Biomedical subjects

F Possmayer

Publications and source records attributed to F Possmayer.

At least 19 recordsLinked to original sources

PubMed2004

Comparison of inhibitory effects of oxygen radicals and calf serum protein on surfactant activity.

The effects of the reactive oxygen species (ROS) superoxide anion (O2*-) and hydroxyl radical (*OH) on the surface tension lowering properties of bovine lipid extract surfactant (BLES) were compared to the effects of calf serum protein (CSP) in a captive bubble surfactometer (CBS). O2*- was generated from xanthine/xanthine oxidase (X/XO), and *OH was generated by the Fenton reaction. ROS were demonstrated by electron spin resonance (ESR) using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as the spin trap. Lipid peroxidation was measured using the thiobarbituric acid method. *OH had broad inhibitory effects on surface tension parameters, including adsorption, minimum surface tension, percentage film area change and film compressibility. O2*- showed inhibitory effects on adsorption, film area change and film compressibility but had no significant effect on minimum surface tension. Both O2*- and *OH treatment were associated with a large 'squeezeout' plateau around 20-25 mN/m in the surface tension-area relation, indicating poor film organization during the compression phase. At the concentrations used, ROS were associated with lipid peroxidation of BLES, which also demonstrated radical scavenging properties. Calf serum protein produced inhibitory effects on adsorption, minimum surface tension and percentage film area change that were quantitatively similar to those produced by *OH. The effects on film compression were significantly greater and qualitatively different from those seen with either O2*- or *OH. We conclude that the inhibition of BLES surface activity by ROS and inhibitory proteins can be distinguished in the captive bubble surfactometer and, particularly, by changes in the film compressibility modulus.

Adsorption↗

PubMed2001

Pulmonary lipid phosphate phosphohydrolase in plasma membrane signalling platforms.

Lipid phosphate phosphohydrolase (LPP) has recently been proposed to have roles in signal transduction, acting sequentially to phospholipase D (PLD) and in attenuating the effects of phospholipid growth factors on cellular proliferation. In this study, LPP activity is reported to be enriched in lipid-rich signalling platforms isolated from rat lung tissue, isolated rat type II cells and type II cell-mouse lung epithelial cell lines (MLE12 and MLE15). Lung and cell line caveolin-enriched domains (CEDs), prepared on the basis of their detergent-insolubility in Triton X-100, contain caveolin-1 and protein kinase C isoforms. The LPP3 isoform was predominantly localized to rat lung CEDs. These lipid-rich domains, including those from isolated rat type II cells, were enriched both in phosphatidylcholine plus sphingomyelin (PC+SM) and cholesterol. Saponin treatment of MLE15 cells shifted the LPP activity, cholesterol, PC+SM and caveolin-1 from lipid microdomains to detergent-soluble fractions. Elevated LPP activity and LPP1/1a protein are present in caveolae from MLE15 cells prepared using the cationic-colloidal-silica method. In contrast, total plasma membranes had a higher abundance of LPP1/1a protein with low LPP activity. Phorbol ester treatment caused a 3.8-fold increase in LPP specific activity in MLE12 CEDs. Thus the activated form of LPP1/1a may be recruited into caveolae/rafts. Transdifferentiation of type II cells into a type I-like cell demonstrated enrichment in caveolin-1 levels and LPP activity. These results indicate that LPP is localized in caveolae and/or rafts in lung tissue, isolated type II cells and type II cell lines and is consistent with a role for LPP in both caveolae/raft signalling and caveolar dynamics.

Animals↗

PubMed2001

Formation of folds and vesicles by dipalmitoylphosphatidylcholine monolayers spread in excess.

Lipid monolayers exist in several biological systems, including the stratum corneum of the skin, the fluid tear film of the eye, the Eustachian tube of the ear, and airway and alveolar pulmonary surfactants. In this paper, the monolayer-to-bilayer transition was studied using dipalmitoylphosphatidylcholine (DPPC) as the model. Depositing DPPC organic solvent solutions in excess at an air:buffer interface led to the formation of elongated structures which could be imaged on carbon grids by transmission electron microscopy. The structures appeared to be DPPC folds protruding into the sol. The structures were frequently ordered with respect to one another, suggesting that they arose during lateral compression due to excess DPPC and are characteristic of a type of monolayer collapse phase. In some cases, series of short folds in an extended line and series of vesicles in line or parallel to the folds were observed. This suggests the elongated folds are unstable and can resolve by forming vesicles. Fold formation occurred at defined lipid concentrations above which more vesicles were observed. Surfactant protein-A did not influence fold or vesicle formation but bound to the edges of these structures preferentially. It is concluded that DPPC monolayers can form bilayers spontaneously in the absence of surfactant apoproteins, other proteins or agents.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2001

Surface activity in situ, in vivo, and in the captive bubble surfactometer.

For studies of the mechanical effects of lung surfactants, the captive bubble surfactometer (CBS) combines the advantages of the continuous film of Pattle's bubbles with the feasibility of the Langmuir-Wilhelmy balance to produce surface tension-area hysteresis loops. The CBS allows the compression of films to very low and stable surface tensions of 1-2 mN/m. Such low and stable surface tensions are in line with results obtained from pressure-volume studies on excised lungs. In addition, the CBS is useful to test other essential physical properties of the surfactant system, including: (1) rapid film formation (within seconds) through adsorption from the hypophase; (2) low film compressibility with a fall in surface tension to very low (<2 mN/m) values during surface compression; and (3) effective replenishment of the surface film on expansion by the incorporation of surfactant material from material associated with the surface (the surface associated surfactant reservoir). Morphological observations of films fixed in situ or in vitro reveal frequently their multilayered structure, which is consistent with the concept of the surface reservoir. The deviation of the bubbles from a Laplacian shape at very low surface tension and the morphological observations suggest that the surfactant film cannot be considered a simple monolayer.

Animals↗

PubMed2001

Surface activity in vitro: role of surfactant proteins.

Pattle, who provided some of the initial direct evidence for the presence of pulmonary surfactant in the lung, was also the first to show surfactant was susceptible to proteases such as trypsin. Pattle concluded surfactant was a lipoprotein. Our group has investigated the roles of the surfactant proteins (SP-) SP-A, SP-B, and SP-C using a captive bubble tensiometer. These studies show that SP-C>SP-B>SP-A in enhancing surfactant lipid adsorption (film formation) to the equilibrium surface tension of approximately 22-25 mN/m from the 70 mN/m of saline at 37 degrees C. In addition to enhancing adsorption, surfactant proteins can stabilize surfactant films so that lateral compression induced through surface area reduction results in the lowering of surface tension (gamma) from approximately 25 mN/m (equilibrium) to values near 0 mN/m. These low tensions, which are required to stabilize alveoli during expiration, are thought to arise through exclusion of fluid phospholipids from the surface monolayer, resulting in an enrichment in the gel phase component dipalmitoylphosphatidylcholine (DPPC). The results are consistent with DPPC enrichment occurring through two mechanisms, selective DPPC adsorption and preferential squeeze-out of fluid components such as unsaturated phosphatidylcholine (PC) and phosphatidylglycerol (PG) from the monolayer. Evidence for selective DPPC adsorption arises from experiments showing that the surface area reductions required to achieve gamma near 0 mN/m with DPPC/PG samples containing SP-B or SP-A plus SP-B films were less than those predicted for a pure squeeze-out mechanism. Surface activity improves during quasi-static or dynamic compression-expansion cycles, indicating the squeeze-out mechanism also occurs. Although SP-C was not as effective as SP-B in promoting selective DPPC adsorption, this protein is more effective in promoting the reinsertion of lipids forced out of the surface monolayer following overcompression at low gamma values. Addition of SP-A to samples containing SP-B but not SP-C limits the increase in gamma(max) during expansion. It is concluded that the surfactant apoproteins possess distinct overlapping functions. SP-B is effective in selective DPPC insertion during monolayer formation and in PG squeeze-out during monolayer compression. SP-A can promote adsorption during film formation, particularly in the presence of SP-B. SP-C appears to have a superior role to SP-B in formation of the surfactant reservoir and in reinsertion of collapse phase lipids.

Animals↗

PubMed2001

Surfactant levels after reversible tracheal occlusion and prenatal steroids in experimental diaphragmatic hernia.

BACKGROUND/PURPOSE: In normal lungs, fetal tracheal occlusion (TO) induces lung growth but decreases the number of type II cells; this is remedied if TO is released (TR) before delivery. In the current study, the effects of TO with or without TR on pulmonary structure and surfactant were assessed in the ovine model in which lung hypoplasia was induced by creation of a diaphragmatic hernia (CDH). METHODS: A left-sided CDH was created in fetal lambs at 80 days gestation; TO was done at 108 days; and TR at 129 days. All ewes were given 1 dose of glucocorticoids at 135 days. At 136 days, the fetus was delivered. Lung weight to body weight ratio, mean terminal bronchiole density, type II cell density, bronchoalveolar lavage fluid (BAL) phosphatidylcholine (PC), BAL surfactant protein A (SP-A) and B (SP-B), and lung tissue SP-A and SP-B were assessed in CDH, CDH with TO, CDH with TO and TR, and controls. RESULTS: CDH lungs were hypoplastic and structurally immature, but had increased type II cell density. TO with or without TR caused lung growth with normalization of lung parenchymal architecture and type II cell density. Although the BAL SP-A and BAL SP-B were similar in all 4 groups, the BAL PC was low in CDH with or without TO or TR. Also, lung tissue SP-B levels were low in CDH with or without TO or TR. However, lung tissue SP-A levels were normal in CDH, but low in CDH with TO with or without TR. CONCLUSIONS: Despite the finding that lung morphology was improved in CDH with TO with or without TR animals, surfactant content and composition remained abnormal. Although surfactant secreted early by the fetus into alveolar spaces contained normal levels of BAL SP-A and BAL SP-B, the low levels of BAL PC and low lung tissue stores of SP-B indicate that these experimental lambs may experience respiratory insufficiency soon after birth. This implies that prophylactic surfactant at birth might be beneficial for CDH.

Analysis of Variance↗

PubMed2001

Surfactant protein interactions with neutral and acidic phospholipid films.

The captive bubble tensiometer was employed to study interactions of phospholipid (PL) mixtures of dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) at 50 microg/ml with physiological levels of the surfactant protein (SP) A SP-B, and SP-C alone and in combination at 37 degrees C. All surfactant proteins enhanced lipid adsorption to equilibrium surface tension (gamma), with SP-C being most effective. Kinetics were consistent with the presence of two adsorption phases. Under the conditions employed, SP-A did not affect the rate of film formation in the presence of SP-B or SP-C. Little difference in gamma(min) was observed between the acidic POPG and the neutral POPC systems with SP-B or SP-C with and without SP-A. However, gamma(max) was lower with the acidic POPG system during dynamic, but not during quasi-static, cycling. Considerably lower compression ratios were required to generate low gamma(min) values with SP-B than SP-C. DPPC-POPG-SP-B was superior to the neutral POPC-SP-B system. Although SP-A had little effect on film formation with SP-B, surface activity during compression was enhanced with both PL systems. In the presence of SP-C, lower compression ratios were required with the acidic system, and with this mixture, SP-A addition adversely affected surface activity. The results suggest specific interactions between SP-B and phosphatidylglycerol, and between SP-B and SP-A. These observations are consistent with the presence of a surface-associated surfactant reservoir which is involved in generating low gamma during film compression and lipid respreading during film expansion.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2001

Molecular cloning and expression of pulmonary lipid phosphate phosphohydrolases.

Pulmonary lipid phosphate phosphohydrolase (LPP) was shown previously to hydrolyze phosphatidic acid and lysophosphatidic acid in purified rat lung plasma membranes. To better investigate the nature of pulmonary LPP isoforms and their role in the lung, LPPs were cloned by RT-PCR from both adult rat lung and type II cell RNA. The RT-PCR generated LPP1 (849 bp), up to three LPP1 variants, and LPP3 (936 bp) cDNAs. The three LPP1 variants include LPP1a (852 bp) and two novel isoforms, LPP1b (697 bp) and LPP1c (1004 bp). The pulmonary LPP1 and LPP3 isoforms are essentially identical to the previously cloned rat liver and intestinal LPPs, respectively, and the LPP1a isoform has 80% sequence identity to the human homolog. The LPP2 isoform was not detected in lung by RT-PCR. Northern analyses revealed that the mRNAs for LPP1 and LPP3 increase in fetal rat lung in late gestation to day 1 after birth. These mRNAs decrease somewhat during the neonatal period but increase slightly during postnatal development. Expression of LPP1, LPP1a, and LPP3 cDNAs in HEK 293 cells established that they encode functional LPP. In contrast, the novel isoforms LPP1b and LPP1c contain frameshifts that would result in premature termination, producing putative catalytically inactive polypeptides of 30 and 76 amino acids, respectively. Further investigation of the LPP1b isoform revealed that it was present across a variety of tissues, although at lower levels than LPP1/1a. Transient mammalian expression of LPP1b failed to increase phosphatidate phosphohydrolase activity in HEK 293 cells.

Amino Acid Sequence↗

PubMed2001

Dipalmitoylphosphatidylcholine and cholesterol in monolayers spread from adsorbed films of pulmonary surfactant.

Pulmonary surfactant forms a surface film that consists of a monolayer and a monolayer-associated reservoir. The extent to which surfactant components including the main component, dipalmitoylphosphatidylcholine (DPPC), are adsorbed into the monolayer, and how surfactant protein SP-A affects their adsorptions, is not clear. Transport of cholesterol to the surface region from dispersions of bovine lipid extract surfactant [BLES(chol)] with or without SP-A at 37 degrees C was studied by measuring surface radioactivities of [4-(14)C]cholesterol-labeled BLES(chol), and the Wilhelmy plate technique was used to monitor adsorption of monolayers. Results showed that transport of cholesterol was lipid concentration dependent. SP-A accelerated lipid adsorption but suppressed the final level of cholesterol in the surface. Surfactant adsorbed from a dispersion with or without SP-A was transferred via a wet filter paper to a clean surface, where the surface radioactivity and surface tension were recorded simultaneously. It was observed that 1) surface radioactivity was constant over a range of dispersion concentrations; 2) cholesterol and DPPC were transferred simultaneously; and 3) SP-A limited transfer of cholesterol. These results indicate that non-DPPC components of pulmonary surfactant can be adsorbed into the monolayer. Studies in the transfer of [1-(14)C]DPPC-labeled BLES(chol) to an equal or larger clean surface area revealed that SP-A did not increase selective adsorption of DPPC into the monolayer. Evaluation of transferred surfactant with a surface balance indicated that it equilibrated as a monolayer. Furthermore, examination of transferred surfactants from dispersions with and without prespread BLES(chol) monolayers revealed a functional contiguous association between adsorbed monolayers and reservoirs.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2000

Three-dimensional structure of rat surfactant protein A trimers in association with phospholipid monolayers.

Surfactant protein A (SP-A) is a C-type lectin found primarily in the lung and plays a role in innate immunity and the maintenance of surfactant integrity. To determine the three-dimensional (3D) structure of SP-A in association with a lipid ligand, we have used single particle electron crystallography and computational 3D reconstruction in combination with molecular modeling. Recombinant rat SP-A, containing a deletion of the collagen-like domain, was incubated with dipalmitoylphosphatidylcholine:egg phosphatidylcholine (1:1, wt/wt) lipid monolayers in the presence of calcium, negatively stained, and examined by transmission electron microscopy. Images of SP-A-lipid complexes with different angular orientations were used to reconstruct the 3D structure of the protein. These results showed that SP-A subunits readily formed trimers and interacted with lipid monolayers exclusively via the globular domains. A homology-based molecular model of SP-A was generated and fitted into the electron density map of the protein. The plane of the putative lipid-protein interface was relatively flat and perpendicular to the hydrophobic neck region, and the cleft region in the middle of the trimer had no apparent charge clusters. Amino acid residues that are known to affect lipid interactions, Glu(195) and Arg(197), were located at the protein-lipid interface. The molecular model indicated that the hydrophobic neck region of the SP-A did not interact with lipid monolayers but was instead involved in intratrimeric subunit interactions. The glycosylation site of SP-A was located at the side of each subunit, suggesting that the covalently linked carbohydrate moiety probably occupies the spaces between the adjacent globular domains, a location that would not sterically interfere with ligand binding.

Animals↗

PubMed2000

Effects of antenatal endotoxin and glucocorticoids on the lungs of preterm lambs.

OBJECTIVE: We hypothesized that the proinflammatory response to intra-amniotic endotoxin would induce lung maturation in preterm lambs. STUDY DESIGN: Ewes were randomly assigned to receive 20 mg Escherichia coli endotoxin by intra-amniotic injection, maternal betamethasone (0.5 mg/kg), or sodium chloride solution. Preterm lambs were delivered at 125 days' gestation and underwent ventilation to assess lung function. Lung gas volume, surfactant concentrations, and inflammation were subsequently evaluated, with data analyzed by analysis of variance. RESULTS: Fetal endotoxin exposure 6 days before delivery increased compliance by 59%, increased lung gas volume 2.3-fold, increased concentrations of surfactant lipids, increased surfactant A and B protein levels, and increased messenger ribonucleic acid expressions for surfactant proteins (all P <.01, vs control group). Betamethasone exposure resulted in less consistent effects. White blood cell counts were increased in fetal membranes and lungs after endotoxin exposure, but there was no severe inflammation. CONCLUSION: A single fetal exposure to endotoxin resulted in large improvements in postnatal lung function and increases in surfactant concentrations after preterm delivery. These effects were qualitatively larger than those achieved with betamethasone.

Animals↗

PubMed2000

Ovine surfactant protein cDNAs: use in studies on fetal lung growth and maturation after prolonged hypoxemia.

cDNAs for ovine surfactant-associated protein (SP) A, SP-B, and SP-C have been cloned and shown to possess strong similarity to cDNAs for surfactant apoproteins in other species. These reagents were employed to examine the effect of fetal hypoxia on the induction of surfactant apoprotein expression in the fetal lamb. Postnatal lung function is dependent on adequate growth and maturation during fetal development. Insulin-like growth factor (IGF) I and IGF-II, which are present in all fetal tissues studied, possess potent mitogenic and proliferative actions, and their effects can be modulated by IGF-specific binding proteins (IGFBPs). Hypoxia can lead to increases in circulating cortisol and catecholamines that can influence lung maturation. Therefore, the effects of mild hypoxia in chronically catheterized fetal lambs at gestational days 126-130 and 134-136 (term 145 days) on the expression of pulmonary surfactant apoproteins and IGFBPs were examined. Mild hypoxia for 48 h resulted in an increase in plasma cortisol that was more pronounced at later gestation, and in these animals, there was a twofold increase in SP-A mRNA. SP-B mRNA levels also increased twofold, but this was not significant. SP-C mRNA was not altered. No significant changes in apoprotein mRNA were observed with the younger fetuses. However, these younger animals selectively exhibited reduced IGFBP-5 mRNA levels. IGF-I mRNA was also reduced at 126-130 days, although this conclusion is tentative due to low abundance. IGF-II levels were not affected at either gestational age. We conclude that these data suggest that mild prolonged fetal hypoxia produces alterations that could affect fetal cellular differentiation early in gestation and can induce changes consistent with lung maturation closer to term.

Amino Acid Sequence↗

PubMed2000

Alveolar environment influences the metabolic and biophysical properties of exogenous surfactants.

Several factors have been shown to influence the efficacy of exogenous surfactant therapy in the acute respiratory distress syndrome. We investigated the effects of four different alveolar environments (control, saline-lavaged, N-nitroso-N-methylurethane, and hydrochloric acid) on the metabolic and functional properties of two exogenous surfactant preparations: bovine lipid extract surfactant and recombinant surfactant-associated protein (SP) C drug product (rSPC) administered to each of these groups. The main difference between these preparations was the lack of SP-B in the rSPC. Our results demonstrated differences in the large aggregate pool sizes recovered from each of the experimental groups. We also observed differences in SP-A content, surface area cycling characteristics, and biophysical activities of these large aggregate forms after the administration of the two exogenous surfactant preparations. We conclude that the alveolar environment plays a critical role, influencing the overall efficacy of exogenous surfactant therapy. Thus further preclinical studies are warranted to investigate the specific factors within the alveolar environment that lead to the differences observed in this study.

Animals↗

PubMed1999

Dissociation of surfactant protein B from canine surfactant large aggregates during formation of small surfactant aggregates by in vitro surface area cycling.

Pulmonary surfactant isolated by lavage can be separated into large aggregates (LA) and small aggregates (SA). Pulse labeling experiments have shown that the LA subtype is the precursor of the SA subtype. Conversion of LA to SA can be demonstrated in vitro using the technique of surface area cycling. The precise mechanisms of surfactant subtype conversion remain unknown. We have previously reported a decline in surfactant-associated protein B (SP-B) during in vitro subtype conversion of canine surfactant. This led to the hypothesis that SP-B may be degraded by a serine protease 'convertase' during cycling. The current studies used a quantitative slot-blot assay to investigate the fates of SP-A and SP-B during in vitro cycling. These studies confirmed some SP-A is present in SA, but SP-B is confirmed to LA. Conversion leads to an apparent loss of SP-B during cycling. However, SP-B can be recovered from the walls of polypropylene and Teflon tubes by washing with chloroform:methanol. Recovered SP-B migrated on non-reducing tricine gels as a single band with an apparent molecular weight of 17 kDa, corresponding to intact SP-B dimer. Reconstitution studies demonstrated that the recovered SP-B retained its surface active properties as determined on a pulsating bubble surfactometer. We conclude in vitro surface area cycling of canine LA results in the dissociation of SP-B from surfactant lipids resulting in an apparent decline in SP-B levels.

Animals↗

PubMed1999

Correlated atomic force and transmission electron microscopy of nanotubular structures in pulmonary surfactant.

Pulmonary surfactant stabilizes the lung by reducing surface tension at the air-water interface of the alveoli. Surfactant is present in the lung in a number of morphological forms, including tubular myelin (TM). TM is composed of unusual 40 x 40 nm square elongated proteolipid tubes. Atomic force microscopy (AFM) was performed on polymer-embedded Lowicryl and London Resin-White (LR-White) unstained thin sections. AFM was used in imaging regions of the sections where TM was detected by transmission electron microscopy (EM) of corresponding stained sections. Tapping- and contact-mode AFM imaging of the unstained sections containing TM indicated a highly heterogeneous surface topography with height variations ranging from 10 to 100 nm. In tapping-mode AFM, tubular myelin was seen as hemispherical protrusions of 30-70 nm in diameter, with vertical dimensions of 5-8 nm. In contact-mode AFM and with phase imaging using a sharper (>10 nm nominal radius) probe, square open-ended tubes which resembled typical electron micrographs of such regions were observed. The cross-hatch structures observed inside the tubes using EM were not observed using AFM, although certain multilobe structures and topographic heterogeneity were detected inside some tubes. Other regions of multilamellar bodies and some regions where such bilayer lamella appear to fuse with the tubes were found in association with TM using AFM. EM of acetone-delipidated tubes in LR-White revealed rectangular tubular cores containing cross-hatched structures, presumably protein skeletons. AFM surface topography of these regions showed hollow depressions at positions at which the protein was anticipated instead of the protrusions seen in the lipid-containing sections. Gold-labeled antibody to surfactant protein A was found associated somewhat randomly within the regions containing the protein skeletons. The topography of the gold particles was observed as sharp peaks in contact-mode AFM. This study suggests a method for unambiguous detection of three-dimensional nanotubes present in low abundance in a biological macromolecular complex. Only limited detection of proteins and lipids in surfaces of embedded tubular myelin was possible. EM and AFM imaging of such unusual biological structures may suggest unique lipid-protein associations and arrangements in three dimensions.

Acetone↗

PubMed1999

Characterization of GAPCenA, a GTPase activating protein for Rab6, part of which associates with the centrosome.

The Rab6 GTPase regulates intracellular transport at the level of the Golgi apparatus, probably in a retrograde direction. Here, we report the identification and characterization of a novel human Rab6-interacting protein named human GAPCenA (for 'GAP and centrosome-associated'). Primary sequence analysis indicates that GAPCenA displays similarities, within a central 200 amino acids domain, to both the yeast Rab GTPase activating proteins (GAPs) and to the spindle checkpoint proteins Saccharomyces cerevisiae Bub2p and Schizosaccharomyces pombe Cdc16p. We demonstrate that GAPCenA is indeed a GAP, specifically active in vitro on Rab6 and, to a lesser extent, on Rab4 and Rab2 proteins. Immunofluorescence and cell fractionation experiments showed that GAPCenA is mainly cytosolic but that a minor pool is associated with the centrosome. Moreover, GAPCenA was found to form complexes with cytosolic gamma-tubulin and to play a role in microtubule nucleation. Therefore, GAPCenA may be involved in the coordination of microtubule and Golgi dynamics during the cell cycle.

Amino Acid Sequence↗

PubMed1999

Cation-mediated conformational variants of surfactant protein A.

Surfactant protein A (SP-A) is the major protein of pulmonary surfactant. This protein is implicated in regulating surfactant secretion, alveolar processing, recycling, and in non-serum-induced immune response. An increasing body of work indicates the importance of cations, particularly calcium, on SP-A function. However, little information exists on the effects of cations on SP-A quaternary structure. Here, the quaternary organisation of bovine surfactant protein A in the presence of cations has been quantitatively and systematically studied by transmission electron microscopy. The conformation of SP-A is altered by the presence of cations, especially calcium, then sodium, and to a small extent, magnesium. There is a transition concentration, unique for each cation, at which a conformational switch occurs. These transition concentrations are: 5 mM for CaCl2, 100 mM for NaCl and 1 mM for MgCl2. Below these concentrations, SP-A exists primarily in an opened form with a large head diameter of 20 nm; above it, SP-A is mostly in a closed form due to a compaction of the headgroups resulting in a head diameter of 11 nm. There is a corresponding increase in particle length from 17 nm for opened SP-A to 20 nm for closed SP-A. The fact that the transition concentrations are within physiological range suggests that cation-mediated conformational changes of SP-A could be operative in vivo.

Calcium Chloride↗

PubMed1999

Influence of phospholipid composition on the properties of reconstituted surfactants.

The influence of phospholipids on the ultrastructure and metabolism of reconstituted surfactants has not been well defined. The aim of this study was to determine if changes in the phospholipid composition of reconstituted surfactants altered their biophysical properties, ultrastructure, and conversion to light subtype by cycling. We prepared various surfactants containing radiolabeled dipalmitoylphosphatidylcholine ([14C]DPPC). The addition of phosphatidylglycerol (PG) or dipalmitoylphosphatidic acid (PA) to DPPC increased conversion to light subtype. In contrast, the addition of dipalmitoylphosphatidylglycerol (DPPG) to DPPC markedly reduced conversion to light subtype on cycling. DPPC and DPPC+PG produced large liposomes ( approximately 1,000 nm), whereas DPPC+PA or DPPC+DPPG formed multilamellar membranes. Mixtures of DPPC and PA were highly surface active in vitro, whereas the surface activity of DPPC+DPPG was similar to that of DPPC. In conclusion, the ultrastructure, metabolism, and surface active properties of DPPC+PG mixtures were influenced markedly by alterations in the fatty acid composition or polar head group of PG.

1,2-Dipalmitoylphosphatidylcholine↗