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L Moens

Publications and source records attributed to L Moens.

At least 37 records · Page 2Linked to original sources
PubMed2001

Two-dimensional gel electrophoresis of selenized yeast and autoradiography of 75Se-containing proteins.

Two-dimensional high-resolution gel electrophoresis (2DE) has been applied to the fractionation of 75Se-containing proteins in yeast, grown in 75Se-containing medium, and autoradiography was used for detection of the 75Se-containing proteins. Gel filtration and ultrafiltration were used to check whether the selenium side-chains were stable in the presence of the chemicals used for lysis and 2DE. The mass distribution of the selenium-containing proteins was estimated by use of gel filtration and the results were compared with the distribution obtained by 2DE. A 2DE map of selenium-containing proteins in yeast is presented, and compared with a total protein map of yeast.

Autoradiography↗
PubMed2001

The use of sector field ICP-mass spectrometry for Rb-Sr geochronological dating.

Rb-Sr dating, one of the most important tools in geochronology, requires determination of the Rb/Sr concentration ratios and the 87Sr/86Sr isotope ratios in co-genetic minerals or rocks and is traditionally performed by thermal ionization mass spectrometry (TIMS). In this work we investigated whether sector field inductively coupled plasma mass spectrometry (ICP-MS), which is characterized by a high sample throughput and straight-forward sample introduction, could be used as an alternative to TIMS. To avoid spectral overlap of the ion signals of the isobaric nuclides 87Sr and 87Rb, Sr was separated from Rb by cation-exchange chromatography. A mathematical correction was applied to take into account the small amount of Rb that can be present in the Sr fraction. The isotope ratio accuracy and precision attainable with ICP-MS were evaluated by analysis of several reference materials from the US Geological Survey. The results of this evaluation show that excellent accuracy could be achieved; the internal precision (repeatability) of the isotope ratio (expressed as the relative standard deviation for 10 successive 1-min measurements) was 0.04-0.12%. An attempt was made to calculate the total or combined uncertainty on the isotope ratio results, by also taking into account other possible error sources (corrections for mass discrimination, detector dead time, blank signal and Rb fraction). Finally, the same procedure was used for dating two rock formations that were 2,500 Ma and 350 Ma old, according to age determinations previously performed by the Rb-Sr laboratory of the University of München (Germany) using TIMS. The ICP-MS results (2,520 +/- 150 Ma and 379 +/- 48 Ma) obtained for these formations compare well with the corresponding TIMS data (2,509 +/- 120 Ma and 357 +/- 25 Ma).

Journal Article↗
PubMed2001

Cadmium-binding to transferrin in the plasma of the common carp Cyprinus carpio.

In this study, it was investigated by autoradiography with radioactive cadmium after Western blotting of two-dimensional electrophoresis gels, to which proteins cadmium is mainly bound in plasma of common carp Cyprinus carpio. The obtained results demonstrate that in carp plasma, cadmium is primarily bound to two high molecular weight proteins. Relative small amounts are bound to a protein with M(r) approximately 60000. The other metal-binding protein, with M(r) approximately 70000 and pI approximately 6.7 was identified as transferrin. The conditional equilibrium constants for the binding of cadmium ions to the two metal-binding sites of this protein were calculated as logK(1)=5.40+/-0.12 and logK(2)=4.66+/-0.21, which are comparable to those of human transferrin under the same experimental conditions. Transport of cadmium in plasma of carp was found to be different from that of brown trout Salmo trutta and man, where cadmium is mainly bound to albumin and transferrin. The prominent binding of cadmium to transferrin can be explained by the absence or at least the very low concentrations in which albumin is present in carp plasma.

Animals↗
PubMed2000

Ligand binding in the ferric and ferrous states of Paramecium hemoglobin.

The unicellular protozoan Paramecium caudatum contains a monomeric hemoglobin (Hb) that has only 116 amino acid residues. This Hb shares the simultaneous presence of a distal E7 glutamine and a B10 tyrosine with several invertebrate Hbs. In the study presented here, we have used ligand binding kinetics and resonance Raman spectroscopy to characterize the effect of the distal pocket residues of Paramecium Hb in stabilizing the heme-bound ligands. In the ferric state, the high-spin to low-spin (aquo-hydroxy) transition takes place with a pK(a) of approximately 9.0. The oxygen affinity (P(50) = 0.45 Torr) is similar to that of myoglobin. The oxygen on- and off-rates are also similar to those of sperm whale myoglobin. Resonance Raman data suggest hydrogen bonding stabilization of bound oxygen, evidenced by a relatively low frequency of Fe-OO stretching (563 cm(-1)). We propose that the oxy complex is an equilibrium mixture of a hydrogen-bonded closed structure and an open structure. Oxygen will dissociate preferentially from the open structure, and therefore, the fraction of open structure population controls the rate of oxygen dissociation. In the CO complex, the Fe-CO stretching frequency at 493 cm(-1) suggests an open heme pocket, which is consistent with the higher on- and off-rates for CO relative to those in myoglobin. A high rate of ligand binding is also consistent with the observation of an Fe-histidine stretching frequency at 220 cm(-1), indicating the absence of significant proximal strain. We postulate that the function of Paramecium Hb is to supply oxygen for cellular oxidative processes.

Amino Acid Sequence↗
PubMed2000

Multielement analysis of polyethylene using solid sampling electrothermal vaporization ICP mass spectrometry

Next to laser ablation (LA) also electrothermal vaporization (ETV) from a graphite furnace as a means of sample introduction opens possibilities for direct analysis of solid samples using inductively coupled plasma mass spectrometry (ICPMS). In this paper, it is demonstrated that solid sampling ETV-ICPMS is very well suited for the determination of metal traces in polyethylene. A limited multielement capability is often cited as an important drawback of ETV-ICPMS. However, by studying the effect of monitoring an increasing number of mass-to-charge ratios on the signal profile (integrated signal intensity and repeatability) of selected analyte elements, the multielement capability of (solid sampling) ETV-ICPMS was systematically evaluated, and the results obtained suggest that, with a quadrupole-based ICPMS instrument, at least 11 elements can be determined "simultaneously" (from the same vaporization step), in essence without compromising the sensitivity or the precision of the results obtained. In this work, the "simultaneous" determination of Al, Ba, Cd, Cu, Mn, Pb, and Ti in a polyethylene candidate reference material has been accomplished, despite the large variation in analyte concentration (from 5 ng/g for Mn to 500 microg/g for Ti) and in furnace behavior (volatility) they exhibit. To avoid premature losses of Cd during thermal pretreatment of the samples, Pd was used as a chemical modifier. Two different calibration methods--external calibration using an aqueous standard solution and single standard addition--were studied and the results obtained were compared with those obtained using neutron activation analysis (NAA) and/or with the corresponding (candidate) certified values (if available). Single standard addition was shown to be preferable (average deviation between ICPMS result and reference value < 3%), although--except for Ba--acceptable results could also be obtained with external calibration.

Journal Article↗
PubMed2000

Isohemoglobin differentiation in the bimodal-breathing amazon catfish Hoplosternum littorale.

The bimodal gill(water)/gut(air)-breathing Amazonian catfish Hoplosternum littorale that frequents hypoxic habitats uses "mammalian" 2,3-diphosphoglycerate (DPG) in addition to "piscine" ATP and GTP as erythrocytic O(2) affinity modulators. Its electrophoretically distinct anodic and cathodic hemoglobins (Hb(An) and Hb(Ca)) were isolated for functional and molecular characterization. In contrast to Hb(An), phosphate-free Hb(Ca) exhibits a pronounced reverse Bohr effect (increased O(2) affinity with decreasing pH) that is obliterated by ATP, and opposite pH dependences of K(T) (O(2) association constant of low affinity, tense state) and the overall heat of oxygenation. Dose-response curves indicate small chloride effects and pronounced and differentiated phosphate effects, DPG < ATP < GTP < IHP. Hb(Ca)-O(2) equilibria analyzed in terms of the Monod-Wyman-Changeux model show that small T state bond energy differences underlie the differentiated phosphate effects. Synthetic peptides, corresponding to N-terminal fragment of the cytoplasmic domain of trout band 3 protein, undergo oxygenation-linked binding to Hb(Ca), suggesting a metabolic regulatory role for this hemoglobin. The amino acid sequences for the alpha and beta chains of Hb(Ca) obtained by Edman degradation and cDNA sequencing show unusual substitutions at the phosphate-binding site that are discussed in terms of its reverse Bohr effect and anion sensitivities.

2,3-Diphosphoglycerate↗
PubMed2000

A novel two-over-two alpha-helical sandwich fold is characteristic of the truncated hemoglobin family.

Small hemoproteins displaying amino acid sequences 20-40 residues shorter than (non-)vertebrate hemoglobins (Hbs) have recently been identified in several pathogenic and non-pathogenic unicellular organisms, and named 'truncated hemoglobins' (trHbs). They have been proposed to be involved not only in oxygen transport but also in other biological functions, such as protection against reactive nitrogen species, photosynthesis or to act as terminal oxidases. Crystal structures of trHbs from the ciliated protozoan Paramecium caudatum and the green unicellular alga Chlamydomonas eugametos show that the tertiary structure of both proteins is based on a 'two-over-two' alpha-helical sandwich, reflecting an unprecedented editing of the classical 'three-over-three' alpha-helical globin fold. Based on specific Gly-Gly motifs the tertiary structure accommodates the deletion of the N-terminal A-helix and replacement of the crucial heme-binding F-helix with an extended polypeptide loop. Additionally, concerted structural modifications allow burying of the heme group and define the distal site, which hosts a TyrB10, GlnE7 residue pair. A set of structural and amino acid sequence consensus rules for stabilizing the fold and the bound heme in the trHbs homology subfamily is deduced.

Amino Acid Motifs↗
PubMed2000

Modulation of the oxygen affinity of cobalt-porphyrin by globin.

We have combined two extreme effects which influence the oxygen affinity to obtain a cobalt-based oxygen carrier with an affinity similar to that of human adult hemoglobin (HbA). The goal was to obtain an oxygen transporter with a lower oxidation rate. Exchange of the heme group (Fe-protoporphyrin IX) in Hb with a cobalt-porphyrin leads to a reduction in oxygen affinity by over a factor of 10, an oxygen affinity too low for use as a blood substitute. At the other extreme, certain globin sequences are known to provide a very high oxygen affinity; for example, Hb Ascaris displays an oxygen affinity 1000 times higher than HbA. We demonstrate here that these opposing effects can be additive, yielding an oxygen affinity similar to that of HbA, but with oxygen binding to a cobalt atom. We have tested the effect of substitution of cobalt-porphyrin for heme in normal HbA, sperm whale (SW) Mb (Mb), and high affinity globins for leghemoglobin, two trematode Hbs: Paramphistomum epiclitum (Pe) and Gastrothylax crumenifer (Gc). As for HbA or SW Mb, the transition from heme to cobalt-porphyrin in the trematode Hbs leads to a large decrease in the oxygen affinity, with oxygen partial pressures for half saturation (P(50)) of 5 and 25 mm Hg at 37 degrees C for cobalt-Pe and cobalt-Gc, respectively. A critical parameter for Hb-based blood substitutes is the autoxidation rate; while both metals oxidize to an inactive state, we observed a decrease in the oxidation rate of over an order of magnitude for cobalt versus iron, for similar oxygen affinities. The time constants for autoxidation at 37 degrees C were 250 and 100 h for Pe and Gc, respectively.

Animals↗
PubMed2000

A hemoglobin with an optical function.

Hemoglobins are best known as oxygen transport proteins. Here we describe a hemoglobin from the parasitic nematode Mermis nigrescens (Mn-GLB-E) that has an optical, light shadowing function. The protein accumulates to high concentration as intracellular crystals in the ocellus of mature phototactic adult females while also being expressed at low concentration in other tissues. It differs in sequence and expression pattern from Mn-GLB-B, a second Mermis globin. It retains the structure and oxygen-binding and light-absorbing properties typical of nematode hemoglobins. As such, recruitment to a shadowing role in the eye appears to have occurred by changes in expression without modification of biochemistry. Both globins are coded by genes interrupted by two introns at the conserved positions B12.2 and G7.0, which is in agreement with the 3exon/2intron pattern model of globin gene evolution.

Amino Acid Sequence↗
PubMed1999

Daphnia pulex didomain hemoglobin: structure and evolution of polymeric hemoglobins and their coding genes.

The high-molecular-weight extracellular hemoglobin of Daphnia pulex is composed of at least three different didomain globin chains. The primary structure of one of these chains was determined at the protein and cDNA levels. Each globin domain of the polypeptide chain displays the standard structural characteristics. The first domain is preceded by a 30-residue extension containing an 18-residue unprecedented threonine-rich segment and a 12-residue preA segment which is homologous to the preA segments of other nonvertebrate globin chains. Both domains are linked together by a preA' segment, which is homologous to other preA segments and lacks the threonine-rich segment. Dimerization of the globin chains by the formation of a disulphide bridge linking the unique cysteines near the amino-termini results in a covalent, vertebrate-like tetradomain structure. The flexible amino-terminal extension most likely facilitates dimerization. The gene coding for this globin chain is interrupted by six small introns. Each domain displays two intradomain introns at the conserved positions B12.2 and G7.0. A precoding intron occurs at position preA(-27.0) and a bridge intron at occurs preA'(-13.2). We propose a crossover event as the most likely mechanism for duplication. Arthropod globin trees reflect the added effects of gene diversification, gene duplication, and species evolution. The position of monodomain intracellular globins in the tree suggests that they resemble the ancestral globin more than the derived didomain extracellular globins do.

Amino Acid Sequence↗
PubMed1998

Structural, functional, and genetic characterization of Gastrophilus hemoglobin.

Hemoglobin of Gastrophilus intestinalis (Insecta, Diptera), was purified and characterized. At least two isoforms have been identified by isoelectrofocusing, mass spectrometry, and genomic Southern blotting. Functional studies show a high oxygen affinity due to a low ligand dissociation rate (koff = 2.4 s-1) and a relatively high autoxidation rate (t1/2 = 1.6/h). The globins were separated under denaturing conditions, and the sequence of Hb1 (Mr = 17,965 +/- 2) was determined at the protein and DNA level. The open reading frame codes for a polypeptide of 150 amino acids. Although the globin is distantly related to globins from other species, it has a low penalty score against globin templates. Freshly isolated hemoglobin was crystallized from polyethylene glycol. Crystals contain two hemoglobin molecules per asymmetric unit. Solution of the three-dimensional structure by molecular replacement could not be achieved, possibly due to the presence of three protein isoforms in the crystals. In order to determine its three-dimensional structure, G. intestinalis Hb1 was overexpressed in Escherichia coli, resulting in a fully functional molecule as confirmed by ligand binding affinity. The globin gene contains two introns at positions D7.0 and G7.0. The D7.0 intron is unprecedented, suggesting that globin gene evolution is much more complex than originally thought.

Amino Acid Sequence↗
PubMed1998

Determination of the uptake of [Pt(NH3)4](NO3)2 by grass cultivated on a sandy loam soil and by cucumber plants, grown hydroponically.

Two cultivation experiments were carried out in order to answer the question to what extent platinum can enter the food chain by accumulation in plants, when the platinum is present in a bio-available form: (i) cucumber plants (Cucumis sativus) were grown hydroponically in nutrient solutions containing [Pt(NH3)4](NO3)2 (from 0.5 to 50 micrograms Pt/l solution); and (ii) a water-soluble platinum compound--[Pt(NH3)4](NO3)2--was added in increasing amounts to a sandy loam soil (from 0.5 to 50 mg Pt/kg soil) and rye grass (Lolium perenne) was grown on it. The roots on the one hand and the green plant fractions in the other hand of the cucumber plants and the rye grass were digested using a high-pressure asher. The platinum concentration was determined by means of a quadrupole-based (VG PQ I) or a double focusing sector field ICP-mass spectrometer (Finnigan MAT, Element), depending on the platinum concentration in the sample solution. The detection limit for platinum obtained with the VG PQ I was observed to be 6 ng/1, while with the 'Element' the detection limit could be improved to 0.5 ng/1 Pt. Accumulation factors were calculated as the ratio of the platinum concentration in the plant to that in the soil or the nutrient solution. The grass grown on spiked soil accumulated platinum only to a slight degree (accumulation factors between 0.008 and 0.032). The hydroponically grown cucumber plants, however, strongly accumulated it (accumulation factors of 11-42 in the shoot and 1700-2100 in the roots). There are three possible causes for the large differences in the accumulation factors: (i) Cucumber plants are dicotyledons; grass, however, is a monocotyledon. Other cultivation experiments already showed that dicotyledons accumulate metals to a higher extent than monocotyledons. (ii) In the grass cultivation experiment, the platinum compound was only added once to the sandy loam soil, namely 2 days before grass was cultivated on it. The nutrient solutions of the cucumber plants were changed twice a week. Consequently, the total amount of platinum that the plants were exposed to during the cultivation of the cucumber plants was higher than during the cultivation of the grass. (iii) Immobilization of the platinum compound in the soil most likely occurred.

Biological Availability↗
PubMed1998

Characterization of the myoglobin and its coding gene of the mollusc Biomphalaria glabrata.

A cDNA clone isolated from a Biomphalaria glabrata (Mollusca, Gastropoda) neural cDNA library was identified as encoding a myoglobin-like protein of 148 amino acids with a single domain and a calculated mass of 16,049.29. Alignment with globin sequences with known tertiary structure confirms its overall globin nature. The expressed myoglobin was identified in the radular muscle and isolated. Oxygen equilibrium measurements on the protein reveal a high oxygen affinity. Val-B10 and Gln-E7, important residues for the determination of the oxygen affinity, are strikingly different from the standard molluscan pattern (Conti, E., Moser, C., Rizzi, M., Mattevi, A., Lionetti, C., Coda, A., Ascenzi, P., Brunori, M., Bolognesi, M. (1993) J. Mol. Biol. 233, 498-508). The single gene encoding the globin chain is interrupted by three introns at positions A3.2, B12.2, and G7.0. Comparison with other nonvertebrate globin genes reveals on the one hand conservation (B12.2 and G7.0) and on the other hand variability of the insertion positions (A3.2). The Biomphalaria myoglobin sequence was used together with all other molluscan globin sequences available to assess the origin and phylogeny of the phylum. Our results confirm the doubts raised about monophyletic origin of the Mollusca, which was first observed using SSU rRNA as a molecular marker.

Amino Acid Sequence↗
PubMed1998

A hierarchy of disulfide-bonded subunits: the quaternary structure of Eudistylia chlorocruorin.

The quaternary structure of the cysteine-rich, approximately 3500-kDa chlorocruorin (Chl) from the marine polychaete Eudistylia vancouverii was investigated using maximum entropy deconvolution of the electrospray ionization mass spectra (ESIMS). The native Chl provided two groups of peaks, at approximately 25 and approximately 33 kDa, and one peak at approximately 66 kDa. ESIMS of the reduced and reduced and carbamidomethylated Chl and of its subunits obtained by HPLC provided the complete subunit composition of the Chl. Two groups of nonglobin linker chains were observed: L1a-f (25 000.4, 25 017.9, 25 039.6, 25 057.0, 25 074.4 and 25 096.8 Da) and L2a-d (25 402.7, 25 446.0, 25 461.6 and 25 478.3Da) (+/-2.5 Da), with relative intensities L1:L2 = 5:2. Six globin chains were found, a1, a2, and b1-4, with reduced masses of 16 051.5, 16 172.4, 16 853.5, 17 088.9, 17 161.2 and 17 103.6 (+/-1.0 Da) and relative intensities of 8:4:1:4:2:1, respectively. Disulfide-bonded dimers and a tetramer of globin chains were identified: D1 = a1 + b3 at 33 207.1; D2 at 33 374.1, which had a cysteinylated Cys (a2 + b2 + Cys); and D3 = a1 + b4 at 33 149.4 Da (+/-3.0 Da), with relative intensities D1:D2:D3 = 5:4:1 and T = a1 + a2 + b1 + b2 at 66 154.8 +/- 4.0 Da. A 206-kDa dodecamer subunit obtained by dissociation of the Chl in 4 M urea [Qabar, A. N., et al. (1991) J. Mol. Biol. 222, 1109-1129], was found to consist only of tetramers T. A model was proposed for the Chl, based on a dimer:tetramer ratio of 2:1: four 206-kDa dodecamers (trimer of tetramers) and 48 dimers tethered to a framework of 30 L1 and 12 L2 linker chains. The 144 globin chains (2480 kDa) and 42 linker chains (1059 kDa) provide a total mass of 3539 kDa, in good agreement with the 3480 +/- 225 kDa determined previously by STEM mass mapping. The hierarchy of disulfide-bonded globin subunits observed for Eudistylia Chl provides a built-in heterogeneity of hexagonal bilayer structures.

Animals↗
PubMed1998

Trematode hemoglobins show exceptionally high oxygen affinity.

Ligand binding studies were made with hemoglobin (Hb) isolated from trematode species Gastrothylax crumenifer (Gc), Paramphistomum epiclitum (Pe), Explanatum explanatum (Ee), parasitic worms of water buffalo Bubalus bubalis, and Isoparorchis hypselobagri (Ih) parasitic in the catfish Wallago attu. The kinetics of oxygen and carbon monoxide binding show very fast association rates. Whereas oxygen can be displaced on a millisecond time scale from human Hb at 25 degrees C, the dissociation of oxygen from trematode Hb may require a few seconds to over 20 s (for Hb Pe). Carbon monoxide dissociation is faster, however, than for other monomeric hemoglobins or myoglobins. Trematode hemoglobins also show a reduced rate of autoxidation; the oxy form is not readily oxidized by potassium ferricyanide, indicating that only the deoxy form reacts rapidly with this oxidizing agent. Unlike most vertebrate Hbs, the trematodes have a tyrosine residue at position E7 instead of the usual distal histidine. As for Hb Ascaris, which also displays a high oxygen affinity, the trematodes have a tyrosine in position B10; two H-bonds to the oxygen molecule are thought to be responsible for the very high oxygen affinity. The trematode hemoglobins display a combination of high association rates and very low dissociation rates, resulting in some of the highest oxygen affinities ever observed.

Animals↗
PubMed1998

Determination of butyltins in harbour sediment and water by aqueous phase ethylation GC-ICP-MS and hydride generation GC-AAS.

Sediment and water samples have been collected between 1992 and 1995 for evaluating butyltin contamination in two marinas from the coastal area in The Netherlands, two years after the ban of TBT. Sediments extracts were analysed by capillary gas chromatography-inductively coupled plasma-mass spectrometry. Sediment concentrations for TBT showed no trend of decrease between 1992 and 1995 and were extremely high in the marina secluded from tidal action; 17.5 +/- 8.0 microg g(-1) and much lower for the marina with tidal action; 0.117 +/- 0.073 microg g(-1). High ratios between TBT and DBT in the sediments indicate that degradation mechanisms in the sediments are of minor importance. Dissolved butyltin compounds were analysed in water by means of gas chromatography-atomic absorption spectrometry. Water concentrations of TBT showed no clear trend of decrease between 1992 and 1995 and were high in the marina secluded from tidal action; 139 +/- 166 ng litre(-1) but much lower for the marina with tidal action; 48 +/- 98 ng litre(-1). An active degradation mechanism during summer periods was indicated by low (<1) TBT/DBT ratios in the water phase of the marina secluded from tidal action.

Journal Article↗
PubMed1998

Uptake of ingested uranium after low "acute intake".

The uptake of uranium, ingested as a soluble compound, was studied by monitoring the uranium level in urine by inductively coupled plasma mass spectrometry and through measurement of an isotopic tracer. The high sensitivity of this method allows measurement of uranium levels in urine samples from each voiding, therefore more detailed biokinetic studies are possible. To simulate low "acute intake," five volunteers with "normal" levels (5-15 ng L(-1)) of uranium in urine ingested a grapefruit drink spiked with 100 microg of uranium (235U/238U = 0.245%) as uranyl nitrate, and the level of uranium in their urine after ingestion was monitored. Two techniques were applied to estimate the extent of exposure: a) uranium levels above the normal level for each volunteer; and b) the deviation from natural isotopic ratio. Results were normalized relative to the creatinine concentration, which served as an indicator of urine dilution, to reduce effects due to diurnal changes. The results clearly indicate that currently accepted bio-kinetic models overestimate the time between ingestion of dissolved uranium and its excretion in urine, the maximum of which was found to be around 6-10 h. The uptake fraction was in agreement with recent studies, i.e., 0.1-0.5% of the ingested uranium for four of the subjects but above 1.5% for the fifth, and well below the 5% reported in International Commission on Radiation Protection Publication 54. Finally, partial results from the isotope dilution study indicate that uranium absorbed through the intestine interchanges with uranium retained in body organs. The time scale of this process is quite short, and the acute exposure led to a minimum in the isotopic ratio within hours, while recovery back to natural abundance due to low chronic exposure takes several days.

Adult↗