lipids detected appeared to be in a similar environment in sensitive and reverting cells. In membrane-enriched fractions, mobile lipid levels were not significantly different in the sensitive and reverting cell lines but decreased in resistant ones. Moreover, lipid droplets stained with a fluorescent Nile red lipophilic probe showed the presence of highly fluorescent particles in the samples in which NMR detected high levels of mobile lipids. These results suggest the participation of cytosolic lipid droplets in NMR signals in drug sensitive and reverting cells and open the question of the relative roles of these droplets and of the membrane lipids in the lipid metabolic pathways associated with drug resistance in cancer cells.

Solution structure of a non-palindromic 16 base-pair DNA related to the HIV-1 kappa B site: evidence for BI-BII equilibrium inducing a global dynamic curvature of the duplex.

1H and 31P NMR spectroscopy have been used together with molecular modelling to determine the fine structure of a non-palindromic 16 bp DNA containing the NF-kappa B binding site. Much emphasis has been placed upon NMR optimization of both two-dimensional 31P NMR techniques to extract structural information defining the phosphodiester backbone conformation and selective homonuclear 2D COSY experiments to determine sugar conformations. NMR data show evidence for a dynamic behaviour of steps flanking the ten base-pairs of the NF-kappa B binding site. A BI-BII equilibrium at these steps is demonstrated and two models for each extreme conformation are proposed in agreement with NMR data. In the refined BII structures, the NF-kappa B binding site exhibits an intrinsic curvature towards the major groove that is magnified by the four flanking steps in the BII conformation. Furthermore, the base-pairs are translated into the major groove. Thus, we present a novel mode of dynamic intrinsic curvature compatible with the DNA curvature observed in the X-ray structure of the p50-DNA complex.

Absolute pitch and sex effect event-related potential activity for a melodic interval discrimination task.

Absolute pitch is a special ability which allows for special perceptual/cognitive strategies. Studies have shown differences in event-related scalp potentials between absolute-pitch (AP) and relative-pitch (RP) subjects of equal musical training. In this study, highly trained musicians (15 females/15 males) performed a melodic interval discrimination task, using intervals on-pitch in equal-tempered tuning (A4 = 440 Hz) and tuned a half-semitone sharp. Subjects identified target intervals (probability 0.2) in a series of 400 randomly transposed intervals. AP subjects were expected to perform differently across intonation conditions, whereas RP subjects were not. Event-related potentials (ERPs) were recorded from three midline sites and two lateral sites. ERPs were analyzed by principal component analysis of variance. Sex was also considered as an independent subject variable. Performance was not significantly different either by absolute pitch or sex. Reaction times did not reveal any significant interactions involving AP or sex, but showed a significant effect by response type (target/nontarget). Strong P3 activity appeared to the target melodic intervals regardless of subject group or intonation. PCA factors with maxima at 352, 511, and 709 ms were sensitive to task relevance. Males showed greater positivity than females along the midline. A significant intonation by response type by sex interaction indicated a greater spread of values for females than males, and greater similarity in response by sex for the sharp than the on-pitch intervals. AP subjects showed reduced P3 activity along the midline, but increased over lateral sites. In a difficult musical task, the ERPs were sensitive to the sex of the listeners, as well as to whether they had absolute pitch.

Sex differences in memory for timbre: an event-related potential study.

Although female/male cognitive differences have been studied for some time, little is known about such differences relative to music. Highly-trained musicians (15 females and 15 males) performed a memory task for musical timbre modeled after the missing-displaced visual object test known to favor female performance. Subjects were tested on memory for a timbre missing from a previously presented set of synthesized instrumental timbres, and a control series of white noise bursts at two different intensity levels. Subjects were given the missing-displaced visual object test and ERPs were recorded from three midline sites and two lateral sites. Waveforms were subjected to a principal component analysis and analysis of variance. Females and males performed equally well on both of the auditory series and the visual object test. Both auditory series elicited several ERP components: a strong early peak to the white noise, and both early (P3a) and late (P3b) peaks to the timbre series. PCA factor, maximum at 675 ms (P3b), showed a main effect for sex across both series with males > females. PCA factor, maximum at 336 ms (P3a), showed larger Fz to Pz differential for females than males for both auditory series. Females show a greater differentiation between targets and nontargets in the white noise series, suggesting greater sensitivity to changes in intensity. Interactions involving absolute pitch also appeared in the sensory processing time frames. Although the behavioral measures did not show significant sex differences, the ERP measures did show reliable task-related sex differences.

P3 event-related potentials and performance of young and old subjects for music perception tasks.

Event-related potentials and performance data were recorded from young and old subjects performing six tasks involving auditory discrimination of musical stimuli. Tasks included pure tone, timbre, rhythm, and interval discrimination, detection of a meter shift, and discrimination of open and closed harmonic endings for chord progressions. P3 latencies were generally longer for the old subjects. P3 amplitude and performance differences between subject groups were not significant. Our results provide a quantitative probe of the neural and behavioral significance of the influence of aging and stimulus complexity on the processing of some of the elementary constituents of music. In particular, pure tone and timbre discrimination appear to correspond to behaviorally and neurally simpler processing than does discrimination of the other musical constituents tested in our study.

Neural processing of musical timbre by musicians, nonmusicians, and musicians possessing absolute pitch.

Cognitive event-related potentials (ERPs) were measured during a timbre discrimination task from three subject groups varying in musical experience. The P3 component of the ERP was recorded from musicians with absolute pitch, musicians without absolute pitch, and nonmusicians during a task comprising timbres of varying difficulty. The three-timbre series, all of which consisted of the same pitch, were (1) string instruments in the same family (cello and viola), (2) flutes made of different materials (silver and wood), and (3) instruments of slightly different size (B-flat versus F tubas). The amplitude and latency of the P3 component varied systematically as a function of musical experience and type of timbre discrimination. The difficult timbre task resulted in mean P3 amplitudes which were larger for musicians relative to nonmusicians, however P3 amplitudes were similar for the two additional timbre series. The mean P3 latencies for musicians were shorter when compared to nonmusicians across all three series. In comparison, the AP subjects displayed the shortest mean P3 latencies, but had smaller P3 amplitudes relative to both musicians and nonmusicians. The implications of these findings suggest that perceptual tasks involving one of the fundamental building blocks of music, namely timbre, does elicit differential brain activity from memory or information processing systems from subjects with varying degrees of musical training.

Effects of musical training and absolute pitch ability on event-related activity in response to sine tones.

The neural correlates of music perception have received relatively little scientific attention. The neural activity of listeners without musical training (N = 11), highly trained musicians (N = 14), and musicians possessing "absolute pitch" (AP) ability (N = 10) have been measured. Major differences were observed in the P3, an endogenous event-related potential (ERP), which is thought to be a neurophysiological manifestation of working memory processing. The P3 was elicited using the classical "oddball" paradigm with a sine-tone series. Subjects' musical backgrounds were evaluated with a survey questionnaire. AP ability was verified with an objective pitch identification test. The P3 amplitude, latency and wave shape were evaluated along with each subjects' performance score and musical background. The AP subjects showed a significantly smaller P3 amplitude than either the musicians or nonmusicians, which were nearly identical. The P3 latency was shortest for the AP subjects, and was longer for the nonmusicians. Performance scores were uniformly high in all three groups. It is concluded that AP subjects do indeed exhibit P3 ERPs, albeit with smaller amplitudes and shorter latencies. The differences in neural activity between the musicians and AP subjects were not due to musical training, as the AP subjects had similar musical backgrounds to the musician group. It is also concluded that persons with the AP ability may have superior auditory sensitivity at cortical levels and/or use unique neuropsychological strategies when processing tones.

[Basedow's neuropathy]. / Neuropathie basedowienne.

A case report of grave's neuropathy. The symptoms appeared in a treated patient when there was a biologic hypothyroidy. A total remission was observed after corticotherapy. Nevertheless, a recurrence of disease happened for reduced doses, so an orbital radiotherapy was requisited. Student of differents treatments for dysthyroid neuropathy.

The effect of loperamide on the thermal behavior of dimyristoylphosphatidylcholine large unilamellar vesicles.

The effect of loperamide, a drug belonging to the opiate family, on dimyristoyl phosphatidylcholine large unilamellar vesicles (DMPC LUV) was investigated by quasielastic light scattering (QLS) and Fourier transform infrared spectroscopy (FT-IR). Both techniques show that, in the presence of loperamide, DMPC LUV undergoes a two step transition in cooling: one step around the transition point of pure lipid vesicles, the other at a lower temperature. The temperature of the latter step transition is different for the head and tail regions of the drug-containing vesicles: FT-IR spectra demonstrate that the hydrophobic acyl chains transition starts at a temperature well above that of the interfacial region whereas the transition of the entire vesicle, explored by QLS, is broad and covers both temperature ranges. These transitions are thermally reversible in the FT-IR which measures local order but aggregation effects prevent the thermal reversibility of the QLS results. The nature of the drug-lipid interaction is also discussed.

Effect of ethanol on dimyristoylphosphatidylcholine large unilamellar vesicles investigated by quasi-elastic light scattering and vibrational spectroscopy.

The gel-like liquid phase transition of dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles prepared by reverse phase evaporation has been investigated in buffers containing ethanol by quasi-elastic light scattering (QLS) and vibrational (infrared and Raman) spectroscopy. With the QLS technique, the relative change in the vesicles area (which is related to the molecular cross-sectional area of lipid molecules) was followed versus both temperature and ethanol concentration. When the latter was low, the depression of the transition point was a linear function of the alcohol concentration, c, but the vesicles area was practically unmodified. At alcohol concentration 10% v/v, an abrupt change of the vesicles area was observed and for c greater than 10% the depression of the transition point was a non-linear function of c. The infrared and Raman spectra showed a perturbation of the hydrophobic regions, including the terminal methyl groups of the acyl tails.

The osmotic response of large unilamellar vesicles studied by quasielastic light scattering.

Large unilamellar vesicles of two phosphatidylcholines, one saturated (DMPC) and the other unsaturated (DOPC), prepared by the reverse-phase evaporation method were studied using the quasielastic light scattering technique. The accurate sizing obtained by this technique showed an osmotic response for the two kinds of vesicles when the salinity of the external medium was diluted. The elastic moduli of lipid vesicles bilayers in the liquid phase were then estimated according to the elasticity theory of spherical shells taking into account salt leakage data known from the literature.

Conformational change of core particles studied by quasielastic light scattering.

The diffusion translational coefficient Dt of core particles in monodisperse solutions has been measured by the quasielastic light scattering method in a large scale of salinities over the range 6.10(-4) to 2M Na+ or K+. The observed values of DT are independent of particle concentration in the range 0.1-2 mg/ml and do not vary with the scattering vector q corresponding to scattering angles between 40 degrees -120 degrees. When the salinity is progressively raised an increase of DT from 1.9.10(-7) cm2s-1 to 3.2.10(-7) cm2s-1 was observed at about 2.10(-3) M NaCl followed by a decrease of DT beyond 0.6 M NaCl.