Biomedical subjects

Luc Moens

Publications and source records attributed to Luc Moens.

At least 55 records · Page 3Linked to original sources

PubMed2004

Validated measurements of the uranium isotopic signature in human urine samples using magnetic sector-field inductively coupled plasma mass spectrometry.

Increased interest in measuring uranium isotope ratios in environmental samples (biological materials, soils, dust particles, water) has come from the necessity to assess the health impact of the use of depleted uranium (DU) based ammunitions during recent military conflicts (e.g., Gulf war, Kosovo) and from the need to identify nondeclared nuclear activities (nuclear safeguards). In this context, very important decisions can arise which have to be based on measurement data of nondisputable uncertainty. The present study describes the certification to 2.5% (k = 2) relative combined uncertainty of n(235U)/n(238U) at ultralow uranium levels (approximately 5-20 pg g(-1)) in human urine samples. After sample decomposition and matrix separation, the isotope ratios were measured by means of a single-detector magnetic sector-field inductively coupled plasma mass spectrometry instrument fitted with an ultrasonic nebulizer. Correction for mass discrimination effects was obtained by means of the certified isotopic reference material IRMM-184. The analytical procedure developed was validated in three complementary ways. First, all major sources of uncertainty were identified and propagated together following the ISO/GUM guidelines. Second, this quality was controlled with a matrix matching NUSIMEP-3 sample (approximately 0.06-0.7% difference from certified). Third, the instrumental part of the procedure was proven to be reproducible from the confirmation of the results obtained for three samples remeasured 7 months later (approximately 1.5% difference). The results obtained for 33 individuals indicated that none seemed to have been exposed to contamination by DU.

Humans↗

PubMed2004

The human brain hexacoordinated neuroglobin three-dimensional structure.

Neuroglobin, mainly expressed in vertebrate brain and retina, is a recently identified member of the globin superfamily. Augmenting O2 supply, neuroglobin promotes survival of neurons upon hypoxic injury, potentially limiting brain damage. In the absence of exogenous ligands, neuroglobin displays a six-coordinated heme. O2 and CO bind to the heme-iron, displacing the endogenous HisE7 heme distal ligand. Hexacoordinated human neuroglobin displays a classical globin fold, adapted to host the reversible bis-histidyl heme complex, and an elongated protein matrix cavity, held to facilitate O2 diffusion to the heme. The structure of neuroglobin suggests that the classical globin fold is endowed with striking adaptability, indicating that hemoglobin and myoglobin are just two examples within a wide and functionally diversified protein homology superfamily.

Globins↗

PubMed2004

Coupling of the heme and an internal disulfide bond in human neuroglobin.

Neuroglobin displays a hexacoordination His-Fe-His in the absence of external ligands such as oxygen. The observed oxygen affinity therefore depends on the binding rates of both oxygen and the competing distal histidine. Furthermore, the binding properties depend on the presence of an internal disulfide bond. In the case of human neuroglobin, cysteines at positions CD7 and D5 are sufficiently close to form an internal disulfide bond. For cytoglobin, the cysteine residues at positions A7 and GH4 may also form a disulfide bond. Mass spectrometry, ligand binding, and thiol accessibility studies were used to study the role influence of these disulfide bonds. Mutation of specific cysteines, or reduction to break the S-S bond, led to a large decrease in the observed oxygen affinity of human neuroglobin, mainly due to a decrease in the histidine dissociation rate. This suggests a novel mechanism for the oxygen binding; reduction of the disulfide bond would provoke the release of oxygen.

Cysteine↗

PubMed2004

Neuroglobin and other hexacoordinated hemoglobins show a weak temperature dependence of oxygen binding.

Mouse and human neuroglobins, as well as the hemoglobins from Drosophila melanogaster and Arabidopsis thaliana, were recombinantly expressed in Escherichia coli, and their ligand-binding properties were studied versus temperature. These globins have a common feature of being hexacoordinated (via the distal histidine) under deoxy conditions, as evidenced by a large amplitude for the alpha absorption band at 560 nm and the Soret band at 426 nm. The transition from the hexacoordinated form to the CO bound species is slow, as expected for a replacement reaction Fe-His --> Fe --> FeCO. The intrinsic binding rates would indicate a high oxygen affinity for the pentacoordinated form, due to rapid association and slow (100 ms-1 s) dissociation. However, the competing protein ligand results in a much lower affinity, on the order of magnitude of 1 torr. In addition to decreasing the affinity for external ligand, the competitive internal ligand leads to a weaker observed temperature dependence of the ligand affinity, since the difference in equilibrium energy for the two ligands is much lower than that of ligand binding to pentacoordinated hemoglobin. This effect could be of biological relevance for certain organisms, since it could provide a globin with an oxygen affinity that is nearly independent of temperature.

Animals↗

PubMed2003

The redox state of the cell regulates the ligand binding affinity of human neuroglobin and cytoglobin.

Neuroglobin and cytoglobin reversibly bind oxygen in competition with the distal histidine, and the observed oxygen affinity therefore depends on the properties of both ligands. In the absence of an external ligand, the iron atom of these globins is hexacoordinated. There are three cysteine residues in human neuroglobin; those at positions CD7 and D5 are sufficiently close to form an internal disulfide bond. Both cysteine residues in cytoglobin, although localized in other positions than in human neuroglobin, may form a disulfide bond as well. The existence and position of these disulfide bonds was demonstrated by mass spectrometry and thiol accessibility studies. Mutation of the cysteines involved, or the use of reducing agents to break the S-S bond, led to a decrease in the observed oxygen affinity of human neuroglobin by an order of magnitude. The critical parameter is the histidine dissociation rate, which changes by about a factor of 10. The same effect is observed with human cytoglobin, although to a much lesser extent (less than a factor of 2). These results suggest a novel mechanism for the regulation of oxygen binding; contact with an appropriate electron donor would provoke the release of oxygen. Hence the oxygen affinity would be directly linked to the redox state of the cell.

Cysteine↗

PubMed2003

Determination of ultra-trace amounts of Fe in AgNO3 solutions by means of isotope dilution analysis applying an inductively coupled plasma mass spectrometer equipped with a dynamic reaction cell.

The development of an ICP-MS method for the determination of ultra-trace amounts of Fe in AgNO(3) solutions using isotope dilution for calibration is described. AgNO(3) solutions are used as raw materials in the production of traditional photographic materials, and it is known that contamination with metal traces can influence the quality of the films thus produced. After adding an appropriate amount of an (54)Fe-enriched spike and permitting isotopic equilibration to take place, Ag was selectively removed from the solutions by precipitation as AgBr. Although to some extent, co-precipitation of Fe is possible under the given circumstances, an incomplete recovery of the analyte element did not affect the accuracy of the results, owing to the use of isotope dilution for calibration. NH(3) was used as a reaction gas in a quadrupole-based ICP-MS instrument, equipped with a dynamic reaction cell (DRC), providing interference-free measurement of the (54)Fe/(56)Fe ratio. The limit of detection (LOD) obtained using this procedure was approximately 0.01 micro g g(-1). This is an excellent value in comparison with the detection limit obtained with the more traditional approach: sample dilution and external calibration with a Fe standard solution (LOD ~1 micro g g(-1)). To validate the method, recovery experiments were carried out. In all instances, a quantitative recovery was established. Finally, the method was applied to the analysis of AgNO(3) solutions. A large variation in Fe concentration was observed. Depending on the Fe content in the samples, relative standard deviations typically ranged between 1 and 14%.

Journal Article↗

PubMed2003

Overcoming spectral overlap in isotopic analysis via single- and multi-collector ICP-mass spectrometry.

For isotope ratio applications where an internal isotope ratio precision >0.05-0.1% relative standard deviation suffices, single-collector inductively coupled plasma mass spectrometry (ICPMS) is fit-for-purpose, but for detecting more subtle variations in the natural isotopic composition of a target element, only multi-collector ICPMS (MC-ICPMS) can compete with thermal ionization mass spectrometry (TIMS). While as a result of the extensive sample preparation (analyte isolation) preceding TIMS and the "softer" ionization in vacuum, spectral interferences only seldom occur with this technique, their occurrence is recognized to be the most important drawback of ICPMS. This paper discusses high mass resolution and chemical resolution in a collision or dynamic reaction cell as powerful and versatile means to overcome spectral overlap and illustrates how their introduction has led to a substantial extension of the application range of ICPMS for isotope ratio applications. High mass resolution is the most elegant and straightforward way to overcome the problem of spectral overlap. Offering the possibility to operate the mass analyzer at a higher mass resolution, while at the same time preserving the flat-topped or trapezoidal peak shape required for highly precise isotope ratio measurements, was a challenge for the manufacturers of MC-ICPMS instrumentation. It will be discussed how these apparently contradicting requirements could be fulfilled simultaneously and an overview of the current situation will be given. Chemical resolution in a collision or dynamic reaction cell is an alternative to high mass resolution for overcoming spectral overlap. Real-life examples will be given to illustrate how also this approach can be used to advantage in isotope ratio work. Despite the greater flexibility and straightforwardness of high mass resolution, some situations will be discussed where chemical resolution is to be preferred. Finally, some desires as to future instrumentation are formulated.

Journal Article↗

PubMed2003

Solution 1h NMR characterization of equilibrium heme orientational disorder with functional consequences in mouse neuroglobin.

The solution 1H NMR spectrum of oxidized (met) mouse neuroglobin, metNgb, demonstrates that it is low-spin and hexacoordinate with strong spectral similarities to ferricytochrome b5. The axial ligands are identified as His(F8) and His(E7), with the latter exhibiting an unstrained Fe-His bond. The presence of two sets of resonances is shown to arise from equilibrium heme orientational isomers ( approximately 2:1). The ligation of cyanide is shown to be extraordinarily slow with a factor approximately 2 difference in rate for the two heme orientations. Not only is Ngb the first mamalian globin with equilibrium heme disorder, but the disorder also has additional functional consequences.

Animals↗

PubMed2003

New insight into the haemoglobin superfamily: preliminary crystallographic characterization of human cytoglobin.

Human cytoglobin, present in almost all tissue types, is a newly identified member of the Hb superfamily. A double mutant, having both cysteines replaced by serines, has been overexpressed in Escherichia coli, purified and crystallized. A highly redundant SAD data set has been collected at the haem Fe-atom absorption edge (lambda = 1.720 A) to 2.60 A resolution. The crystals belong to the orthorhombic P2(1)2(1)2(1) space group, with unit-cell parameters a = 46.8, b = 73.1, c = 98.9 A and two molecules per asymmetric unit. The anomalous difference Patterson map clearly reveals the position of the haem Fe-atom sites, thus paving the way for SAD structure determination.

Binding Sites↗

PubMed2003

A globin in the nucleus!

Cytoglobin and neuroglobin are recently discovered members of the globin family. In situ hybridization localized neuroglobin mainly in brain and retina, while cytoglobin was expressed ubiquitously in all analyzed tissues. In the present study, polyclonal antibodies were raised against both proteins and the distribution of them was studied by immunocytochemistry at tissue and subcellular level. Cytoglobin immunoreactivity was uniformly distributed and found in all tissues studied. At the subcellular level, cytoglobin immunoreactivity was exclusively detected in the cell nucleus. In contrast, neuroglobin immunoreactivity was detected in specific brain regions with varying intensities and in the islet of Langerhans in the pancreas. The immunoreactivity was restricted to the cytoplasm of neurons and endocrine beta cells. The nuclear localization of cytoglobin opens new perspectives for possible function(s) of globin-folded proteins as transcriptional regulators.

Animals↗

PubMed2003

Kinetic modulation in carbonmonoxy derivatives of truncated hemoglobins: the role of distal heme pocket residues and extended apolar tunnel.

Truncated hemoglobins (trHbs), are a distinct and newly characterized class of small myoglobin-like proteins that are widely distributed in bacteria, unicellular eukaryotes, and higher plants. Notable and distinctive features associated with trHbs include a hydrogen-bonding network within the distal heme pocket and a long apolar tunnel linking the external solvent to the distal heme pocket. The present work compares the geminate and solvent phase rebinding kinetics from two trHbs, one from the ciliated protozoan Paramecium caudatum (P-trHb) and the other from the green alga Chlamydomonas eugametos (C-trHb). Unusual kinetic patterns are observed including indications of ultrafast (picosecond) geminate rebinding of CO to C-trHb, very fast solvent phase rebinding of CO for both trHbs, time-dependent biphasic CO rebinding kinetics for P-trHb at low CO partial pressures, and for P-trHb, an increase in the geminate yield from a few percent to nearly 100% under high viscosity conditions. Species-specific differences in both the 8-ns photodissociation quantum yield and the rebinding kinetics, point to a pivotal functional role for the E11 residue. The response of the rebinding kinetics to temperature, ligand concentration, and viscosity (glycerol, trehalose) and the viscosity-dependent changes in the resonance Raman spectrum of the liganded photoproduct, together implicate both the apolar tunnel and the static and dynamic properties of the hydrogen-bonding network within the distal heme pocket in generating the unusual kinetic patterns observed for these trHbs.

Animals↗

PubMed2003

Structure and function of the globin and globin gene from the Antarctic mollusc Yoldia eightsi.

The mechanism of adaptation of haemoglobin from the Antarctic mollusc Yoldia eightsi to its low-temperature environment is a decrease in the oxygen affinity via an increased ligand-dissociation rate. At 2 degrees C this haemoglobin has an oxygen affinity similar to other haemoglobins at 25 degrees C. At 25 degrees C, Yoldia haemoglobin shows a low oxygen affinity, resembling that of human deoxyhaemoglobin. The mechanism involves a lower binding energy to oxygen, suggesting a loss or weakening of the usual hydrogen bond, leading to a higher oxygen-dissociation rate. However, Yoldia haemoglobin has the usual distal and proximal histidines, so the primary structure alone does not provide an obvious explanation for the low affinity. The CO-binding kinetics are biphasic, with the fraction of slow phase increasing at higher protein concentrations, indicating the formation of dimers or a higher level of polymerization. The protein-protein interaction appears to be of hydrophobic nature, since it can be partially reversed by addition of ethylene glycol as co-solvent. While the CO-association rates differ by a factor of 10, the oxygen equilibrium data could be simulated with a single affinity. The Yoldia haemoglobin gene contains three introns, interrupting the coding region at position NA1.2, B12.2 and G7.0. The conservation of the B12.2 and G7.0 introns is in contrast with the unprecedented NA1.2 intron. Phylogenetic analyses reveal a gene tree where the Yoldia haemoglobin gene is separated from other mollusc globin genes, confirming the specific adaptation of the Yoldia haemoglobin.

Amino Acid Sequence↗

PubMed2003

Detection of metals in proteins by means of polyacrylamide gel electrophoresis and laser ablation-inductively coupled plasma-mass spectrometry: application to selenium.

The capabilities of laser ablation-inductively coupled plasma-mass spectrometry for the detection of trace elements in a gel after gel electrophoresis were systematically studied. Figures of merit, such as limit of detection, linearity, and repeatability, were evaluated for various elements (Li, V, Cr, Mn, Ni, Cu, Zn, As, Se, Mo, Pd, Ag, Cd, Pt, Tl, Pb). Two ablation strategies were followed: single hole drilling, relevant for ablation of spots after two-dimensional (2-D) separations, and ablation with translation, i.e., on a line, relevant for one-dimensional (1-D) separations. This technique was applied to the detection of selenoproteins in red blood cells extracts after a 1-D separation (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and the detection of selenium-containing proteins in yeast after 2-D electrophoresis (2-DE). The detection procedure was further improved by using the dynamic reaction cell technology, which allowed the removal of the Ar_2(+) interference and hence the use of the most abundant Se isotope, (80)Se. Reaction gases were compared (methane, carbon monoxide, ammonia, oxygen and the combination of argon (collision gas) and hydrogen (reaction gas)). In each instance, the reaction cell parameters were optimized in order to obtain the lowest detection limit for Se (as (80)Se(+), (82)Se(+) or (77)Se(+); and as (80)Se(16)O(+), (82)Se(16)O(+) or (77)Se(16)O(+) with O(2) as the reaction gas). Carbon monoxide was found to offer the best performance. The detection limit with the use of DRC and He as transport gas was 0.07 microg Se g(-1) gel with single hole drilling and 0.15 microg Se g(-1) gel for ablation with translation.

Animals↗

PubMed2003

Human brain neuroglobin structure reveals a distinct mode of controlling oxygen affinity.

Neuroglobin, mainly expressed in vertebrate brain and retina, is a recently identified member of the globin superfamily. Augmenting O(2) supply, neuroglobin promotes survival of neurons upon hypoxic injury, potentially limiting brain damage. In the absence of exogenous ligands, neuroglobin displays a hexacoordinated heme. O(2) and CO bind to the heme iron, displacing the endogenous HisE7 heme distal ligand. Hexacoordinated human neuroglobin displays a classical globin fold adapted to host the reversible bis-histidyl heme complex and an elongated protein matrix cavity, held to facilitate O(2) diffusion to the heme. The neuroglobin structure suggests that the classical globin fold is endowed with striking adaptability, indicating that hemoglobin and myoglobin are just two examples within a wide and functionally diversified protein homology superfamily.

Amino Acid Sequence↗

PubMed2003

Raman spectroscopy of different types of Mexican copal resins.

Dispersive Raman spectra of six copal resins, that were purchased in local markets in Mexico, are presented. The spectra were interpreted and compared with each other. For all these spectra, the relative intensity of the Raman band at approximately 1645 cm(-1), attributed to the exomethylene nu(C=C) stretching vibration, was rather low, especially as fresh samples are involved. In one resin, viz. Incienso, CaCO3 was detected. Probably this inorganic pigment was added as a whitener. In the spectrum of Lágrima a starch fraction was present. Raman spectra of a sample from an Aztec figurine were recorded. It was shown that its composition was inhomogeneous at the micrometer level. Here, too, CaCO3 was observed. It was not possible to identify the resin applied in the antique figurine due to material degradation by age and environmental exposure.

Fabaceae↗

PubMed2003

Micro-Raman spectroscopy of natural and synthetic indigo samples.

In this work indigo samples from three different sources are studied by using Raman spectroscopy: the synthetic pigment and pigments from the woad (Isatis tinctoria) and the indigo plant (Indigofera tinctoria). 21 samples were obtained from 8 suppliers; for each sample 5 Raman spectra were recorded and used for further chemometrical analysis. Principal components analysis (PCA) was performed as data reduction method before applying hierarchical cluster analysis. Linear discriminant analysis (LDA) was implemented as a non-hierarchical supervised pattern recognition method to build a classification model. In order to avoid broad-shaped interferences from the fluorescence background, the influence of 1st and 2nd derivatives on the classification was studied by using cross-validation. Although chemically identical, it is shown that Raman spectroscopy in combination with suitable chemometric methods has the potential to discriminate between synthetic and natural indigo samples.

Journal Article↗

PubMed2003

1H NMR study of the molecular structure and magnetic properties of the active site for the cyanomet complex of O2-avid hemoglobin from the trematode Paramphistomum epiclitum.

The solution molecular and electronic structures of the active site in the extremely O2-avid hemoglobin from the trematode Paramphistomum epiclitum have been investigated by 1H NMR on the cyanomet form in order to elucidate the distal hydrogen-bonding to a ligated H-bond acceptor ligand. Comparison of the strengths of dipolar interactions in solution with the alternate crystal structures of methemoglobin establish that the solution structure of wild-type Hb more closely resembles the crystal structure of the recombinant wild-type than the true wild-type met-hemoglobin. The distal Tyr66(E7) is found oriented out of the heme pocket in solution as found in both crystal structures. Analysis of dipolar contacts, dipolar shift and paramagnetic relaxation establishes that the Tyr32(B10) hydrogen proton adopts an orientation that allows it to make a strong H-bond to the bound cyanide. The observation of a significant isotope effect on the heme methyl contact shifts confirms a strong contact between the Tyr32(B10) OH and the ligated cyanide. The quantitative determination of the orientation and anisotropies of the paramagnetic susceptibility tensor reveal that the cyanide is tilted approximately 10 degrees from the heme normal so as to avoid van der Waals overlap with the Tyr32(B10) Oeta. The pattern of heme contact shifts with large low-field shifts for 7-CH3 and 18-CH3 is shown to arise not from the 180 degrees rotation about the alpha-gamma-meso axis, but due to the approximately 45 degrees rotation of the axial His imidazole ring, relative to that in mammalian globins.

Animals↗

PubMed2003

Testosterone metabolism in the estuarine mysid Neomysis integer (Crustacea; Mysidacea) following tributyltin exposure.

Current evidence suggests that the biocide tributyltin (TBT) causes the development of imposex, a state of pseudohermaphrodism in which females exhibit functional secondary male characteristics, by altering the biotransformation or elimination of testosterone. Imposex in gastropods following TBT exposure is the most complete example of the effects of an endocrine disrupter on marine invertebrates. Previous studies have demonstrated that the estuarine mysid Neomysis integer converts testosterone into multiple polar and nonpolar metabolites resulting from both phase I and phase II biotransformations. In this study, the effects of TBT chloride (TBTCl) on the phase I and II testosterone metabolism of N. integer were evaluated. The TBTCl was highly toxic to N. integer (96-h median lethal concentration [LC50] of 164 ng/L). To assess the effects on testosterone metabolism, mysids were exposed for 96 h to different concentrations of TBTCl (control, 10, 100, and 1,000 ng/L), and testosterone elimination as polar hydroxylated, nonpolar oxido-reduced, and glucose- and sulfate-conjugated metabolites was examined. The TBTCl differentially affected testosterone metabolism. The effect of TBTCl on phase I metabolism was unclear and has been shown to vary among species, likely depending on the inducibility or presence of certain P450 isozyme families. Reductase activity and metabolic androgenization were induced in the 10-ng/L treatment, whereas higher concentrations resulted in a reduction of sulfate conjugation. The exact mechanisms underlying TBT-induced imposex and alterations in the steroid metabolism need to be further elucidated.

Animals↗