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Osteochondrosis (OC) is a disturbance in the process of endochondral ossification, a process in which cartilage is mineralised and transformed into bone. In this process different biochemical events occur, of which the cartilage component has been studied so far almost exclusively. In this study we concentrated on the biochemical characterisation of normal and osteochondrotic subchondral bone, by analysis of enzyme activities, DNA content and phospholipids (PL). In subchondral bone, lysyl oxidase and both total and bone alkaline phosphatase activity were significantly increased in all degrees of OC. DNA content was increased only in the most established grade of OC investigated (grade 4). Furthermore, lactate dehydrogenase activity was significantly lower in grades 2 and 3 OC, but was normal in grade 4 OC, indicating that severe cell damage is not probable. Nonbuffer extractable PL content was substantially higher in osteochondrotic subchondral bone. The phosphatidylethanolamine (PE) to phosphatidylcholine (PC) ratio in both normal and OC subchondral bone was very low (typically 0.21 w/w, PE/PC), which indicates that these PLs were not from cellular origin and could be important in the maturation process of mineralised cartilage into bone and hence in the pathogenesis of OC.

Pigs can be infected with both human and avian influenza A virus (IAV) strains and are therefore considered to be important intermediates in the emergence of new IAV strains due to mixing of viral genes derived from human, avian, or porcine influenza viruses. These reassortant strains may have potential to cause pandemic influenza outbreaks in humans. The innate immune response against IAV plays a significant role in containment of IAV in the airways. We studied the interactions of IAV with porcine surfactant protein D (pSP-D), an important component of this first line defense system. Hemagglutination inhibition analysis shows that the distinct interactions of pSP-D with IAV mediated by the N-linked carbohydrate moiety in the carbohydrate recognition domain of pSP-D depend on the terminal sialic acids (SAs) present on this carbohydrate. Analysis by both lectin staining and by cleavage with linkage-specific sialidases shows that the carbohydrate of pSP-D is exclusively sialylated with alpha(2,6)-linked SAs, in contrast to surfactant protein A, which contains both alpha(2,3)- and alpha(2,6)-linked SAs on its N-linked carbohydrate. Enzymatic modification of the SA-linkages present on pSP-D demonstrates that the type of SA-linkage is important for its hemagglutination-inhibitory activity, and correlates with receptor-binding specificity of the IAV strains. The SAs present on pSP-D appear especially important for interactions with poorly glycosylated IAV strains. It remains to be elucidated to what extent the unique sialylation profile of pSP-D is involved in host range control of IAV in pigs, and whether it facilitates adaptation of avian or human IAV strains that can contribute to the production of reassortant strains in pigs.

Collectins are a family of collagenous calcium-dependent defense lectins in animals. Their polypeptide chains consist of four regions: a cysteine-rich N-terminal domain, a collagen-like region, an alpha-helical coiled-coil neck domain and a C-terminal lectin or carbohydrate-recognition domain. These polypeptide chains form trimers that may assemble into larger oligomers. The best studied family members are the mannan-binding lectin, which is secreted into the blood by the liver, and the surfactant proteins A and D, which are secreted into the pulmonary alveolar and airway lining fluid. The collectins represent an important group of pattern recognition molecules, which bind to oligosaccharide structures and/or lipid moities on the surface of microorganisms. They bind preferentially to monosaccharide units of the mannose type, which present two vicinal hydroxyl groups in an equatorial position. High-affinity interactions between collectins and microorganisms depend, on the one hand, on the high density of the carbohydrate ligands on the microbial surface, and on the other, on the degree of oligomerization of the collectin. Apart from binding to microorganisms, the collectins can interact with receptors on host cells. Binding of collectins to microorganisms may facilitate microbial clearance through aggregation, complement activation, opsonization and activation of phagocytosis, and inhibition of microbial growth. In addition, the collectins can modulate inflammatory and allergic responses, affect apoptotic cell clearance and modulate the adaptive immune system.

The anticancer drug hexadecylphosphocholine (HePC), an alkyl-lysophospholipid analog (ALP), has been shown to induce apoptosis and inhibit the synthesis of phosphatidylcholine (PC) in a number of cell lines. We investigated whether inhibition of PC synthesis plays a major causative role in the induction of apoptosis by HePC. We therefore directly compared the apoptosis caused by HePC in CHO cells to the apoptotic process in CHO-MT58 cells, which contain a genetic defect in PC synthesis. HePC-provoked apoptosis was found to differ substantially from the apoptosis observed in MT58 cells, since it was (i) not accompanied by a large decrease in the amount of PC and diacylglycerol (DAG), (ii) not preceded by induction of the pro-apoptotic protein GADD153/CHOP, and (iii) not dependent on the synthesis of new proteins. Furthermore, lysoPC as well as lysophosphatidylethanolamine (lysoPE) could antagonize the apoptosis induced by HePC, whereas only lysoPC was able to rescue MT58 cells. HePC also induced a rapid externalisation of phosphatidylserine (PS). These observations suggest that inhibition of PC synthesis is not the primary pathway in HePC-induced apoptosis.

Influenza A virus (IAV) infections are a major cause of respiratory disease of humans and animals. Pigs can serve as important intermediate hosts for transmission of avian IAV strains to humans, and for the generation of reassortant strains; this may result in the appearance of new pandemic IAV strains in humans. We have studied the role of the porcine lung collectins surfactant proteins D and A (pSP-D and pSP-A), two important components of the innate immune response against IAV. Hemagglutination inhibition assays revealed that both pSP-D and pSP-A display substantially greater inhibitory activity against IAV strains isolated from human, swine, and horse, than lung collectins from other animal species. The more potent activity of pSP-D results from interactions mediated by the asparagine-linked oligosaccharide located in the carbohydrate recognition domain of pSP-D, which is absent in SP-Ds from other species characterized to date. Presence of this sialylated oligosaccharide moiety enhances the anti-influenza activity of pSP-D, as demonstrated by assays of viral aggregation, inhibition of infectivity, and neutrophil response to IAV. The greater hemagglutination inhibitory activity of pSP-A is due to porcine-specific structural features of the conserved asparagine-linked oligosaccharide in the carbohydrate recognition domain of SP-A. A more efficient lung collectin-mediated immune response against IAV in pigs may play a role in providing conditions by which pigs can act as “mixing vessel” hosts that can lead to the production of reassortant, pandemic strains of IAV.

Inhibition of de novo synthesis of phosphatidylcholine (PC) by some anti-cancer drugs such as hexadecylphosphocholine leads to apoptosis in various cell lines. Likewise, in MT58, a mutant Chinese hamster ovary (CHO) cell line containing a thermo-sensitive mutation in CTP:phosphocholine cytidylyltransferase (CT), an important regulatory enzyme in the CDP-choline pathway, inhibition of PC synthesis causes PC depletion. Cellular perturbations like metabolic insults and unfolded proteins can be registered by the endoplasmic reticulum (ER) and result in ER stress responses, which can lead eventually to apoptosis. In this study we investigated the effect of PC depletion on the ER stress response and ER-related proteins. Shifting MT58 cells to the non-permissive temperature of 40 degrees C resulted in PC depletion via an inhibition of CT within 24 h. Early apoptotic features appeared in several cells around 30 h, and most cells were apoptotic within 48 h. The temperature shift in MT58 led to an increase of pro-apoptotic CCAAT/enhancer-binding protein-homologous protein (CHOP; also known as GADD153) after 16 h, to a maximum at 24 h. Incubation of wild-type CHO-K1 or CT-expressing MT58 cells at 40 degrees C did not induce differences in CHOP protein levels in time. In contrast, expression of the ER chaperone BiP/GRP78, induced by an increase in misfolded/unfolded proteins, and caspase 12, a protease specifically involved in apoptosis that results from stress in the ER, did not differ between MT58 and CHO-K1 cells in time when cultured at 40 degrees C. Furthermore, heat-shock protein 70, a protein that is stimulated by accumulation of abnormal proteins and heat stress, displayed similar expression patterns in MT58 and K1 cells. These results suggest that PC depletion in MT58 induces the ER-stress-related protein CHOP, without raising a general ER stress response.

The determinants for the formation of multilayers upon compression of surfactant monolayers were investigated by compressing films, beyond the squeeze-out plateau, to a surface tension of 22 millinewtons/m. Atomic force microscopy was used to visualize the topography of lipid films containing varying amounts of native surfactant protein B (SP-B). These films were compared with films containing synthetic peptides based on the N terminus of human SP-B: monomeric mSP-B-(1-25) or dimeric dSP-B-(1-25). The formation of typical hexagonal network structures as well as the height of protrusions were shown to depend on the concentration of SP-B. Protrusions of bilayer height were formed from physiologically relevant concentrations of 0.2-0.4 mol % (4.5-8.5 wt %) SP-B upwards. Much higher concentrations of SP-B-(1-25) peptides were needed to obtain network structures, and protrusion heights were not equal to those found for films with native SP-B. A striking observation was that while protrusions formed in films of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dipalmitoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (DPPG) (80/20) had single bilayer thickness, those formed in DPPC/1-palmitoyl-2-oleoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (80/20) had various heights of multilayers, whereas those seen in DPPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/DPPG (60/20/20) were mainly of bilayer height. For the first time direct observations by atomic force microscopy show (i) that a certain minimal concentration of SP-B is required for the formation of layered protrusions upon film compression, (ii) that protrusion height depends on whether the phospholipids contain an unsaturated fatty acyl chain, and (iii) that protrusion height also depends on whether the unsaturated acyl chain is present in phosphatidylcholine or in phosphatidylglycerol.

Infection with gram-positive bacteria is a major cause of pneumonia. Surfactant proteins A (SP-A) and D (SP-D) are thought to play an important role in the innate immunity of the lung. Both proteins can bind to gram-positive bacteria. Until now, it was not known with which surface component(s) of gram-positive bacteria SP-A and SP-D interact. Lipoteichoic acid (LTA) and peptidoglycan (PepG) are components of the cell wall of gram-positive bacteria. By use of a solid phase-based binding assay, LTA of Bacillus subtilis was shown to be bound by SP-D but not by SP-A. Unmodified PepG of Staphylococcus aureus was bound by SP-D. SP-D binding to both LTA and PepG was calcium dependent and carbohydrate inhibitable. These results indicate that SP-D interacts with gram-positive bacteria via binding to the cell wall components LTA and PepG and that the carbohydrate recognition domain is responsible for this binding.

Surfactant protein B (SP-B) enhances lipid insertion into the alveolar air/liquid interface upon inhalation. The aim of this study was (i) to apply a palette of tests for a detailed biochemical and biophysical characterization of SP-B and (ii) to use these tests to compare native SP-B with a fluorescent (Bodipy) SP-B analog. The method of labeling was fast and resulted in a covalent fluorophore–protein bond. The ability of both proteins to spread a surfactant film on top of a buffer surface was determined in a spreading tray using the Wilhelmy plate technique to allow detection of alterations in surface tension and calculation of spreading velocities. In a captive bubble surfactometer surface tensions of spread films were measured. Similar biophysical properties were found for both native and Bodipy-labeled SP-B. It is concluded that the combination of tests used allows detection of small differences in structure and activity between the two proteins.

A pressure-driven captive bubble surfactometer was used to determine the role of surfactant proteins in refinement of the surface film. The advantage of this apparatus is that surface films can be spread at the interface of an air bubble with a different lipid/protein composition than the subphase vesicles. Using different combinations of subphase vesicles and spread surface films a clear correlation between dipalmitoylphosphatidylcholine (DPPC) content and minimum surface tension was observed. Spread phospholipid films containing 50% DPPC over a subphase containing 50% DPPC vesicles did not form stable surface films with a low minimum surface tension. Addition of surfactant protein B (SP-B) to the surface film led to a progressive decrease in minimum surface tension toward 1 mN/m upon cycling, indicating an enrichment in DPPC. Surfactant protein C (SP-C) had no such detectable refining effect on the film. Surfactant protein A (SP-A) had a positive effect on refinement when it was present in the subphase. However, this effect was only observed when SP-A was combined with SP-B and incubated with subphase vesicles before addition to the air bubble containing sample chamber. Comparison of spread films with adsorbed films indicated that refinement induced by SP-B occurs by selective removal of non-DPPC lipids upon cycling. SP-A, combined with SP-B, induces a selective adsorption of DPPC from subphase vesicles into the surface film. This is achieved by formation of large lipid structures which might resemble tubular myelin.

CTP:phosphoethanolamine cytidylyltransferase (ET) is a key regulatory enzyme in the CDP-ethanolamine pathway for phosphatidylethanolamine synthesis. As a first step in the elucidation of the structure-function relationship and the regulation of ET, an ET cDNA was cloned from rat liver. The cloned cDNA encodes a protein of 404 amino acid residues with a calculated molecular mass of 45.2 kDa. The deduced amino acid sequence is very similar to that of human ET (89% identity). Furthermore, it shows less, but significant, similarity to yeast ET as well as to other cytidylyltransferases, including rat CTP:phosphocholine cytidylyltransferase and Bacillus subtilis glycerol-3-phosphate cytidylyltransferase. Like human and yeast ET, rat ET has a large repetitive internal sequence in the N- and C-terminal halves of the protein. Both parts of the repeat contain the HXGH motif, the most conserved region in the N-terminal active domain of other cytidylyltransferases, indicating the existence of two catalytic domains in ET. The hydropathy profile revealed that rat ET is largely hydrophilic and lacks a hydrophobic stretch long enough to span a bilayer membrane. There was no prediction for an amphipathic α-helix. Transfection of COS cells with the cDNA clone resulted in an 11-fold increase in ET activity, corresponding to an increase in the amount of ET protein as detected on a Western blot. Determination of the ET activity during liver development showed a 2.5-fold increase between day 17 of gestation and birth (day 22) and the amount of ET protein changed accordingly. Northern blot analysis showed that this was accompanied by an increase in the amount of ET mRNA. Between day 17 of gestation and birth, the amount of mRNA in fetal rat liver increased approx. 6-fold, suggesting the regulation of ET at both pretranslational and post-translational levels during rat liver development.

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. —Brouwers, J. F. H. M., E. A. A. M. Vernooij, A. G. M. Tielens, and L. M. G. van Golde. Rapid separation and identification of phosphatidylethanolamine molecular species. J. Lipid Res. 1999. 40: 164–169.

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 α-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.

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).

Previous studies with electropermeabilized cells have suggested the occurrence of metabolic compartmentation and Ca2+-dependent channeling of intermediates of phosphatidylcholine (PC) biosynthesis in C6 rat glioma cells. With a more accessible permeabilization technique, we investigated whether this is a more general phenomenon also occurring in other cell types and whether channeling is involved in phosphatidylethanolamine (PE) synthesis as well. C6 rat glioma cells, C3H10T½ fibroblasts and rat hepatocytes were permeabilized with Staphylococcus aureus α-toxin, and the incorporation of the radiolabelled precursors choline, phosphocholine (P-choline), ethanolamine and phosphoethanolamine (P-EA) into PC and PE were measured both at high and low Ca2+ concentrations. In glioma cells, permeabilization at high Ca2+ concentration did not affect [14C]choline or [14C]P-choline incorporation into PC. However, reduction of free Ca2+ in the medium from 1.8 mM to < 1 nM resulted in a dramatic increase in [14C]P-choline incorporation into permeabilized cells, whereas [14C]choline incorporation remained unaffected. Also, in fibroblasts, reduction of extracellular Ca2+ increased [14C]P-choline and [14C]P-EA incorporation into PC and PE respectively. In hepatocytes, a combination of α-toxin and low Ca2+ concentration severely impaired [14C]choline incorporation into PC. Therefore, α-toxin-permeabilized hepatocytes are not a good model in which to study channeling of intermediates in PC biosynthesis. In conclusion, our results indicate that channeling is involved in PC synthesis in glioma cells and fibroblasts. PE synthesis in fibroblasts is also at least partly dependent on channeling.

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.

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).

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.

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.

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.

Regulation of CTP:choline-phosphate cytidylyltransferase activity was studied in regenerating rat liver. The formation of phosphatidylcholine from [14C]choline in hepatocytes isolated from regenerating liver at 22 h after surgery was increased 1.9-fold when compared with hepatocytes from sham-operated animals. This effect was accompanied by a 1.4-fold increase in cytosolic cytidylyltransferase activity as well as by a 1.5-fold increase in the amount of immunoreactive cytidylyltransferase protein, and a 1.7-fold increase in [35S]methionine incorporation into cytidylyltransferase protein. Northern blot analysis of cytidylyltransferase mRNA showed two signals at 1.5 and 5.0 kb. Partial hepatectomy caused a significant 2–3-fold increase in the 1.5-kb and 5.0-kb messengers at 12 h after surgery. During the next 10 h after partial hepatectomy cytidylyltransferase mRNA levels slightly decreased, although they were still elevated in comparison with sham-operated rats 20–22 h after surgery. In contrast to the elevated cytidylyltransferase mRNA levels, the amount of acetyl-CoA carboxylase mRNA did not increase between 12 and 22 h after surgery, which is in line with the unchanged activity of this enzyme. In conclusion, our data demonstrate that in regenerating liver phosphatidylcholine biosynthesis and cytidylyltransferase activity are regulated at a pretranslational level.

We studied the metabolism of sphingolipids by oligodendrocytes derived from rat spinal cord by providing lipid vesicles with either N-lissamine-rhodaminyl-ceramide (LRh-Cer) or N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-ceramide (NBD-Cer) to the cells cultured in a chemically-defined medium. With both probes them the fluorescent product turned out to be sphingomyelin (SM). Most or LRh-SM was not cell-associated but recovered from the culture medium, probably due to back-exchange to the lipid vesicles. The accumulation of LRh-SM, both in the cells and in the medium, was inhibited in the presence of monensin or brefeldin A, whereas the production of NBD-SM was much less affected by these Golgi perturbing drugs. With LRh-Cer as substrate, LRh-labelled fatty acid (FA), galactosyl- and sulfogalactosyl-ceramides (GalCer and SGalCer) were also formed. NBD-Cer, however, was metabolized to glucosylecramide (GlcCer) and GalCer but not to SGalCer or NBD-FA. These data demonstrate that chemical modifications of ceramide alter its metabolism in oligodendrocytes and that the metabolites of LRh-Cer reflect the glycolipid composition of myelin more closely than those of NBD-Cer.

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 arylsufatases in seminal plasma for spermatozoa are discussed.

It has been suggested that oligodendrocytes can actively phagocytose myelin debris during active myelination or after injury and experimental demyelination. Therefore, we have used a fluorescent analogue (N-lissamine rhodaminyl-(12-aminododecanoyl) cerebroside 3-sulphate) to study the metabolic fate of sulphatide, a galactosphingolipid that is highly enriched in myelin membranes. The fluorescent sulphatide was incorporated in small unilamellar vesicles and administered to cultured oligodendrocytes. The association of the lipid probe to the cells in culture was saturable in time and with the concentration of the probe. The processes of association, internalization and subcellular distribution were followed by confocal scanning laser microscopy and appeared to be very rapid. Within 20 min a marked perinuclear staining was seen. After prolonged incubation the fluorescence distributed gradually over the cytoplasm and into cellular branches along structures suggestive of cytoskeletal elements. Lipid analysis demonstrated that ceramide was the major metabolite present in the cells but galactosylceramide, sphingomyelin and free fatty acid were also detected. In the culture medium only free fatty acid and sphingomyelin were found. Monensin did not affect the cellular association and internalization of the fluorescent sulphatide but markedly reduced its conversion to metabolic products. These results indicate that exogenous sulphatide is targeted to the Golgi apparatus prior to its lysosomal degradation.

Okadaic acid, a specific inhibitor of protein phosphatase 1 and 2A, inhibited the synthesis of phosphatidylethanolamine via the CDPethanolamine pathway in isolated hepatocytes. Pulse-chase experiments and measurement of the enzyme activity demonstrated that the inhibition of phosphatidylethanolamine synthesis was not caused by an inhibition of CTP:phosphoethanolamine cytidylyltransferase, the putative regulatory enzyme. However, okadaic acid decreased the cellular diacylglycerol level to 30% of that in control cells. The data suggest that the availability of diacylglycerol limits phosphatidylethanolamine synthesis in okadaic acid-treated hepatocytes.

Effects of single (0.4 ppm for 3, 6, or 12 hr) and repeated (0.4 ppm, 12 hr/day for 3 or 7 days) in vivo ozone exposures on rat and mouse alveolar macrophage functions and cell number were investigated. Single ozone exposure of rats resulted in a small (approximately 15%) decrease in Fc-receptor-mediated phagocytosis and phorbol ester-induced superoxide production by the alveolar macrophages and was followed by recovery above control levels within 12 hr of exposure. Repeated exposures of rats for up to 7 days did not alter alveolar macrophage functions, with the exception of the effects of 3 days of exposure on superoxide production (71 +/- 9% as compared with the controls). In mice, significant changes in alveolar macrophage functions were not observed until 12 hr of exposure (at that timepoint phagocytosis was 74 +/- 2%). Repeated ozone exposures of mice did not cause a further decrease in phagocytosis (at Day 7, 74 +/- 14%). Both after 3 and 7 days of repeated ozone exposure of mice, superoxide production by the alveolar macrophages was inhibited approximately 50%. In rats and mice, repeated ozone exposures led to an increase in the number of alveolar macrophages. In mice, this increase appeared at a later time point (at Day 7 vs Day 3) and was less pronounced (at Day 7, 139 +/- 9% vs 179 +/- 17%) as compared with rats. In summary, our data show that rat and mouse alveolar macrophages have different susceptibilities to both single and repeated in vivo ozone exposures.

Arylsulfatases A (EC 3.1.6.1) and B (EC 3.1.6.12) are lysosomal enzymes that can remove sulfate groups from sulfatides and sulfo-glycosaminoglycans, respectively. The activities of these enzymes in cerebral cortex and in spinal cord of developing rat pups were measured. The tissues were homogenized and the arylsulfatases A and B in the soluble fraction were separated from each other by anion exchange chromatography on DE-52 cellulose. Subsequently, the enzyme activities were assayed with p-nitrocatechol sulfate as substrate at 37 degrees C and pH 5.6. We observed a developmental profile of arylsulfatase A, similar to that previously reported for cerebroside sulfatase (EC 3.1.6.8; (Van der Pal et al. (1990) Biochim. Biophys. Acta 1043, 91-96]. The activity of arylsulfatase A increased gradually during development, whereas arylsulfatase B rose more steeply, peaked around day 15 and declined thereafter. As a consequence the ratio between B and A forms of arylsulfatase dropped from about 4 in 1-week-old pups to 2.2 (cortex) and 0.7 (cord) in 7-week-old rat pups.

The in vivo metabolism of sulfatides was studied in spinal cord and cerebral cortex of developing rat pups. Developmental changes in the rate of sulfolipid synthesis were measured after the intraperitoneal injection of 35 SO 4 2− . We also measured the accumulation of sulfatides, as well as the profiles of cerebroside sulfotransferase, cerebroside sulfatase and arylsulfatase A in both brain regions as a function of postnatal development. The accumulation of sulfatides was higher in spinal cord than in cerebral cortex. In addition, sulfatide metabolism was more active in spinal cord. In both brain regions, the developmental pattern of 35 SO 4 2− incorporation into sulfolipids was closely correlated to the activities of cerebroside sulfotransferase and of arylsulfatase A. The activity of these enzymes was initially low, increased during the period of active myelination and declined thereafter. However, the activity of cerebroside sulfatase, measured with its physiological substrate, [ 35 S]sulfatide, increased during development and did not decline. An explanation for the difference between the developmental profiles of the arylsulfatase A and cerebroside sulfatase reactions (which are supposed to be catalysed by the same enzyme) is proposed.

Spinal cords and cerebra from 7-day-old rat pups were compared as tissue sources for the isolation of oligodendrocytes and for studies on the development of these cells in culture. After 1 day in culture the serum-containing medium was replaced by a chemically-defined medium, which contained a cocktail of hormones that stimulated oligodendrocyte development. The cultures were characterized with various immunocytochemical markers; monoclonal A2B5 for bipotential glial progenitor cells, anti-galactocerebroside (GC) serum for oligodendrocytes, and anti-glial fibrillary acidic protein (GFAP) serum for astrocytes. The number of positive cells was counted and expressed as a percentage of total cells. At 1 day in culture the cell cultures from spinal cord contained 30% GC+ cells, increasing to 90% after 7 days in culture. In cultures derived from cerebra the percentage of GC+ cells was always lower than in cultures from spinal cord. In cerebral cultures GFAP+ cells increased from 15% at 1 day in culture to 30% at 7 days in culture, whereas it remained low in spinal cord cultures. The activity of oligodendroglial marker enzyme 2',3'-cyclic-nucleotide 3'-phosphodiesterase was followed during development in culture. The specific activity increased rapidly in both types of culture but was more than threefold higher in cultures derived from spinal cord. This procedure yields, within one week and without subculture, primary glial cultures from rat spinal cord, that are highly enriched in oligodendrocytes (greater than or equal to 90%; 3.10(5) oligodendrocytes per rat pup).

Pulmonary surfactant protein (SP)-D is an important component of the innate immune system of the lung, which is thought to function by binding to specific carbohydrates on the surface of viruses and unicellular pathogens. SP-D has been shown to have a relatively high affinity for the monosaccharides mannose, glucose, and fucose. However, there is limited information on SP-D binding to complex carbohydrate structures, and binding of SP-D to fucose in the context of an oligosaccharide has not yet been investigated. In this study, we used surface plasmon resonance spectroscopy to examine the potential of SP-D to bind to various synthetic fucosylated oligosaccharides, and identified Fucalpha1-3GalNAc and Fucalpha1-3GlcNAc elements as strong ligands. These types of fucosylated glycoconjugates are presented at the surface of Schistosoma mansoni, a parasitic worm that, during development, transiently resides in the lung. In line with the findings by surface plasmon resonance, we found that SP-D can bind to larval stages of S. mansoni, demonstrating for the first time that SP-D interacts with multicellular lung pathogens.

Surfactant protein C (SP-C) is a small lipopeptide of which the main part consists of a typical valyl-rich transmembrane domain. The protein is expressed as a propeptide (proSP-C) which is processed and sorted via the regulated secretory pathway to the lamellar body, where mature SP-C is stored before secretion into the alveolar space. In this study we investigated the identity of the compartment to which proSP-C is sorted in cells that do not have a regulated secretory pathway, such as CHO cells. By electron microscopy we determined that proSP-C was localized in an uncommon membrane compartment with very regular morphology, which was not present in control cells. This membrane compartment is not influenced by the palmitoylation of proSP-C and is probably derived from the endoplasmic reticulum. However, proSP-C chimeras with artificial transmembrane domains induced a membrane compartment with a different morphology. Therefore we propose that the typical amino acid sequence of the transmembrane domain of proSP-C plays a role in membrane formation and morphology, which may be relevant under physiological conditions.

Pulmonary surfactant forms a monolayer of lipids and proteins at the alveolar air/liquid interface. Although cholesterol is a natural component of surfactant, its function in surface dynamics is unclear. To further elucidate the role of cholesterol in surfactant, we used a captive bubble surfactometer (CBS) to measure surface activity of spread films containing dipalmitoylphosphatidylcholine/1-palmitoyl-2-oleoylphosphatidylcholine/1-palmitoyl-2-oleoylphosphatidylglycerol (DPPC/POPC/POPG, 50/30/20 molar percentages), surfactant protein B (SP-B, 0.75 mol %), and/or surfactant protein C (SP-C, 3 mol %) with up to 20 mol % cholesterol. A cholesterol concentration of 10 mol % was optimal for reaching and maintaining low surface tensions in SP-B-containing films but led to an increase in maximum surface tension in films containing SP-C. No effect of cholesterol on surface activity was found in films containing both SP-B and SP-C. Atomic force microscopy (AFM) was used, for the first time, to visualize the effect of cholesterol on topography of SP-B- and/or SP-C-containing films compressed to a surface tension of 22 mN/m. The protrusions found in the presence of cholesterol were homogeneously dispersed over the film, whereas in the absence of cholesterol the protrusions tended to be more clustered into network structures. A more homogeneous dispersion of surfactant lipid components may facilitate lipid insertion into the surfactant monolayer. Our data provide additional evidence that natural surfactant, containing SP-B and SP-C, is superior to surfactants lacking one of the components, and furthermore, this raises the possibility that the cholesterol found in surfactant of warm-blooded mammals does not have a function in surface activity.

Pulmonary surfactant, a mixture of lipids and proteins, reduces the surface tension at the air-water interface of the lung alveoli by forming a surface active film. This way, it prevents alveoli from collapsing and facilitates the work of breathing. Surfactant protein C (SP-C) plays an important role in this surfactant function. SP-C is expressed as a proprotein (proSP-C), which becomes posttranslationally modified with palmitate and undergoes several rounds of proteolytical cleavage. This results in the formation of mature SP-C, which is stored in the lamellar bodies (LB) and finally secreted into the alveolar space. Recently, new insights into the sorting, processing and palmitoylation of proSP-C have been obtained by mutagenesis studies. Moreover, reports on the association of development of lung disease with SP-C deficiency have led to new insights into the importance of SP-C for proper surfactant homeostasis. In addition, new information has become available on the role of the palmitoyl chains of SP-C in surface activity. This review summarizes these recent developments in the processing and function of SP-C, with particular emphasis on the signals for and role of palmitoylation of SP-C.

Surfactant protein D (SP-D) belongs to a subgroup of mammalian collagenous Ca(2+)-dependent lectins known as the collectins. It is thought to play a significant role in the innate immune response against microorganisms within the lungs and at other mucosal surfaces. This report documents the isolation and characterization of SP-D purified from porcine lung lavage using mannan affinity chromatography and gel filtration. Ultrastructural analysis shows both dodecameric and higher order oligomeric complexes of SP-D. The molecular mass of monomeric porcine SP-D (50 kD) is larger than that of SP-D from humans (43 kD). The difference in mass is due to the presence of an Asparagine-linked glycosylation in the carbohydrate recognition domain of porcine SP-D, which is absent in SP-D of other species investigated so far. Analysis of this carbohydrate moiety indicates that it is a highly heterogeneous, complex type oligosaccharide which is sialylated. The heterogeneity of oligosaccharide sialylation results in the existence of many differently charged porcine SP-D isoforms. The removal of the carbohydrate moiety reduces the inhibitory effect of porcine SP-D on influenza A virus haemagglutination. Therefore, the carbohydrate moiety may influence interactions with pathogens.