Biomedical subjects

L Moens

Publications and source records attributed to L Moens.

At least 37 records · Page 2Linked to original sources

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↗

PubMed1997

Biochemical characterization of cardiotin, a sarcoplasmic reticulum associated protein.

The further biochemical characterization and subcellular localization of cardiotin, a high molecular weight (300 kDa) constituent of cardiac muscle, is described. Immunofluorescence assays revealed a colocalization of cardiotin and the Ca2+ pump SERCA2a in the longitudinal sarcoplasmic reticulum (SR). However, in contrast to SERCA2a, cardiotin is not detected in the junctional SR. Differential centrifugation experiments show that cardiotin cosediments with the microsomal fraction of swine heart, while differential extraction demonstrates that cardiotin is associated with the SR membranes. In the SR enriched cell fraction a 60 and a 100 kDa protein band are detected. Microsequence analyses of these two fragments showed a common amino-terminus of 14 amino acids, with great homology to amino acid positions 11-24 of human skeletal muscle alpha-actinin. Second generation antibodies directed to these specific fragments show the typical cardiotin pattern in cardiomyocytes and cross-reactivity amongst the respective antigens. Cardiotin did not colocalize with alpha-actinin, and alpha-actinin could not be detected in the microsomal SR fraction. Cardiotin therefore represents a new SR associated constituent.

Actinin↗

PubMed1997

Trematode myoglobins, functional molecules with a distal tyrosine.

The myoglobins of two trematodes, Paramphistomum epiclitum and Isoparorchis hypselobagri, were isolated to homogeneity. The native molecules are monomeric with Mr 16,000-17,000 and pI 6.5-7.5. In each species, at least four different globin isoforms occur. Primary structure was determined at the protein level. The globin chains contain 147 amino acid residues. Although major determinants of the globin fold are conserved, characteristic substitutions are present. A Tyr residue occurs at the helical positions B10 and E7 (distal position). This is confirmed by NMR measurements (Zhang, W., Rashid, K. A., Haque, M., Siddiqi, A. H., Vinogradov, S. N., Moens, L. & La Mar, G. N. (1997) J. Biol. Chem. 272, 3000-3006). A distal Tyr normally provokes oxidation of the iron atom and the inability to bind oxygen, whereas a Tyr-B10 is indicative for a high oxygen affinity. In contrast, trematode myoglobins are functional molecules with a high oxygen affinity. Molecular modeling predicts two possible positions for the aromatic ring of Tyr-E7: one being outside the heme pocket making it freely accessible to the ligand and one within the heme pocket potentially able to form a second hydrogen bond with the iron-bound oxygen. A hydrogen bond between Tyr-B10 and the bound oxygen as in the Ascaris hemoglobin is predicted as well. The predicted structure may explain the high oxygen affinity of the trematode myoglobins.

Amino Acid Sequence↗

PubMed1997

Solution of 1H NMR structure of the heme cavity in the oxygen-avid myoglobin from the trematode Paramphistomum epiclitum.

A two-dimensional 1H NMR study has been carried out on the heme cavity of the extreme oxygen-avid and autoxidation-resistant oxy-myoglobin complex from the trematode Paramphistomum epiclitum, and the residues were identified which potentially provide hydrogen bond stabilization for the bound oxygen. Complete assignment of the heme core resonances allows the identification of 10 key heme pocket residues, 4 Phe, 4 Tyr, and 2 upfield ring current aliphatic side chains. Based solely on the conserved myoglobin folding topology that places the E helix-heme crossover and the completely conserved Phe(CD1)-heme contact at opposing meso positions, the heme orientation in the cavity and the E helix alignment were unambiguously established that place Tyr66 at position E7. Moreover, all eight aromatic and the two aliphatic side chains were shown to occupy the positions in the heme cavity predicted by amino acid sequence alignment with globins of known tertiary structure. The dipolar contacts for the Tyr32(B10) and Tyr66(E7) rings indicate that both residues are oriented into the heme cavity, which is unprecedented in globins. The ring hydroxyl protons for both Tyr are close to each other and in a position to provide hydrogen bonds to the coordinated oxygen, as supported by strong retardation of their exchange rate with bulk solvent. A more crowded and compact structure increases the dynamic stability of the distal pocket and may contribute to the autoxidation resistance of this myoglobin.

Amino Acid Sequence↗

PubMed1997

Computer analysis of two-dimensional electrophoresis gels: a new segmentation and modeling algorithm.

The complexity of the spot patterns of two-dimensional electrophoresis gels made it necessary to use image processing techniques to analyze the gels. An important issue in the analysis is the detection and quantification of the protein spots. In this paper we describe a new technique to segment and model the different spots. For the segmentation of a gel into its different spot regions we apply a watershed technique, which is robust and efficient. For the quantification of the spots, a new spot model is constructed, based on diffusion principles. Besides the advantage of having a physical interpretation, the model is demonstrated to be superior to the commonly used Gaussian models.

Algorithms↗

PubMed1996

Globin and globin gene structure of the nerve myoglobin of Aphrodite aculeata.

The globin of the nerve cord of the polychaete annelid Aphrodite aculeata was isolated and purified to homogeneity. The native molecule has a pI of 6.3 and acts as a dimer of two identical Mr 15, 644.5 polypeptide chains as determined by electrospray mass spectrometry. It has an average affinity for oxygen (P50 = 1.24 torr) resulting from fast association (kon = 170 X 10(6) M-1 . s-1) and dissociation rates (koff = 360 s-1). The partial primary structure of this nerve globin was determined at the protein level and completed and confirmed by translation of the cDNA sequence. The globin chain has 150 amino acid residues and a calculated Mr of 15, 602.69 strongly suggesting that the amino terminus is acetylated. The absence of a leader sequence and the lack of Cys at the positions NA2 and H9 needed for the formation of the high Mr complexes found in extracellular annelid globins classify the Aphrodite globin with the cellular globin species. The Aphrodite nerve globin is unlikely to represent a separate globin family, as cDNA derived primers detect globin messenger RNA in muscle, gut, and pharynx tissue as well. The gene encoding this globin species is interrupted by a single intron, inserted at position G7.0. Comparison to other globin gene structures strongly suggest that introns can be lost independently, rather than simultaneously as a result of a single conversion event as suggested previously (Lewin, R. (1984) Science 226, 328).

Amino Acid Sequence↗

PubMed1996

Time-dependent pseudo-activation of hepatic glycogen synthase b by glucose 6-phosphate without involvement of protein phosphatases.

During a 30 min incubation at 25 degrees C in the presence of 5-10 mM glucose 6-phosphate, pure glycogen-bound glycogen synthase b from dog liver was progressively converted into a form that was fully catalytically active in the presence of 10 mM Na2SO4 plus 0.5 mM glucose 6-phosphate. The latter enzyme was unlike synthase a (which does not require glucose 6-phosphate for activity), and unlike synthase b (which is strongly inhibited by sulphate). The conversion was insensitive to various inhibitors of Ser/Thr-protein phosphatases and alkaline phosphatases, and was therefore termed 'pseudo-activation'. Kinetically, pseudo-activation increased the V(max) 4-fold without affecting the K(m) for the substrate UDP-glucose. Pseudo-activation appeared to be an irreversible process, but several lines of evidence argue against a limited proteolysis. Pseudo-activation of glycogen synthase occurred also readily in a rat liver cytosol, but it was not observed with purified synthase from skeletal muscle. These observations have important implications for the assay of liver gycogen-synthase phosphatase; the possible physiological implications remain to be explored.

Animals↗

PubMed1996

Determination of low amounts of platinum in environmental and biological materials using thermospray nebulization inductively coupled plasma-mass spectrometry.

A description is given of the experimental arrangement for thermospray nebulization of discrete liquid samples (100 microl) into an inductively coupled plasma-mass spectrometer (ICP-MS). The determination of ultratrace levels of platinum in environmental and biological materials is described. This method comprises an off-line sample enrichment by adsorptive chromatography, elution with ethanol and subsequent analysis of the evaporated eluate. The quantification of the concentration was performed with isotope dilution. The recovery, precision, accuracy and detection limit of the method are discussed.

Journal Article↗