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Biomedical subjects

L Moens

Publications and source records attributed to L Moens.

At least 19 recordsLinked to original sources
PubMed2001

Crystallization and preliminary X-ray analysis of neural haemoglobin from the nemertean worm Cerebratulus lacteus.

The nemertean worm Cerebratulus lacteus neural tissue haemoglobin (109 amino acids, the shortest known haemoglobin) has been overexpressed in Escherichia coli, purified and crystallized. A highly redundant native data set has been collected at the Cu K(alpha) wavelength to 2.05 A resolution. The crystals belong to the orthorhombic P2(1)2(1)2(1) space group, with unit-cell parameters a = 42.5, b = 43.1, c = 60.2 A and one molecule per asymmetric unit. The anomalous difference Patterson map clearly reveals the position of the haem Fe atom, thus paving the way for MAD/SAD structure determination.

Animals↗
PubMed2001

Biochemical characterization and ligand binding properties of neuroglobin, a novel member of the globin family.

Neuroglobin is a recently discovered member of the globin superfamily that is suggested to enhance the O(2) supply of the vertebrate brain. Spectral measurements with human and mouse recombinant neuroglobin provide evidence for a hexacoordinated deoxy ferrous (Fe(2+)) form, indicating a His-Fe(2+)-His binding scheme. O(2) or CO can displace the endogenous protein ligand, which is identified as the distal histidine by mutagenesis. The ferric (Fe(3+)) form of neuroglobin is also hexacoordinated with the protein ligand E7-His and does not exhibit pH dependence. Flash photolysis studies show a high recombination rate (k(on)) and a slow dissociation rate (k(off)) for both O(2) and CO, indicating a high intrinsic affinity for these ligands. However, because the rate-limiting step in ligand combination with the deoxy hexacoordinated form involves the dissociation of the protein ligand, O(2) and CO binding is suggested to be slow in vivo. Because of this competition, the observed O(2) affinity of recombinant human neuroglobin is average (1 torr at 37 degrees C). Neuroglobin has a high autoxidation rate, resulting in an oxidation at 37 degrees C by air within a few minutes. The oxidation/reduction potential of mouse neuroglobin (E'(o) = -129 mV) lies within the physiological range. Under natural conditions, recombinant mouse neuroglobin occurs as a monomer with disulfide-dependent formation of dimers. The biochemical and kinetic characteristics are discussed in view of the possible functions of neuroglobin in the vertebrate brain.

Animals↗
PubMed2001

Very high resolution structure of a trematode hemoglobin displaying a TyrB10-TyrE7 heme distal residue pair and high oxygen affinity.

Monomeric hemoglobin from the trematode Paramphistomum epiclitum displays very high oxygen affinity (P(50)<0.001 mm Hg) and an unusual heme distal site containing tyrosyl residues at the B10 and E7 positions. The crystal structure of aquo-met P. epiclitum hemoglobin, solved at 1.17 A resolution via multiwavelength anomalous dispersion techniques (R-factor=0.121), shows that the heme distal site pocket residue TyrB10 is engaged in hydrogen bonding to the iron-bound ligand. By contrast, residue TyrE7 is unexpectedly locked next to the CD globin region, in a conformation unsuitable for heme-bound ligand stabilisation. Such structural organization of the E7 distal residue differs strikingly from that observed in the nematode Ascaris suum hemoglobin (bearing TyrB10 and GlnE7 residues), which also displays very high oxygen affinity. The oxygenation and carbonylation parameters of wild-type P. epiclitum Hb as well as of single- and double-site mutants, with residue substitutions at positions B10, E7 and E11, have been determined and are discussed here in the light of the protein atomic resolution crystal structure.

Amino Acid Sequence↗
PubMed2001

The primary structure of globin and linker chains from the chlorocruorin of the polychaete Sabella spallanzanii.

Annelid hemoglobins are organized in a very complex supramolecular network of interacting polypeptides, the structure of which is still not wholly resolved. We have separated by two-dimensional electrophoresis the 4-MDa chlorocruorin of Sabella spallanzanii and identified its components by amino-terminal sequencing. This work reveals a high rate of heterogeneity of constituent chains in a single animal as well as in the Sabella population. Using a cDNA library prepared from the hematopoietic tissue of this worm, we have isolated and fully sequenced most globin and linker cDNAs. The primary structure features of these polypeptides have been characterized by comparison with model globin and linker sequences.

Amino Acid Sequence↗
PubMed2001

The evolution of extracellular hemoglobins of annelids, vestimentiferans, and pogonophorans.

The evolution of extracellular hemoglobins of annelids, vestimentiferans, and pogonophorans was investigated by applying cladistic and distance-based approaches to reconstruct the phylogenetic relationships of this group of respiratory pigments. We performed this study using the aligned sequences of globin and linker chains that are the constituents of these complex molecules. Three novel globin and two novel linker chains of Sabella spallanzanii described in an accompanying paper (Pallavicini, A., Negrisolo, E., Barbato, R., Dewilde, S., Ghiretti-Magaldi, A., Moens, L., and Lanfranchi, G. (2001) J. Biol. Chem. 276, 26384--26390) were also included. Our results allowed us to test previous hypotheses on the evolutionary pathways of these proteins and to formulate a new most parsimonious model of molecular evolution. According to this novel model, the genes coding for the polypeptides forming these composite molecules were already present in the common ancestor of annelids, vestimentiferans, and pogonophorans.

Amino Acid Sequence↗
PubMed2001

Stir bar sorptive extraction for the determination of ppq-level traces of organotin compounds in environmental samples with thermal desorption-capillary gas chromatography--ICP mass spectrometry.

The extraction and preconcentration capabilities of a new extraction technique, stir bar sorptive extraction, were combined with the separation power of capillary gas chromatography (CGC) and the low limits of detection (LODs) of inductively coupled plasma mass spectrometry (ICPMS) for the determination of the organotin compounds tributyltin (TBuT) and triphenyltin (TPhT) in aqueous standard solutions, harbor water, and mussels (after digestion with tetramethylammonium hydroxide). Throughout, tripropyltin for TBuT and tricyclohexyltin for TPhT were used as internal standards to correct for variations in the derivatization and extraction efficiency. Calibration was accomplished by means of single standard addition. Derivatization to transform the trisubstituted compounds into sufficiently volatile compounds was carried out with sodium tetraethylborate. The compounds were extracted from their aqueous matrix using a stir bar of 1-cm length, coated with 55 microL of poly(dimethylsiloxane) (PDMS). After 15 min of extraction, the stir bar was desorbed in a thermal desorption unit at 290 degrees C for 15 min, during which the compounds were cold-trapped on a precolumn at -40 degrees C. Flash heating was used to rapidly transfer the compounds to the GC where they were separated on a capillary column with a PDMS coating. After separation, the compounds were transported to the ICP by means of a homemade heated (270 degrees C) transfer line. Monitoring of the 120Sn+ signal by ICPMS during the run of the GC provided extremely low LODs for TPhT in water: 0.1 pg L(-1) (procedure) and 10 fg L(-1) (instrumental) and a repeatability of 12% RSD (n = 10). In harbor water, concentrations of 200 pg L(-1) for TBuT and 22 pg L(-1) for TPhT were found. In fresh mussels, a concentration of 7.2 ng g(-1) (dry weight) TPhT was found. The accuracy of the method was checked by the determination of TPhT in CRM477 (mussel tissue) and comparison of the result to that of an analysis of the same material with a classical liquid/liquid extraction with isooctane.

Animals↗
PubMed2001

Protein and gene structure of a chlorocruorin chain of Eudistylia vancouverii.

The polychaete annelid, Eudistylia vancouverii, contains as oxygen carrier a hexagonal bilayer (HBL) chlorocruorin. One of the globin chains, chain a1, has 142 amino acids (Mr 16,054.99) and its sequence deviates strongly from other nonvertebrate globin sequences. Unprecedented, it displays a Phe at the distal position E7 as well as at position B10, creating a very hydrophobic heme pocket probably responsible for the low oxygen affinity of the native molecule. Phylogenetic analysis of annelid globin chains clearly proves that globin chain a1 belongs to type I of globin chains having a pattern of 3 cysteine residues essential for the aggregation into a HBL structure. The gene coding for globin chain a1 is interrupted by 2 introns at the conserved positions B12.2 and G7.0. Based on protein and gene structure it can therefore be concluded that the globin chains of chlorocruorins are not fundamentally different from other annelid globin chains.

Amino Acid Sequence↗
PubMed2001

Determination of rare earth elements in environmental matrices by sector-field inductively coupled plasma mass spectrometry.

In the framework of an international certification campaign, sector-field inductively coupled plasma mass spectrometry (sector-field ICP-MS) was used for the accurate determination of the rare earth elements in five candidate reference materials: aquatic plant, calcareous soil, mussel tissue, river sediment, and tuna muscle. All samples were taken into solution by use of microwave-assisted or mixed microwave-assisted / open beaker acid digestion. Subsequently, the samples were appropriately diluted and subjected to ICP-MS analysis. Except for Sc, all the elements involved were determined at low mass resolution (R = 300). For Sc, application of a higher resolution setting (R = 3,000) was required to separate the analyte signal from those of several molecular ions which gave rise to spectral overlap at low mass resolution. Some of the heavier REE can also suffer from spectral overlap attributed to the occurrence of oxide ions (MO+) of the lighter REE and Ba. This spectral overlap could be successfully overcome by mathematical correction. Matrix effects were overcome by use of two carefully selected internal standards, such that external calibration could be used. On each occasion, a geological reference material was analyzed as a quality-control sample and the reliability of all results obtained was additionally checked by means of chondrite normalization. For tuna muscle the content of all REE was below the limit of detection. For calcareous soil and river sediment, low to sub microg g(-1) values were observed, whereas the REE content of aquatic plant and mussel tissue was considerably lower (low to sub ng g(-1)). Overall, the results obtained were in excellent agreement with the average values, calculated on the basis of all "accepted" values, obtained in different laboratories using different techniques.

Journal Article↗
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↗