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Biomedical subjects

L M van Golde

Publications and source records attributed to L M van Golde.

At least 19 recordsLinked to original sources
PubMed1999

Production of surfactant protein C in the baculovirus expression system: the information required for correct folding and palmitoylation of SP-C is contained within the mature sequence.

Surfactant protein C (SP-C) is synthesized in the alveolar type II cells of the lung as a 21 kDa propeptide which is proteolytically processed to a 4.2 kDa mature active form. The main function of this extremely hydrophobic protein is to enhance lipid insertion into the air/liquid interface in the lung upon inhalation. This is necessary to maintain a relatively low surface tension at this interface during breathing. In this report we describe the production of mature human SP-C in the baculovirus expression system. The recombinant protein contains a secondary structure with a high alpha-helical content (73%), comparable to native SP-C, as determined by circular dichroism and attenuated total reflection Fourier transform infrared analysis. The expressed protein is a mixture of dipalmitoylated (15%) and non-palmitoylated SP-C. This suggests that the information required for palmitoylation is contained within the sequence of the mature protein. The activity of the protein to insert phospholipids into a preformed monolayer of lipids at an air/liquid interface was determined with a captive bubble surfactometer. Recombinant SP-C significantly reduced the surface tension at the air/liquid interface during dynamic expansion and compression. We conclude that correctly folded, dipalmitoylated and active SP-C can be expressed in the baculovirus expression system. Our results may facilitate investigations into the relation between structure and function of SP-C and into protein palmitoylation in general.

Animals↗
PubMed1999

Rapid separation and identification of phosphatidylethanolamine molecular species.

A novel high performance liquid chromatography method is presented for the separation and identification of intact molecular species of phosphatidylethanolamine (PE). After isocratic separation, detection of species can be achieved by measurement of UV absorbance as well as by the quantitative method of light scattering detection. A mathematical relationship exists between i) the relative retention time of a PE molecular species and ii) the number of carbon atoms and double bonds in the aliphatic groups of the species. This relationship can aid in the identification of the species. Furthermore, the absence of non-volatile components in the solvent allows the use of electrospray mass spectrometry to identify the eluting components and to establish the position of the individual radyl groups at the glycerol backbone. Using this method, samples of bovine heart PE (rich in plasmalogens) and rat liver PE (rich in diacyl species) have been analyzed.

Animals↗
PubMed1998

Ether lipids and their possible physiological function in adult Schistosoma mansoni.

Schistosomes have lost the capability to synthesize fatty acids de novo, but they can modify fatty acids by chain elongation. This has a profound effect on the molecular species composition of the two main phospholipid fractions of schistosomes, phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Molecular species of phospholipids are increasingly recognized as important mediators, or precursors thereof, in signal transduction, immune response modulation, and events like membrane fusion. As these are all important aspects of schistosome membranes and of the tegumental membranes in particular, we analysed the PE and PC molecular species of the tegumental membranes, the worm body and the blood of the host. With the aid of on-line mass spectrometry, we unequivocally identified a large number of PC and PE species in schistosomes, among which considerable amounts of plasmalogen species. This was unexpected, as this lipid subclass has been assumed to be absent in the parasite. Species, like (20:1-16:0) diacyl PC and (16:0-20:1) plasmalogen PE, found to be main constituents in schistosomes, were absent from the blood of the host. Large differences were also found between the molecular species composition of the tegumental membranes and the membranes of the worm body. In the tegumental membranes, 1-hexadecyl 2-palmitoyl PC was detected, which could possibly function as a precursor for platelet activating factor (PAF).

Animals↗
PubMed1998

5-Octadecenoic acid: evidence for a novel type of fatty acid modification in schistosomes.

The lipid metabolism of schistosomes is characterized by several intriguing adaptations to a parasitic way of living. The surface of the parasite consists of two closely apposed phospholipid bilayers, a structure unique to blood flukes. Schistosomes do not synthesize fatty acids de novo, but are able to modify fatty acids, which they obtain from the host, by chain elongation. Here we present evidence that schistosomes are capable of another type of fatty acid modification, resulting in the formation of 5-octadecenoic acid [C18:1(5)]. This highly unusual fatty acid, which is absent in the blood of the host, was shown to be almost exclusively located in the outer membrane complex of the schistosome. Within these membranes, it was almost exclusively present in one molecular phospholipid species, 1-palmitoyl-2,5-octadecenoyl phosphatidylcholine [C16:0-18:1(5)PtdCho]. Apart from dipalmitoyl phosphatidylcholine, this was the most abundant phosphatidylcholine species in the outer membrane complex. The specific synthesis by the schistosome of C18:1(5) and the highly specific localization of this fatty acid to the tegumental membranes suggest an important tegument-mediated role for this lipid.

Animals↗
PubMed1998

A microtiter plate assay for the determination of uronic acids.

The amount of uronic acid residues in samples containing glycosaminoglycans or pectin is an important parameter in the quantitative and structural analysis of these complex carbohydrates. This paper describes a method to determine the content of uronic acids in biological samples, using conventional polystyrene microtiter plates and microtiter plate-reading equipment with standard interference filters (i.e., 540 or 492 nm). This assay is a modification of a commonly used procedure, viz. hydrolysis of uronic acid containing carbohydrate polymers in 80% sulfuric acid containing tetraborate ions at 80 degrees C followed by a coloring step with an m-hydroxydiphenyl reagent at room temperature. The use of microtiter plates has several practical advantages: (i) less risk of handling hot, concentrated sulfuric acid is present; (ii) an accurate estimate of background absorbance by multiple reading of the plates is possible; and (iii) many samples can be assayed in one series without errors due to fading of the final color. The validity of the assay was checked for the quantification of hyaluronic acid in equine synovial fluid samples. We consider this the method of choice when a large number of samples must be analyzed for their content of uronic acid residues.

Animals↗
PubMed1998

Loading-induced changes in synovial fluid affect cartilage metabolism.

The purpose of this study was to determine whether changes in the synovial fluid (SF) induced by in vivo loading can induce an alteration in the metabolic activity of chondrocytes in vitro. Therefore, SF was collected from ponies after a period of box rest and after they had exercise for a week. Normal, unloaded articular cartilage explants were cultured in 20% solutions of these SFs for 4 days and chondrocyte activity was determined by glycosaminoglycan (GAG) turnover. In explants cultured in post-exercise SF, GAG synthesis was enhanced and GAG release was diminished when compared to cultures in pre-exercise SF. SF analysis showed that levels of insulin-like growth factors (IGF-I and IGF-II) tended to be higher in post-exercise SF, while no differences were found in metalloproteinase activity, hyaluronic acid and protein concentrations. This study showed that anabolic effects of joint loading on cartilage are, at least partially, mediated by alterations in the SF.

Animals↗
PubMed1998

Use of lectins to characterize plasma membrane preparations from boar spermatozoa: a novel technique for monitoring membrane purity and quantity.

The object of this study was to develop a method to quantify the amount of outer acrosomal membrane material in isolated plasma membranes from boar sperm cells. The cells were fractionated by nitrogen cavitation, and plasma membranes were isolated by subsequent differential centrifugation steps. Marker enzyme measurement showed that the plasma membrane isolates were enriched in plasma membrane markers and did not contain nuclei, inner acrosomal membranes, or mitochondria. Since there is no marker enzyme known for the outer acrosomal membrane, lectins were used for the detection of this membrane. The membrane specificity of a number of lectin conjugates was tested with fluorescence microscopy and transmission electron microscopy. Membrane binding of these lectin conjugates was quantified with flow-cytometry and an enzyme-linked lectin binding assay. Wheat germ agglutinin was specific for the plasma membrane while peanut agglutinin was specific for the outer acrosomal membrane. The use of these lectins made it possible for the first time to discriminate between these two membranes. The isolated plasma membrane fraction was enriched more than 10-fold (17-fold after further purification by a sucrose gradient) in plasma membrane material compared to outer acrosomal membrane material. Highly purified sperm plasma membranes should prove to be useful for research on primary sperm-zona interactions.

Acrosome↗
PubMed1998

Quantitative analysis of phosphatidylcholine molecular species using HPLC and light scattering detection.

A number of HPLC chromatographic procedures can be used to separate intact molecular species of phosphatidylcholine (PC), but on-line quantification has remained problematic due to insensitivity of UV-detection for saturated species. Here, a new method is presented, separating all major PC molecular species from a variety of biological samples in intact form using a single, short and isocratic run. Species were separated on two RP18 reverse-phase columns in series and all species displayed an exponential relation between retention time and the percentage of acetonitrile or triethylamine in the mobile phase, allowing optimization of the mobile phase on a theoretical base, rather than on time-consuming test-runs. The use of triethylamine as a volatile additive instead of choline chloride allowed the use of light scattering detection. On a molar base, the response of the detector was invariant between species and allowed quantification of as little as 50 pmoles. The method was tested using phosphatidylcholines with widely different molecular species patterns, such a PC from rat liver, porcine pulmonary surfactant, bovine heart, boar sperm cells, and the parasite Schistosoma mansoni. As only volatile components are present in the solvents, individual molecular species can easily be recovered in pure form from the column effluent, enabling their further analysis (e.g., scintillation counting).

Acetonitriles↗
PubMed1997

CTP:phosphoethanolamine cytidylyltransferase.

CTP:phosphoethanolamine cytidylyltransferase (ET) catalyzes the conversion of phosphoethanolamine into CDP-ethanolamine. Immunogold electron microscopy studies have demonstrated that, in hepatocytes, ET is localized predominantly in areas of the cytoplasm that are rich in rough endoplasmic reticulum (RER). Within these areas the enzyme shows a bimodal distribution between the cisternae of the RER and the cytosolic space. Studies on the substrate specificity of ET have shown that it can utilize both CTP and dCTP as substrates, but not other trinucleotides. In addition, the enzyme shows a very pronounced specificity for phosphoethanolamine. Under most conditions ET contributes significantly to the overall regulation of the CDP-ethanolamine pathway. Reversible binding of the enzyme to the endoplasmic reticulum could potentially play a key-role in metabolic channeling of phosphatidylethanolamine synthesis. ET has been purified from rat liver. Convincing evidence has been provided that ET and CTP:phosphocholine cytidylyltransferase (CT), the analogous enzyme in the CDP-choline pathway, are separate activities that reside on different proteins. The gene coding for yeast ET has been cloned. The deduced amino acid sequence contained a region in the N-terminal half with significant similarities to the conserved catalytic domain of both yeast and rat CT. The human cDNA for ET was also cloned recently. The predicted amino acid sequence of human ET shows a high degree of similarity (36% identity) to that of yeast ET, but the human protein is longer than the yeast protein, especially at the C-terminal region. Interestingly, both yeast and human ET have a large repetitive sequence in their N-terminal and C-terminal half.

Animals↗
PubMed1997

Short-term ozone exposure affects the surface activity of pulmonary surfactant.

The effects of short-term ozone exposure on the lung function and surface activity of surfactant subtypes isolated from rat lung lavage were studied. Rats were exposed to 0.8 ppm ozone for 2 or 12 hr. The surface activity of surfactant was affected by ozone exposure, whereas distinct morphological changes in bronchoalveolar lavage or in the surfactant subtypes were not observed. Adsorption experiments indicated that bronchoalveolar lavage from rats exposed for 12 hr to ozone remained at lower equilibrium surface pressures than lavage from control rats. These observations suggest interference of inflammatory proteins with the surface film. Extracted surfactant, containing only lipids and surfactant proteins B and C, had a decreased adsorption rate after ozone exposure. These results suggest that the activity of one or both of the hydrophobic surfactant proteins (SP-B and SP-C) was affected by ozone.

Administration, Inhalation↗
PubMed1997

The pulmonary surfactant system: biochemical and clinical aspects.

This article starts with a brief account of the history of research on pulmonary surfactant. We will then discuss the morphological aspects and composition of the pulmonary surfactant system. We describe the hydrophilic surfactant proteins A and D and the hydrophobic surfactant proteins B and C, with focus on the crucial roles of these proteins in the dynamics, metabolism, and functions of pulmonary surfactant. Next we discuss the major disorders of the surfactant system. The final part of the review will be focused on the potentials and complications of surfactant therapy in the treatment of some of these disorders. It is our belief that increased knowledge of the surfactant system and its functions will lead to a more optimal composition of the exogenous surfactants and, perhaps, widen their applicability to treatment of surfactant disorders other than neonatal respiratory distress syndrome.

Bronchoalveolar Lavage↗
PubMed1997

Toxic oxidant species and their impact on the pulmonary surfactant system.

In this review the effects of oxidant inhalation on the pulmonary surfactant system of laboratory animals are discussed. Oxidant lung injury is a complex phenomenon with many aspects. Inhaled oxidants interact primarily with the epithelial lining fluid (ELF), a thin layer covering the epithelial cells of the lung which contains surfactant and antioxidants. In the upper airways this layer is thick and contains high levels of antioxidants. Therefore oxidant injury in this area is rare and is more common in the lower airways where the ELF is thin and contains fewer antioxidants. In the ELF oxidants can react with antioxidants or biomolecules, resulting in inactivation of the biomolecules or in the formation of even more reactive agents. Oxidation of extracellular surfactant constituents may impair its function and affect breathing. Oxidized ELF constituents may promote inflammation and edema, which will impair the surfactant system further. Animal species differences in respiratory tract anatomy, ventilatory rate, and antioxidant levels influence susceptibility to oxidants. The oxidant exposure dose dictates injury, subsequent repair processes, and tolerance induction.

Animals↗
PubMed1997

The incorporation, modification and turnover of fatty acids in adult Schistosoma mansoni.

Schistosoma mansoni is known to be unable to synthesize fatty acids and sterols de novo, but the parasite is capable of synthesizing phospholipids and triacylglycerols from precursors obtained from the host. The present study focuses on the dynamics of the incorporation of fatty acids in adult parasites. This study showed that fatty acids were rapidly metabolized into complex lipids and that oleate (18:1) was efficiently converted to eicosenoate (20:1) by chain elongation, whereas palmitate was not elongated at an appreciable rate. This chain elongation mainly involved fatty acids that were previously esterified to complex lipids. Furthermore it was shown that in adult parasites triacylglycerols do not serve as fatty-acyl donors in phospholipid synthesis as had been suggested to be the case in schistosomula, because: (1) Immediately after pulse-labelling the specific activity of fatty acids in phospholipids was higher than in triacylglycerols; and (2) the specific activity of eicosenoate, which had been formed by chain elongation of incorporated oleate. was higher in phospholipids than in triacylglycerols. Fatty acids that were esterified to phospholipids had a high turnover, in contrast to fatty acids esterified to triacylglycerols, which persisted for extended periods of time in this lipid class (days rather than hours).

Acylation↗
PubMed1997

Relationships between phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin metabolism in cultured oligodendrocytes.

In most cell types the major pathway of sphingomyelin synthesis is the direct transfer of the phosphocholine head group from phosphatidylcholine to ceramide catalyzed by the enzyme L-acylsphingosine:phosphatidylcholine phosphocholinetransferase (SM synthase; EC 2.7.8.-). Although this pathway has been demonstrated in brain tissue, its quantitative importance has been questioned. An alternative biosynthetic pathway for sphingomyelin synthesis in brain tissue has been proposed, viz., the direct transfer of phosphoethanolamine from phosphatidylethanolamine to ceramide, followed by methylation of the ethanolamine moiety to a choline group. We have evaluated various possible biosynthetic pathways of sphingomyelin synthesis in rat spinal cord oligodendrocytes, the myelin-forming cells of the CNS, by labeling cells in culture with radiolabeled choline, ethanolamine, or serine. Our results indicate that, in oligodendrocytes, most of the phosphocholine for the biosynthesis of sphingomyelin is provided by phosphatidylcholine, which is predominantly derived from de novo synthesis. No evidence was found for the operation of the alternative pathway via ceramide-phosphoethanolamine. Furthermore, our results indicate that a small pool of phosphatidylcholine is provided by methylation of phosphatidylethanolamine, which in turn is formed preferentially by decarboxylation of phosphatidylserine.

Animals↗
PubMed1996

Surface properties, morphology and protein composition of pulmonary surfactant subtypes.

Separation of surfactant subtypes is now commonly used as a parameter in assessing the amount of active compared with inactive material in various models of lung injury. The protein content, morphology and surface activity were determined of the heavy and light subtype isolated by differential centrifugation. Here we report the presence of surfactant proteins B and C in the heavy subtype but not in the light subtype. Adsorption studies revealed that separation of fast adsorbing bronchoalveolar lavage resulted in slowly adsorbing heavy and light subtypes. Surfactant, reconstituted from heavy and light fractions, did not show a high adsorption rate. It is concluded that the isolation procedures might result in a loss of fast adsorbing surfactant structures. Surface area cycling was used as a model in vitro for the extracellular surfactant metabolism. The heavy subtype is converted into the light subtype during conversion. Conversion performed with resuspended heavy subtype revealed the generation of a disparate subtype. Furthermore it was found that the conversion was dependent on preparation and handling of the samples before cycling. Finally, adsorption studies at low surfactant concentrations revealed a delayed adsorption of lipid-extracted surfactants compared with natural surfactants. These observations emphasize the importance of the (surfactant-associated protein A-dependent) structural organization of surfactant lipids in the adsorption process.

Adsorption↗
PubMed1996

Surfactant protein B: effects on lipid domain formation and intermembrane lipid flow.

Pulmonary surfactant is a mixture of (phospho)lipids and surfactant specific proteins, lining the alveolar space. During each respiration cycle phospholipids are transferred between the phospholipid monolayer at the air/water interface and a variety of underlying membranes. Surfactant proteins may play a role in facilitating the insertion and removal of phospholipids by affecting the lipid organization of the bilayer and monolayer. The experiments described in this article were carried out in order to investigate the influence of surfactant protein B (SP-B) on the distribution of phospholipids in membranes and on the mixing of lipids between membranes. To determine the distribution of the non-labeled phospholipids in small unilamellar vesicles (SUV), the relative clustering of pyrene-labeled phospholipids was used, by measuring the ratio of excimer-to-monomer (E/M) pyrene fluorescence. In the absence of SP-B it was found that the clustering of the pyrenePC molecules was dependent on the proportion of saturated acyl chains and not on the proportion of negative charges. Addition of the positively charged SP-B to a mixture of DPPC and PG, led to an increase of approximately 20% in E/M ratio, indicating a clustering of the negatively charged PG molecules. This effect was intensified by addition of calcium ions. If pyrenePC-containing SUV were mixed with excess non-labeled SUV in the presence of SP-B and calcium ions, the E/M ratio decreased, corresponding with a flow of the pyrenePC molecules into the acceptor membranes. It is concluded that presence of domains of phospholipids can be detected with the use of pyrene-labeled phospholipids. Furthermore, SP-B showed a concentrating effect on the distribution of the negatively charged phospholipids, a process that could be important in regulating the phospholipid composition of the monolayer.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1995

Sphingomyelin is synthesized at the plasma membrane of oligodendrocytes and by purified myelin membranes: a study with fluorescent- and radio-labelled ceramide analogues.

In most cell types sphingomyelin is synthesized predominantly in the cis-medial compartments of the Golgi stacks whereas the contribution of the plasma membrane is much lower. The aim of this study was to assess the contribution of both compartments to the synthesis of sphingomyelin in myelinating cells. Therefore, oligodendrocytes from rat spinal cord were incubated in culture with fluorescently- or radiolabelled ceramides, and the effects of a block in the vesicular flow (monensin, brefeldin A, low temperature) on surface synthesis of sphingomyelin were evaluated. The results indicate that approximately 50% of the sphingomyelin synthase is present at the plasma and myelin membranes of oligodendrocytes.

Animals↗