Biomedical subjects

L M van Golde

Publications and source records attributed to L M van Golde.

At least 55 records · Page 3Linked to original sources

PubMed1995

A novel procedure for the rapid isolation of surfactant protein A with retention of its alveolar-macrophage-stimulating properties.

Previous studies have shown that surfactant protein A (SP-A) derived from alveolar-proteinosis patients activates rat alveolar macrophages. However, it is not known if normal rat, dog and human SP-A can also stimulate alveolar macrophages. As alveolar-proteinosis SP-A has a slightly different structure from ordinary SP-A, it would be possible that the ascribed alveolar-macrophage-stimulating properties of SP-A are restricted to alveolar-proteinosis SP-A. To clarify this issue, we isolated SP-A from normal rat and dog pulmonary surfactants, using the same isolation technique commonly used for the isolation of alveolar-proteinosis SP-A, i.e. by butanol precipitation. In contrast with human alveolar-proteinosis SP-A, rat and dog SP-A obtained thus could not activate rat alveolar macrophages to produce oxygen radicals or enhance the phagocytosis of fluorescein isothiocyanate-labelled herpes simplex virus. However, rat, dog and normal human SP-A isolated by a novel method, involving extraction from pulmonary surfactant by using n-octyl beta-D-glucopyranoside and subsequent purification by cation-exchange chromatography, were able to elicit an oxidative burst in rat as well as normal human alveolar macrophages. In addition, dog and rat SP-A obtained thus stimulated the phagocytosis of herpes simplex virus by rat alveolar macrophages. These findings indicate that normal human, rat and dog SP-A have the same alveolar-macrophage-stimulating properties as human alveolar proteinosis SP-A. Dog and rat SP-A isolated by this novel method had the same Ca(2+)-dependent self-aggregation and lipid-aggregation properties as SP-A isolated by butanol precipitation. The new and milder isolation procedure yielded SP-A of high purity, as judged by SDS/PAGE and ELISA.

Adult↗

PubMed1995

Neutralization of the positive charges of surfactant protein C. Effects on structure and function.

Pulmonary surfactant protein C (SP-C) is a small, extremely hydrophobic peptide with a highly conservative primary structure. The protein is characterized by two adjacent palmitoylated cysteine residues, two positively charged residues (one arginine residue and one lysine residue) in the N-terminal region, and a long hydrophobic stretch. SP-C enhances the adsorption of phospholipids into an air-water interface. To determine the importance of the positively charged residues, we carried out experiments with natural porcine SP-C and modified porcine SP-C (SP-Cm) in which the positive charges had been blocked by phenylglyoxal. Circular dichroism experiments showed that SP-Cm had an increased content of alpha-helix. Natural SP-C, but not SP-Cm, catalyzed insertion of phospholipids into a monolayer at the airwater interface. This reduced insertion was due to a strong reduction of binding of phospholipid vesicles to the monolayer. The insertion catalyzed by the natural porcine SP-C was decreased by an increased pH of the subphase. In contrast to natural SP-C, SP-Cm induced lipid mixing between phospholipid vesicles. The extent of lipid mixing was a function of the SP-C content. We conclude that the positively charged residues of SP-C are important for the binding of phospholipid vesicles to the monolayer, a process that precedes the insertion of phospholipids into the monolayer.

Animals↗

PubMed1995

Characterization of a dimeric canine form of surfactant protein C (SP-C).

Canine pulmonary surfactant protein C (SP-C) is a small hydrophobic peptide which has one palmitoylated cysteine residue. SP-C enhances the insertion of phospholipids into a monolayer. Two forms of canine SP-C were isolated using Sephadex LH-60 chromatography. It was found that canine SP-C exists in a palmitoylated monomeric form of 3.5 kDa, and a non-acylated dimeric form of 7 kDa. Circular dichroism showed that both forms of SP-C exhibit similar secondary structures at the air/water interface. Both forms of SP-C were able to induce the insertion of phospholipids into a monolayer as measured with the Wilhelmy plate technique. In contrast to the palmitoylated monomeric form of SP-C, the non-acylated dimeric form of SP-C does not require calcium ions to insert phospholipids into a monolayer without the negatively charged phosphatidylglycerol. It is concluded that two forms of canine SP-C exist, but the physiological significance of these different forms remains to be established.

Animals↗

PubMed1995

Pulmonary surfactant subtype metabolism is altered after short-term ozone exposure.

Rats were exposed to 0.8 ppm ozone for 2 or 12 hr. The latter condition resulted in lung damage and inflammation while the former did not. Directly after exposure surfactant was isolated and two morphologically and functionally different surfactant subtypes were obtained by differential centrifugation. Surfactant subtypes isolated from rats exposed to 0.8 ppm ozone for 2 and 12 hr showed an increase in the amount of heavy subtype and a decrease in light subtype. These results suggest that acute ozone exposure of rats can alter surfactant subtype composition. The conversion in vitro of heavy to light subtype was increased in ozone-exposed rats. Degradation of surfactant protein A (SP-A) was observed during in vitro conversion of heavy subtype isolated from ozone-exposed rats. This suggests that oxidation of SP-A may lead to enhanced susceptibility for degradation. The observed effects were more pronounced in rats exposed for 12 hr than those exposed for 2 hr, indicating that proteolytic enzymes from inflammatory cells may aggravate the observed effects. We conclude that extracellular surfactant metabolism is altered by short-term exposures to ozone and that oxidation of SP-A may contribute to the observed alterations.

Animals↗

PubMed1995

Interactions of surfactant protein A with influenza A viruses: binding and neutralization.

The interaction of pulmonary surfactant protein A (SP-A) with influenza A H1N1 and H3N2 viruses was investigated. SP-A is a sialated C type lectin with affinity for mannose residues. Flow cytometry showed that binding of fluorescein isothiocyanate (FITC)-labeled SP-A to H3N2 virus-infected cells was specific and time- and concentration-dependent. Oligosaccharides did not affect the binding of FITC-SP-A to the infected cells. Preincubation of H1N1 and H3N2 with SP-A resulted in a dose-dependent reduction of the infectivity of the viruses to cells. Removal of the carbohydrate moiety of SP-A by N-glycosidase F or cleavage of its sialic acid residues by neuraminidase prevented the interactions of SP-A with the viruses. It is concluded that SP-A binds to influenza A viruses via its sialic acid residues and, thereby, neutralizes the virus.

Animals↗

PubMed1995

Opsonic activities of surfactant proteins A and D in phagocytosis of gram-negative bacteria by alveolar macrophages.

Surfactant proteins A and D (SP-A, SP-D) can interact with lipopolysaccharide (LPS) and stimulate alveolar macrophages. The opsonic activities of SP-A and SP-D for bacteria with different types of LPS and alveolar macrophages were investigated. In flow cytometric studies with fluorescein-labeled rough (J5) and smooth (O111) Escherichia coli and rat alveolar macrophages, SP-A enhanced binding of J5 but not O111 bacteria to macrophages. Most importantly, SP-A enhanced ingestion of J5 bacteria by alveolar macrophages and subsequent bacterial killing. Immunoelectron microscopy demonstrated that J5 bacteria, the interface between the bacterium and the outer membrane of the alveolar macrophage, and ingested bacteria were heavily labeled with SP-A. In contrast, SP-D did not mediate phagocytosis. SP-A acted as an opsonin in the phagocytosis of rough LPS-containing bacteria by alveolar macrophages, emphasizing the possible role for SP-A in the alveolar defense system.

Animals↗

PubMed1995

Potential role of surfactant proteins A and D in innate lung defense against pathogens.

The physiological role of pulmonary surfactant is probably not limited to conferring mechanical stability to the alveoli. Increasing evidence suggests that surfactant components, in particular the hydrophilic surfactant proteins SP-A and SP-D, play potentially important roles in host defense mechanisms. Both SP-A and SP-D are collagenous C-type lectins (collectins) that are structurally and, perhaps, functionally related to collectins in the circulation. As will be discussed in this review, evidence is accumulating that the alveolar collectins SP-A and SP-D could be important components of a first-line defense system against infiltrating pathogenic micro-organisms and viruses.

Animals↗

PubMed1995

Glycolipid migration from the apical to the equatorial subdomains of the sperm head plasma membrane precedes the acrosome reaction. Evidence for a primary capacitation event in boar spermatozoa.

In order to extend the static information of immunolabelling sulphogalactolipids in fixed boar spermatozoa, a fluorescent sulphogalactolipid analogue, galactose(3-sulphate)-beta 1-1'[(N-lissamine rhodaminyl)-12-aminodode-canoyl]-sphingosine, was incorporated into plasma membranes of living spermatozoa and its lateral distribution over the sperm head was studied. The fluorescent lipid was enriched in the apical ridge subdomain of freshly ejaculated sperm cells. After sperm binding to the zona pellucida the lipid redistributed to the equatorial segment of the sperm surface. A similar shift occurred during capacitation in vitro with 2 mM CaCl2 or with 4% (w/v) bovine serum albumin. The desulphated derivative galactose-beta 1-1'[(N-lissamine rhodaminyl)-12-aminododecanoyl]-sphingosine was also incorporated into the plasma membrane of freshly ejaculated sperm cells and clearly stained the apical ridge subdomain and the (pre)-equatorial subdomains of the sperm heads. The desulphogalactolipid analogue showed a slightly faster migration to the equatorial segment of the sperm plasma membrane than did its sulphated counterpart. The measured fluorescence intensity distributions correlated linearly with the spatial probe distribution, which was checked by fluorescence lifetime imaging microscopy. The observed migration of the incorporated glycolipids precedes the acrosome reaction and is one of the underlying molecular events likely to be important in the process of sperm capacitation. The results of this study suggest that lipid phase segregation is an important driving force for the organization of the sperm head plasma membrane into subdomains.

Acrosome↗

PubMed1994

Identification of the non-specific lipid-transfer protein (sterol carrier protein 2) in peroxisomes of lung type II cells.

In view of the proposed role of the non-specific lipid-transfer protein (nsL-TP; sterol carrier protein 2) in the metabolism of pulmonary surfactant lipids (Batenburg et al. (1994) Biochem. J. 298, 223-229), its subcellular localization was studied in the surfactant producing alveolar type II cells. It was shown by immuno-electron microscopy that nsL-TP colocalizes with the peroxisomal marker catalase. The peroxisomal localization of nsL-TP was confirmed by gradient fractionation of type II cell homogenates. As a peroxisomal marker acyl-CoA:dihydroxyacetone-phosphate acyltransferase was assayed. Given this subcellular localization, it is very unlikely that nsL-TP plays a role in the transfer of surfactant lipids from the endoplasmic reticulum to the lamellar bodies. These results strengthen the opinion that peroxisomes are involved in surfactant synthesis.

Acyltransferases↗

PubMed1994

Binding of surfactant protein A to the lipid A moiety of bacterial lipopolysaccharides.

Surfactant protein A (SP-A) enhances the phagocytosis of opsonized and non-opsonized bacteria by alveolar macrophages, but it is not known with which component of the bacterial surface it associates. We investigated the interaction of SP-A with lipopolysaccharides (LPS), which are important biologically active constituents of the outer membranes of Gram-negative bacteria. Flow cytometry was used to study the binding of fluorescein isothiocyanate-labelled SP-A either to LPS of various chain lengths coupled to magnetic beads or to Gram-negative bacteria. The binding of SP-A to LPS-coated beads was saturable, both time- and concentration-dependent, and required both Ca2+ and Na+. SP-A bound to the lipid A moiety of LPS and to LPS from either the Re-mutant of Salmonella minnesota or the J5-mutant of Escherichia coli. In contrast, it did not bind to O111 LPS of E. coli, suggesting that SP-A binds only to rough LPS. The binding of SP-A to LPS was not affected by mannan and heparin or by deglycosylation of the SP-A, indicating that the carbohydrate-binding domain and the carbohydrate moiety of SP-A are not involved in its interaction with LPS. We also observed saturable and concentration-dependent binding of SP-A to the live J5 mutant of whole E. coli, but not to its O111 mutant. In addition, Re LPS aggregated in the presence of SP-A, Ca2+ and Na+. We conclude that SP-A associates with LPS via the lipid A moiety of rough LPS and may be involved in the anti-bacterial defences of the lung.

Bronchoalveolar Lavage Fluid↗

PubMed1994

Ultrastructural localization of CTP:phosphoethanolamine cytidylyltransferase in rat liver.

CTP:phosphoethanolamine cytidylyltransferase (EC 2.7.7.14) (ethanolamine-phosphate cytidylyltransferase, ET) was recently purified to homogeneity from a post-microsomal supernatant of rat liver and subsequently used to raise a polyclonal antibody against the enzyme in rabbits (Vermeulen, P. S., Tijburg, L. B. M., Geelen, M. J. H., and van Golde, L. M. G. (1993) J. Biol. Chem 268, 7458-7464). In the present study, we used the affinity-purified antibody against ET for ultrastructural immunogold labeling studies on rat liver cryosections. Single-label experiments clearly demonstrated that ET label was not randomly distributed in hepatocytes. The ET label was concentrated in areas that contained cisternae of the rough endoplasmic reticulum, whereas other cellular organelles (nuclei, mitochondria, and peroxisomes) were only marginally labeled for ET. Double-label experiments for ET and established markers for either soluble or integral endoplasmic reticulum proteins suggested a bimodal distribution of ET between the RER cisternae and the cytosolic space. Complementary single-label studies for ET and the soluble marker protein showed that the fraction of ET label that was present on RER cisternae was significantly greater than that of the soluble marker, supporting the idea of an uneven distribution. These immunoelectron microscopy studies strongly suggest that the cellular organization of ET differs considerably from that reported recently for the corresponding enzyme in the CDP-choline pathway, CTP:phosphocholine cytidylyltransferase (Wang, Y., Sweiter, T. D., Weinhold, P. A., and Kent, C. (1993) J. Biol. Chem. 268, 5899-5904).

Animals↗

PubMed1994

Substrate specificity of CTP: phosphoethanolamine cytidylyltransferase purified from rat liver.

CTP: phosphoethanolamine cytidylyltransferase was recently purified to homogeneity from rat liver (Vermeulen, P.S. Tijburg, L.B.M., Geelen, M.J.H. and van Golde, L.M.G. (1993) J. Biol. Chem. 268, 7458-7464). The present study focuses on the specificity of this enzyme for phosphorylated bases with a varying degree of N-methylation. The apparent Km for phosphoethanolamine was 0.072 mM. As the number of N-methylated substituents on phosphoethanolamine increased, the apparent Km increased: 0.11 mM for phosphomonomethylethanolamine and 6.8 mM for phosphodimethylethanolamine. Introduction of a third N-methyl group did not further increase the Km value. The effect of N-methyl groups on the reaction velocity was far more pronounced. A decreased Vmax for the reaction was found as the number of N-methyl substituents increased: 1.52 and 0.24 mumol/min per mg protein for phosphoethanolamine and phosphomonomethylethanolamine, and 44 and 0.69 nmol/min per mg protein for phosphodimethylethanolamine and phosphocholine, respectively. Phosphomonomethylethanolamine, phosphodimethylethanolamine and phosphocholine were weak competitive inhibitors of the cytidylyltransferase catalyzed reaction when phosphoethanolamine was used as a substrate, with Ki values of 7.0, 6.8 and 52.9 mM, respectively. The results show that this cytidylyltransferase is highly specific for phosphoethanolamine. Comparison of these data with previously reported information on the substrate specificity of CTP: phosphocholine cytidylyltransferase endorses the view that the two cytidylyltransferases are functionally different.

Animals↗

PubMed1994

Visualization and quantification of glycolipid polarity dynamics in the plasma membrane of the mammalian spermatozoon.

Seminolipid (sulphogalactosylalkylacylglycerol), the glycolipid that is specific for mammalian germ cells, is located exclusively in the outer leaflet of the sperm plasma membrane. In this study the lateral distribution of seminolipid on sperm heads has been investigated by indirect immunofluorescence labelling and detection with digital imaging fluorescence microscopy. In freshly ejaculated sperm cells this glycolipid was present primarily at the apical ridge subdomain of the plasma membrane of the sperm head. After binding the sperm cells to zona-coated coverslips seminolipid migrated, in 40 minutes, from the apical ridge to the equatorial subdomain of the plasma membrane. A similar redistribution of seminolipid was observed during capacitation of sperm cells in vitro induced by Ca2+ or bovine serum albumin. Comparable migration of seminolipid was also found after prolonged storage of ejaculated sperm cells, albeit at a much slower rate. Addition of arylsulphatase A, an enzyme present in seminal plasma that desulphates seminolipid, significantly enhanced the migration of seminolipid during storage of sperm cells. Its breakdown product desulphoseminolipid (galactosylalkylacylglycerol) appeared highly specifically at the equatorial segment. The measured fluorescence intensity over the sperm head surface correlated linearly with the spatial probe distribution as was checked by fluorescence lifetime imaging microscopy. This paper demonstrates and quantifies for the first time the polarity of seminolipid on the surface of the sperm cell and the dynamic alterations that occur in this polarity during post-ejaculatory events.

Animals↗

PubMed1993

Effect of acylation on structure and function of surfactant protein C at the air-liquid interface.

Pulmonary surfactant protein C (SP-C) is a small hydrophobic peptide that is palmitoylated on 2 adjacent cysteine residues. SP-C enhances the adsorption of phospholipids into a monolayer. The function of the acylation is not clear yet. The experiments described in this article were carried out in order to investigate the function of SP-C acylation in (protein-catalyzed) lipid monolayer formation, and in bilayer interactions. Palmitoylated and nonpalmitoylated human recombinant SP-C were used. In addition, a nonacylated SP-C with a Cys-->Ser mutations was included in these studies. In Wilhelmy plate experiments using negatively charged, protein-containing phospholipid monolayers and negatively charged vesicles, CaCl2 was required to obtain a maximal insertion rate of lipids into the monolayer. If the negatively charged phospholipids in the monolayer were replaced by neutral phospholipids, CaCl2 was only required to show a maximal SP-C-catalyzed insertion rate (if the molecule is palmitoylated, but not if nonpalmitoylated proteins were added). In pressure area measurements, the palmitoylated protein showed a different change in pressure as a function of the surface area, as compared with the nonpalmitoylated proteins. Circular dichroism experiments showed that all three proteins had a high content of alpha-helix. All three proteins showed a preferential orientation at the air-water interface, but the palmitoylated protein has an orientation which is more parallel to the monolayer than that of the nonpalmitoylated proteins. It is concluded that acylation of SP-C alters structural and physical properties of this protein.

Acylation↗

PubMed1993

Immunological characterization, lipid dependence, and subcellular localization of CTP:phosphoethanolamine cytidylyltransferase purified from rat liver. Comparison with CTP:phosphocholine cytidylyltransferase.

CTP:phosphoethanolamine cytidylyltransferase (ET) (ethanolamine-phosphate cytidylyltransferase, EC 2.7.7.14), which is generally considered as the rate-regulatory enzyme of phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway, was purified to homogeneity from a rat liver postmicrosomal supernatant. A polyclonal antibody was raised against the enzyme in rabbits and subsequently purified by affinity chromatography. The affinity-purified antibody recognized one single immunoreactive 49.6-kDa protein band on SDS-polyacrylamide gel. The enzyme showed an isoelectric point at a pH of 6.5 and was sensitive to various sulfhydryl reagents. Cross-reactivity experiments of ET and CTP:phosphocholine cytidylyltransferase (CT) (choline-phosphate cytidylyltransferase, EC 2.7.7.15) with their corresponding antibodies showed that these enzymes were immunologically distinct. In contrast with the well known lipid dependence of CT, the activities of both purified and cytosolic ETs were not affected by the presence of various phospholipid preparations. Differential centrifugation studies as well as release experiments with digitonin-permeabilized hepatocytes demonstrated that ET, unlike CT, is not associated with cellular organelles. However, amino acid analysis of ET revealed a high content of hydrophobic amino acids, suggesting a possible association of this enzyme with some kind of cellular structure in the hepatocyte.

Amino Acids↗

PubMed1992

Binding of surfactant protein A (SP-A) to herpes simplex virus type 1-infected cells is mediated by the carbohydrate moiety of SP-A.

Pulmonary surfactant protein A (SP-A) has been shown to act as an opsonin in the phagocytosis of viruses by alveolar macrophages. To determine whether SP-A binds to viral proteins and which part of the SP-A molecule is involved in this interaction, binding studies were undertaken. SP-A was labeled with fluorescein isothiocyanate, and its binding to herpes simplex virus type 1-infected HEp-2 cells, as a model for virus-infected cells in general, was studied using flow cytometry. The binding of SP-A to virus-infected cells was saturable, reversible, and both time- and concentration-dependent, reaching a maximal level after 30 min at an SP-A concentration of 10 micrograms/ml. An approximately 4-fold increase in binding of SP-A to infected cells over control cells was observed. Yeast mannan, a mannose homopolysaccharide, did not influence the binding. However, heparin inhibited binding of SP-A in a concentration-dependent manner. In addition, heparin could also dissociate cell-bound SP-A, indicating that polyanionic oligosaccharides are involved in the binding of SP-A to virus-infected cells. Deglycosylated SP-A, obtained by digestion with N-glycosidase F, did not bind to infected cells. Heparin or deglycosylation of SP-A had no effect on the stimulation of alveolar macrophages by SP-A. It is concluded that the carbohydrate moiety of SP-A is involved in the recognition of viruses by SP-A and may play a role in the antiviral defenses of the lung.

Animals↗

PubMed1992

Characterization of three arylsulfatases in semen: seminolipid sulfohydrolase activity is present in seminal plasma.

Sperm cells and seminal plasma of various mammals contain high levels of arylsulfatase. In the present study, we investigated the composition of soluble AS in these compartments of boar semen by analysing sperm cells and seminal plasma using anion-exchange chromatography. Seminal plasma contained both arylsulfatase B (2.4 units per ml), an enzyme which desulfates sulfoglycosaminoglycans and probably sulfoglycoproteins, and arylsulfatase A (10.2 units per ml), an enzyme which desulfates sulfogalactolipids. Sperm cells contained only arylsulfatase A, which differed biochemically from the extracellular arylsulfatase A of seminal plasma (2.6 units per ml). Both types of arylsulfatase A desulfate seminolipid, the natural sulfolipid substrate in sperm, as well as two brain sulfatides. The possible physiological consequences of the presence of extracellular arylsulfatases in seminal plasma for spermatozoa are discussed.

Animals↗

PubMed1992

Lipid mixing is mediated by the hydrophobic surfactant protein SP-B but not by SP-C.

Pulmonary surfactant contains two families of hydrophobic proteins, SP-B and SP-C. Both proteins are thought to promote the formation of the phospholipid monolayer at the air/fluid interface of the lung. The excimer/monomer ratio of pyrene-labeled PC fluorescence intensities was used to investigate the capacity of the hydrophobic surfactant proteins, SP-B and SP-C, to induce lipid mixing between protein-containing small unilamellar vesicles and pyrene-PC-labeled small unilamellar vesicles. At 37 degrees C SP-B induced lipid mixing between protein-containing vesicles and pyrene-PC-labeled vesicles. In the presence of negatively charged phospholipids (PG or PI) the SP-B-induced lipid mixing was enhanced, and dependent on the presence of (divalent) cations. The extent of lipid mixing was maximal at a protein concentration of 0.2 mol%. SP-C was not capable of inducing lipid mixing at 37 degrees C not even at protein concentrations of 1 mol%. The SP-B-induced lipid mixing may occur during the Ca(2+)-dependent transformation of lamellar bodies into tubular myelin and the subsequent formation of the phospholipid monolayer.

1,2-Dipalmitoylphosphatidylcholine↗