We describe a chemical method for the detection and quantification of surfactant protein C (SP-C)... more We describe a chemical method for the detection and quantification of surfactant protein C (SP-C), a palmitoylated 3.5-kDa protein, based on the release of free-sulfhydryl groups upon depalmitoylation. SP-C was purified from calf lung surfactant extract by gel filtration on LH-20 and LH-60 columns in chloroform:methanol:0.1 N HCl (19:19:2). SP-C was allowed to react with [14C]iodoacetamide in the presence or absence of triethylamine, a deacylating agent. Unreacted iodoacetamide was removed by extraction with 1% KCl. Polyacrylamide gel electrophoresis followed by fluorography demonstrated that the intensity of the 14C-labeled SP-C bands correlated with the chloroform-soluble radioactivity. Under conditions designed to allow maximal incorporation of radioactivity, the concentration of SP-C was calculated and found to be approximately twice that indicated by the Lowry protein assay. The present assay is sensitive to nanogram levels (approximately 100 ng) of SP-C and is not affected by surfactant protein B or components of the organic solvent system. However, it is slightly inhibited by high concentrations of phospholipids (approximately 30% at a phospholipid:SP-C ratio of 700:1). The low interference of other surfactant components makes this method useful for the determination of SP-C in crude organic extracts and partially purified and purified preparations. It can also be used to detect other hydrophobic palmitoylated proteins, such as myelin proteolipid protein.
phosphohydr olase (P A P ase) is a key enzym e inv olv ed in g lycer olip id synthesis w here u P... more phosphohydr olase (P A P ase) is a key enzym e inv olv ed in g lycer olip id synthesis w here u P hosphatid ate it co nvert s ph ospha tidic aci d to diacylg lycero l. P re viou s studies per f orm ed in lung ha ve dem onstrate d the existence of 2 diþ er ent f orm s of P A P ases, namely P A P -1 and P A P -2. T he f or mer pulm onary M g + 2 - dep endent enzyme is N -eth ylm ale imid e (N E M )-sensitiv e, hea t labile , and is invol ved in phosph olipi d bio synthe sis. H ow eve r, the f unctio n of th e la tter lung isozyme is unk now n. P A P -2 activi ty w as selective ly assayed using N E M in th e ab sence of M g + 2 . Studie s em plo ying this assay and ad ult r at lung micro som al pr epa ra tions dem onstra ted tha t P A P -2 activ ity w as inhib ited by am ph iphi lic am ines, sphing oid bases, pro ducts of the P A P -2 re action (monoacy lg lyce rol [M A G ] and diacylg lycero l [D A G ]), and substrate analog s such as lysoph osphat idic acid (lyso-P A ), cera mid e-1-phosphate , and to a lesser extent, sphing osine-1-pho sphate . P uri üed lung plasm a m em br anes, pre pa red using disco ntinuous sucro se and P ercoll g ra dien ts, show ed th at P A P -2 acti vity w as enriche d 6.9 6 1.6-f old ove r th e w hole hom og enate and w as betw een th e enrichm ent f or plasma m em bra ne m ar ker s, 5' -nucle otid ase (14.7 6 0.3) and N a + , K + -A T P ase (4.0 6 0.2). B oth pho sphatid ic aci d and lysopho sphati dic aci d w ere g ood substra tes f or P A P -2 acti vity in th is puri üed pla sm a membra ne f ra ctio n. I n contr ast, sphing osine-1-pho sphate w as a re lativ ely poor substrate . P A P -2 activ ity w as slig htly enriche d in isolate d typ e II cells and low in isola ted r at lung übr oblasts. T his study show s lung cont ains P A P -2 act ivity in plasma membra nes and type I I cells w her e it co uld play a r ole in sig nal tr ansductio n. K eyw or ds phosph atida te pho sphoh ydr ola se-2, ph osphol ipid sig naling , pho sphol ipase D , pla sm a membran es, sphing olip ids, typ e I I cells Phospha tidate phosph ohydro lase (PAPase) was ürst described in plants by K ates in 1955 [ 1 ]. In the early 1960s, the characterization of this enzym e in a
We describe a chemical method for the detection and quantification of surfactant protein C (SP-C)... more We describe a chemical method for the detection and quantification of surfactant protein C (SP-C), a palmitoylated 3.5-kDa protein, based on the release of free-sulfhydryl groups upon depalmitoylation. SP-C was purified from calf lung surfactant extract by gel filtration on LH-20 and LH-60 columns in chloroform:methanol:0.1 N HCl (19:19:2). SP-C was allowed to react with [14C]iodoacetamide in the presence or absence of triethylamine, a deacylating agent. Unreacted iodoacetamide was removed by extraction with 1% KCl. Polyacrylamide gel electrophoresis followed by fluorography demonstrated that the intensity of the 14C-labeled SP-C bands correlated with the chloroform-soluble radioactivity. Under conditions designed to allow maximal incorporation of radioactivity, the concentration of SP-C was calculated and found to be approximately twice that indicated by the Lowry protein assay. The present assay is sensitive to nanogram levels (approximately 100 ng) of SP-C and is not affected by surfactant protein B or components of the organic solvent system. However, it is slightly inhibited by high concentrations of phospholipids (approximately 30% at a phospholipid:SP-C ratio of 700:1). The low interference of other surfactant components makes this method useful for the determination of SP-C in crude organic extracts and partially purified and purified preparations. It can also be used to detect other hydrophobic palmitoylated proteins, such as myelin proteolipid protein.
Pulmonary surfactant (PS) dysfunction because of the leakage of serum proteins into the alveolar ... more Pulmonary surfactant (PS) dysfunction because of the leakage of serum proteins into the alveolar space could be an operative pathogenesis in acute respiratory distress syndrome. Albumin-inhibited PS is a commonly used in vitro model for studying surfactant abnormality in acute respiratory distress syndrome. However, the mechanism by which PS is inhibited by albumin remains controversial. This study investigated the film organization of albumin-inhibited bovine lipid extract surfactant (BLES) with and without surfactant protein A (SP-A), using atomic force microscopy. The BLES and albumin (1:4 w/w) were cospread at an air-water interface from aqueous media. Cospreading minimized the adsorption barrier for phospholipid vesicles imposed by preadsorbed albumin molecules, i.e., inhibition because of competitive adsorption. Atomic force microscopy revealed distinct variations in film organization, persisting up to 40 mN/m, compared with pure BLES monolayers. Fluorescence confocal microscopy confirmed that albumin remained within the liquid-expanded phase of the monolayer at surface pressures higher than the equilibrium surface pressure of albumin. The remaining albumin mixed with the BLES monolayer so as to increase film compressibility. Such an inhibitory effect could not be relieved by repeated compression-expansion cycles or by adding surfactant protein A. These experimental data indicate a new mechanism of surfactant inhibition by serum proteins, complementing the traditional competitive adsorption mechanism.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1969
I. The relative incorporation of a number of radioactive fatty acids into the different glyceroli... more I. The relative incorporation of a number of radioactive fatty acids into the different glycerolipids of rat liver microsomes has been investigated. 2. Studies on the distribution of the radioactivity incorporated into phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid showed that in all three lipids, the majority of the saturated fatty acids were at the r-position while the polyunsaturated fatty acids were largely confined to the a-position. 3. The phosphatidic acid fraction of rat liver was isolated in a pure form, and its fatty acid distribution agreed with the results of the incorporation studies. 4. The incorporation of radioactive fatty acids into different molecular species of phosphatidic acid, separated as dimethyl phosphatidates was investigated.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1974
1. The properties of a stable glycerophosphate acyltransferase preparation obtained by lyophiliza... more 1. The properties of a stable glycerophosphate acyltransferase preparation obtained by lyophilization of rabbit lung microsomal fractions are described. Phosphatidic acid was the principal radioactive product formed in the presence of either sn-[ * "C] glycerol-3-P or 3H-or ' 4C fatty acids plus rat-glycerol-3-P. 2. The subcellular localization of glycerophosphate acyltransferase was examined by comparing the distribution of this enzyme with that of "marker" enzymes. A similarity was observed between the percentage distribution and the relative specific activities of glycerophosphate acyltransferase and NADPH-cytochrome c reductase in the different fractions, which indicates that endopl~mi~ reticulum is the principal organelle responsible for pulmonary glycerolipid synthesis. The mitochondr~ fraction contained glycerophosphate acyltransferase activity which could not be explained through contamination with endoplasmic reticulum. However, under the assay conditions used, the activity associated with the mitochondria could only account for a small proportion of the total lung glycerophosphate acyltransferase activity. 3. The kinetics of the reaction indicated that considerable free fatty acid was either associated with the preparation or produced during the incubations. Treatment of the fresh microsomal preparations with serum albumin removed some of the endogenous fatty acid. 4. The relative incorporation of radioactive fatty acids into phosphatidic acid was in the following order: linoleate > palmitate, oleate and stearate > myristate > laurate > linolenate and arachidonate. A small acylation of endogenous lyso-derivatives was also indicated by the experimental results. 5. Phospholipase AZ degr%dation of the radioactive phosphatidic acids revealed that laurate, myristate, linolenate and arachidonate were preferenti~ly incorporated to the 2-position. Palm&ate, oleate, stearate and linoleate were randomly incorporated.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1988
Two low-molecular-weight hydrophobic proteins with nominal molecular weights M, = 15 000 and M, =... more Two low-molecular-weight hydrophobic proteins with nominal molecular weights M, = 15 000 and M, = 3 500 have been isolated from the lipid extracts of bovine pulmonary smfactant by several methods, including (a) dialysis plus silicic acid chromatography, (b) elution from Waters SEP-PAK silica cartridges with a variety of solvent mixtures, and (c) ultrafiltration. As detailed in the text, these proteins have been designated surfactant-associated protein-BC (SP-BC) (15 kDa: nonreduced), and SP-C (3.5 kDa). The biophysical activities of reconstituted surfactant containing these proteins and the phospholipids present in lung surfactant have been compared with the biophysical activities of bovine lipid extract surfactant on a pulsating bubble surfactometer using a phospholipid concentration of 10 mg/ml. At this concentration, unmodified lipid extract surfactant reduces the surface tension of the pulsating bubble to near 0 within 10 pulsations at 20 cycles per min. Similar biophysical properties were observed with modified lipid extract surfactant in which the relative concentration of hydrophobic protein had been reduced from 1 to 0.4% (w/w) of the phospholipids by addition of dipalmitoylphosphatidylcholine (DPPC) or DPPC plus phosphatidylglycerol. Reconstituted surfactants, which contained partially delipidated SP-BC (15 kDa: nonreduced) obtained by method (a) at a relative concentration of 0.196, were also capable of reducing the surface tension to near 0 mN/m. Preparations of SP-BC (15 kDa: nonreduced) obtained by method (b), which had been subjected to very low pH levels during isolation and were extensively delipidated, exhibited full biophysical activity only at higher protein concentrations and with prolonged pulsation. Extensively delipidated samples of SP-BC obtained by method (c) exhibited impaired biophysical activities, even when prepared with neutral organic solvents. Reconstituted surfactant samples containing SP-C (3.5 kDa) obtained by any of the methods listed above were only able to reduce the surface tension at minimum bubble radius to approx. 20 mN/m. The biophysical activity of SP-C (3.5 kDa) was not significantly affected by low pH or extensive delipidation. Reconstituted samples containing mixtures of SP-BC (15 kDa: nonreduced) and SP-C (3.5 f&a) were more effective than samples containing either protein alone. Furthermore, with samples containing both hydrophobic proteins the final surface tensions at maximum bubble radius were attained within a few bubble
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1990
The surfactant-associated proteins, SP-A, SP-B and SP-C have been isolated from bovine pulmonary ... more The surfactant-associated proteins, SP-A, SP-B and SP-C have been isolated from bovine pulmonary surfactant. The biophysical roles of SP-B and SP-C in reconstituted surfactants, with various phospholipid mixtures subjected to different thermal treatments, have been examined using a pulsating bubble surfactometer. The phospholipid mixtures were: (A) dipalmitoylphosphatidylcholine (DPPC)/egg phosphatidylcholine (PC)/egg phosphatidylglycerol (PG) (6:2:2, w/w); (B) DPPC/PG (9:1); and (C) DPPC/PG (7:3). Thermal treatments involved mixing SP-B or SP-C, at room temperature, with lipids in chloroform/methanol (9:1, v/v) and removing the solvent under N2 by (1) evaporation at room temperature; (2) evaporation at 45 degrees C; or (3) incubation at 45 degrees C overnight prior to evaporation at 45 degrees C. In all cases, 45 degrees C solvent evaporation was the most effective treatment. DPPC/egg PG (7:3) was the most favourable lipid composition. With either a static or a pulsating bubble, SP-C promoted a rapid decrease in surface tension with little change thereafter. This implies that SP-C is effective in enhancing phospholipid adsorption but does not play an important role in the removal of non-DPPC lipid from the monolayer. While SP-B was not as effective in facilitating phospholipid absorption, samples containing this protein could achieve near zero surface tension upon pulsation. A very low surface tension could also be attained during the initial pulsation of DPPC/PG plus SP-B mixtures which had been allowed to adsorb until equilibrium. This observation indicates that SP-B promotes the removal of PG from the monolayer. SP-A alone had only a slight effect on the surface activity of the DPPC/PG (7:3) mixture, and did not accelerate adsorption of samples containing SP-C. However, SP-A facilitated phospholipid adsorption and may also enhance the removal of PG from monolayers in the presence of SP-B.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1993
We have investigated the ontogeny and hormonal regulation of both synthesis rates and cellular ac... more We have investigated the ontogeny and hormonal regulation of both synthesis rates and cellular accumulation of the mRNA for surfactant-associated protein B (SP-B) in rabbit fetal lung. The developmental pattern for SP-B mRNA synthesis increased as a function of gestational age and paralleled that for SP-B mRNA levels except on days 22-26 of gestation where relatively higher levels of gene transcription were observed. Time-course studies with explants from 26- and 30-day fetal lung maintained in culture revealed a gradual increase in mRNA levels and a much smaller increase in gene transcription relative to adult values. Within 48 h of exposure of 26-day explants to dexamethasone at 10(-8) M there was a rapid increase in SP-B mRNA levels to 7-fold adult levels. A similar overall although somewhat slower and attenuated pattern was observed with 30-day explants. Dexamethasone at 10(-8) M had no effect on SP-B gene transcription with explants of either gestational age. We conclude that the major effect of dexamethasone treatment in vitro on SP-B mRNA levels appears to be post-transcriptional and there are small but distinct differences in the effects of glucocorticoids on SP-B mRNA levels with explant cultures from early and late stages of fetal lung maturation.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1994
The effect of exogenous cholesterol on the stability of surface films at 37°C from various surfac... more The effect of exogenous cholesterol on the stability of surface films at 37°C from various surfactants was stiddled with the pulsating bubble surfactometer. Addition of cholesterol (5%,, w/w) to bovine lipid extract surfactant QbLES) or mixtures of dipalmitoylphosphatidylcholine/l-palmitoyl-Z-oleoyl-pbosphatidylglyceroi/SP-B (7 : 3 : 1%) dispersed in 1.5 r&M CaCIJO.9% NaCl resulted in unstable surface films. Although 10% cholesterol only partially impaired the surface activny of bEESi, it virtuaily abolished that of the reconstituted surfactant. The inhibitory effects of cholesterol were significantly repressed by SPA (ifBG, w/w of lipid) and 3 mM CaCI, or 5 mM CaCl, without SPA. Adsorption of cholesterol from various surfaclants into the air/water interface was examined by measuring the surface radioactivity of [14C]cholasteroi. Cholesterol alone dispersed in I.5 mIt4 CaCl,/O.9% NaCl could not adsorb to the interface, but it adsorbed readily when mixed with bLES. ChoiesteroI adsorption was markedly suppressed by W-A in 3 mM CaC1,/0.9% NaCl or 5 m CaC1,/0.9% NaCI without-A. Eiectron microsco=jy revealed striking ultrastructural differences between bLES/S% ch sterol/lO% %P-A in 3 m CaCl,jO.9% NaC1 and bLES/S% cholesterol in 3 or 5 mM CaC1,/0.9% NaCl. The former exhibited large multilayer and small unilameflar vesicles, while the latter displayed condensed patches of aggregates. Adsorption studies showed aggregated patches adsorbed more rapidly than vesicles but attained lower equilibrium surface pressures. These results indicate SPA and calcium hit he adsorption of surfactant cholesterol to the air-water interface.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1984
Rat lung microsomes washed with increasing conceutrations of NaCl show a displacement of protein ... more Rat lung microsomes washed with increasing conceutrations of NaCl show a displacement of protein from microsomes to the wash supe~at~t. Among the proteins removed from the microsoma~ surface 'was the Mg 2 f-dependent phosphatidate phosphohydrolase, while the Mg ' +-independent activity remained associated with the microsomes. The Mg2+-dependent ac tivity could be quantitatively assayed in the wash supernatant. ~icrosomes washed with increasing concen~ations of MaCl showed a progressive impairment in the synthesis of labelled neutral lipid and phosphatidylcholine from f r4C]glycerol 3-phospbate witb a concomitant increase in the labelling of phosphatidic acid. The impairment was sigmoidal and correlated bigbly with the decrease in Mg2+-dependent phosphatidate phosphohydrolase activity. Wheu ~g~+~de~~dent ~hosp~atidate phosphohydrolase from wash supernatant was incubated with microsomes previously washed witb higb salt concentrations, the labelling of neutral lipid and phosphatidylcholine was returned to control levels. Labelling of neutral lipids and phosphatidylcholine could be restored upon addition of a cytosolic Mg"pendent phosphaiidate phosphohydrol~e isolated by gel filtration. Mg'+-independent ~hos~batidate phosphohydrolase isolated from cytosol was incapable of restoring the labelling of neutral lipids and phosphatidylcholine. These findings confirm that the iMg2+-dependent p hosphatidate phosphohydrolase of sat lenpng is involved in pulmona~ glycerolipid biosyn~esis. The role of the Mg*+-inde~ndent phos~hatidate uhosphohydrolase activity remains unknown.
Under optimal conditions, rat lung microsomal cholinephosphotransferase (EC 2.7.8.2) activity is ... more Under optimal conditions, rat lung microsomal cholinephosphotransferase (EC 2.7.8.2) activity is markedly stimulated by exogenously added 1,2-sn-diacylglycerols containing an unsaturated fatty acid at the 2-position. Diacylglycerols containing long-chain saturated fatty acids at the 1- and 2-positions did not stimulate the incorporation of CDP[14C]choline above the incorporation observed with the endogenous diacylglycerols present in the microsomal preparation. 1-Oleoyl,2-palmitoyl-sn-glycerol was also ineffective in stimulating phosphatidylcholine synthesis. Diacylglycerols containing linoleate or linolenate at the 2-position were not as effective as 1-palmitoyl,2-oleoyl-sn-glycerol. Identical stimulations were observed with the latter diacylglycerol and 'natural' diacylglycerols prepared from egg-yolk phosphatidylcholine or pig liver phosphatidylcholine. Marked selectivities were observed with diacylglycerols containing two unsaturated fatty acids. Only minor amounts of 1,...
Pulmonary surfactant facilitates breathing by reducing the surface tension at the air/liquid inte... more Pulmonary surfactant facilitates breathing by reducing the surface tension at the air/liquid interface. We examined the effect of experimental lung transplantation on the phospholipid pool sizes of alveolar surfactant large and small aggregates, the composition of the large aggregates, the surface tension-reducing ability of lipid extract surfactant, and the leakage of serum proteins into the lung. A double-lung block from the donor animal was stored for 2 or 12 h after perfusion with either Euro-Collins solution or University of Wisconsin solution. The right donor lung was lavaged immediately after the storage period to determine the effects of storage on pulmonary surfactant. The left donor lung was transplanted. The recipient animal, containing its own native right lung and the transplanted left lung, was reperfused for 6 h. After the reperfusion period, the transplanted left lung and the native right lung were lavaged. After an ischemic time of 12 h, impaired gas exchange was observed in the transplanted lung as well as the native lung during the 6 h of reperfusion. This impaired gas exchange was associated with several significant changes in pulmonary surfactant: (1) total serum protein in the lung lavage was increased, (2) the small to large surfactant aggregate ratio was increased, (3) sphingomyelin content was increased and phosphatidylglycerol content was decreased in large aggregates, and (4) the surfactant-associated protein A content was decreased in large aggregates. No significant differences were observed between the results obtained with Euro-Collins and University of Wisconsin solutions.(ABSTRACT TRUNCATED AT 250 WORDS)
The lysophosphatidic acid receptor 1 (LPA1), a G-protein coupled receptor, regulates cell prolife... more The lysophosphatidic acid receptor 1 (LPA1), a G-protein coupled receptor, regulates cell proliferation, migration, and cytokine release. Here, we investigate the molecular signature of LPA1 trafficking to the cell surface. The overexpressed LPA1 with a C-terminal V5 tag (LPA1-V5) is majorly expressed on the cell surface, while two deletion mutants (C320 and ∆84-87) failed to be trafficked to the cell surface. Further, site-directed mutagenesis analysis of the LPA1 revealed that Ile325, Tyr85, and Leu87 within these two fragments regulate LPA1 maturation and trafficking to the cell surface. Over-expression of Sar1, a component of coat protein complex II (COPII), enhances glycosylation of LPA1 wild type, but not these mutants. The mutants of LPA1 are majorly localized in the endoplasmic reticulum (ER) and exhibit a higher binding affinity to heat shock protein 70 (Hsp70), when compared to the LPA1 wild type. Further, we found that all these mutants failed to increase phosphorylation ...
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1989
The potential involvement of the glycerophosphorylcholine (GPC) pathway for the synthesis of phos... more The potential involvement of the glycerophosphorylcholine (GPC) pathway for the synthesis of phosphatidylcholine (PC) has been examined in rat liver and lung and in a human line, the A549 cell which possesses characteristics representative of mature alveolar type |l epithelial cells. Although mitochondrial and microsomai fractions from the above sources readily incorporated radioactive g|ycerophosphate into lipids, the only incorporation observed with radioactive GPC w&s a small variable labelling with the mitochondriai and microsomal fractions from rat lung. Even with these fractions, no radioactivity from GPC was incorporated into PC or |ysoPC. Attempts to increase the incorporation of GPC into lipids by manipulating the incubation conditions were unsuccessful. It was concluded that the occurrence of the GPC pathway in liver and lung is unlikely.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1989
The properties of natural bovine surfactant and its lipid extract have been examined with a pulsa... more The properties of natural bovine surfactant and its lipid extract have been examined with a pulsating bubble surfactometer which assesses the ability of surfactant lipids to adsorb to the air/liquid interface and reduce the surface tension to near 0 dynes/cm during dynamic compression. Studies conducted at 1 mg/ml phospholipid revealed that the surface activity (i.e., the ability to produce low surface tensions) of lipid extracts could be enhanced by incubating the sample at 37 degrees C for 120 min or by addition of CaCl2. In contrast, incubation at 37 degrees C only slightly improved the biophysical activity of natural surfactant and the addition of CaCl2 had a more modest effect than with lipid extracts. With 20 mM CaCl2, the surfactant activity of lipid extract surfactant was similar to that of natural surfactant. Incubation with EDTA reduced the biophysical activity of natural surfactant. Experiments in which increasing amounts of lipid extract were replaced by natural surfactant revealed that small amounts of natural surfactant enhanced the surfactant activity of lipid extract. The biophysical activity of lipid extract surfactant was also increased by the addition of soluble surfactant-associated protein-A (SP-A) (28-36 kDa) purified from natural bovine surfactant. These results indicate that SP-A (28-36 kDa) improves the surfactant activity of lipid extracts by enhancing the rate of adsorption and/or spreading of phospholipid at the air/liquid interface resulting in the formation of a stable lipid monolayer at lower bulk concentrations of either phospholipid or calcium.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1987
The effects of levonorgestrel treatment (4 pg/day per kg body weight o.'5 for 18 days) on rate-li... more The effects of levonorgestrel treatment (4 pg/day per kg body weight o.'5 for 18 days) on rate-limiting enzymes of hepatic triacylglycerol synthesis, namely glycerol-3-phosphate acyltransferase and phosphatidic acid phosphatase were investigated in microsomal, mitochondrial and cytosolic fractions of rat liver. Levonorgestrel treatment resulted in a significant reduction (26%) of hepatic microsomal glycerol-3-phosphate acyltransferase specific activity. Hepatic mitochondrial glycerol-3-phosphate acyltransferase specific activity was unchanged. Levonorgestrel treatment also significantly reduced (by 20%) the specific activity of hepatic microsomal magnesium-independent phosphatidic acid phosphatase. However, magnesium-dependent phosphatic acid phosphatase specific activities in microsomal and cytosolic fractions were unaffected. Cytosolic magnesium-independent phosphatidic acid phosphatase activity was also unchanged. These studies are consistent with the view that levonorgestrel lowers serum triacylglycerol levels, at least in part, by inhibition of the glycerol-3-phosphate acyltransferase (EC 2.3.1.15) step in hepatic triacylglycerol synthesis.
Gold (Au) nanoribbons were synthesized in aqueous phase under ambient conditions by using dimethy... more Gold (Au) nanoribbons were synthesized in aqueous phase under ambient conditions by using dimethylene bis(tetradecyldimethyl-ammonium bromide) (14-2-14) as a capping agent as well as a soft template. A two steps seed-growth (S-G) method was used. The first step of S-G method mainly gave nanorods of high aspect ratio along with nanoparticles of other shapes, but the next step produced mainly fine Au nanoribbons of several micrometers long, 50-150 nm wide, and ≈5 nm thick. They were characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, energy dispersive X-ray, and X-ray diffraction analysis. Isotropic liquid crystalline phase of 14-2-14 provided a soft template effect and derived nanorods of high aspect ratios toward nanoribbon formation at room temperature.
We describe a chemical method for the detection and quantification of surfactant protein C (SP-C)... more We describe a chemical method for the detection and quantification of surfactant protein C (SP-C), a palmitoylated 3.5-kDa protein, based on the release of free-sulfhydryl groups upon depalmitoylation. SP-C was purified from calf lung surfactant extract by gel filtration on LH-20 and LH-60 columns in chloroform:methanol:0.1 N HCl (19:19:2). SP-C was allowed to react with [14C]iodoacetamide in the presence or absence of triethylamine, a deacylating agent. Unreacted iodoacetamide was removed by extraction with 1% KCl. Polyacrylamide gel electrophoresis followed by fluorography demonstrated that the intensity of the 14C-labeled SP-C bands correlated with the chloroform-soluble radioactivity. Under conditions designed to allow maximal incorporation of radioactivity, the concentration of SP-C was calculated and found to be approximately twice that indicated by the Lowry protein assay. The present assay is sensitive to nanogram levels (approximately 100 ng) of SP-C and is not affected by surfactant protein B or components of the organic solvent system. However, it is slightly inhibited by high concentrations of phospholipids (approximately 30% at a phospholipid:SP-C ratio of 700:1). The low interference of other surfactant components makes this method useful for the determination of SP-C in crude organic extracts and partially purified and purified preparations. It can also be used to detect other hydrophobic palmitoylated proteins, such as myelin proteolipid protein.
phosphohydr olase (P A P ase) is a key enzym e inv olv ed in g lycer olip id synthesis w here u P... more phosphohydr olase (P A P ase) is a key enzym e inv olv ed in g lycer olip id synthesis w here u P hosphatid ate it co nvert s ph ospha tidic aci d to diacylg lycero l. P re viou s studies per f orm ed in lung ha ve dem onstrate d the existence of 2 diþ er ent f orm s of P A P ases, namely P A P -1 and P A P -2. T he f or mer pulm onary M g + 2 - dep endent enzyme is N -eth ylm ale imid e (N E M )-sensitiv e, hea t labile , and is invol ved in phosph olipi d bio synthe sis. H ow eve r, the f unctio n of th e la tter lung isozyme is unk now n. P A P -2 activi ty w as selective ly assayed using N E M in th e ab sence of M g + 2 . Studie s em plo ying this assay and ad ult r at lung micro som al pr epa ra tions dem onstra ted tha t P A P -2 activ ity w as inhib ited by am ph iphi lic am ines, sphing oid bases, pro ducts of the P A P -2 re action (monoacy lg lyce rol [M A G ] and diacylg lycero l [D A G ]), and substrate analog s such as lysoph osphat idic acid (lyso-P A ), cera mid e-1-phosphate , and to a lesser extent, sphing osine-1-pho sphate . P uri üed lung plasm a m em br anes, pre pa red using disco ntinuous sucro se and P ercoll g ra dien ts, show ed th at P A P -2 acti vity w as enriche d 6.9 6 1.6-f old ove r th e w hole hom og enate and w as betw een th e enrichm ent f or plasma m em bra ne m ar ker s, 5' -nucle otid ase (14.7 6 0.3) and N a + , K + -A T P ase (4.0 6 0.2). B oth pho sphatid ic aci d and lysopho sphati dic aci d w ere g ood substra tes f or P A P -2 acti vity in th is puri üed pla sm a membra ne f ra ctio n. I n contr ast, sphing osine-1-pho sphate w as a re lativ ely poor substrate . P A P -2 activ ity w as slig htly enriche d in isolate d typ e II cells and low in isola ted r at lung übr oblasts. T his study show s lung cont ains P A P -2 act ivity in plasma membra nes and type I I cells w her e it co uld play a r ole in sig nal tr ansductio n. K eyw or ds phosph atida te pho sphoh ydr ola se-2, ph osphol ipid sig naling , pho sphol ipase D , pla sm a membran es, sphing olip ids, typ e I I cells Phospha tidate phosph ohydro lase (PAPase) was ürst described in plants by K ates in 1955 [ 1 ]. In the early 1960s, the characterization of this enzym e in a
We describe a chemical method for the detection and quantification of surfactant protein C (SP-C)... more We describe a chemical method for the detection and quantification of surfactant protein C (SP-C), a palmitoylated 3.5-kDa protein, based on the release of free-sulfhydryl groups upon depalmitoylation. SP-C was purified from calf lung surfactant extract by gel filtration on LH-20 and LH-60 columns in chloroform:methanol:0.1 N HCl (19:19:2). SP-C was allowed to react with [14C]iodoacetamide in the presence or absence of triethylamine, a deacylating agent. Unreacted iodoacetamide was removed by extraction with 1% KCl. Polyacrylamide gel electrophoresis followed by fluorography demonstrated that the intensity of the 14C-labeled SP-C bands correlated with the chloroform-soluble radioactivity. Under conditions designed to allow maximal incorporation of radioactivity, the concentration of SP-C was calculated and found to be approximately twice that indicated by the Lowry protein assay. The present assay is sensitive to nanogram levels (approximately 100 ng) of SP-C and is not affected by surfactant protein B or components of the organic solvent system. However, it is slightly inhibited by high concentrations of phospholipids (approximately 30% at a phospholipid:SP-C ratio of 700:1). The low interference of other surfactant components makes this method useful for the determination of SP-C in crude organic extracts and partially purified and purified preparations. It can also be used to detect other hydrophobic palmitoylated proteins, such as myelin proteolipid protein.
Pulmonary surfactant (PS) dysfunction because of the leakage of serum proteins into the alveolar ... more Pulmonary surfactant (PS) dysfunction because of the leakage of serum proteins into the alveolar space could be an operative pathogenesis in acute respiratory distress syndrome. Albumin-inhibited PS is a commonly used in vitro model for studying surfactant abnormality in acute respiratory distress syndrome. However, the mechanism by which PS is inhibited by albumin remains controversial. This study investigated the film organization of albumin-inhibited bovine lipid extract surfactant (BLES) with and without surfactant protein A (SP-A), using atomic force microscopy. The BLES and albumin (1:4 w/w) were cospread at an air-water interface from aqueous media. Cospreading minimized the adsorption barrier for phospholipid vesicles imposed by preadsorbed albumin molecules, i.e., inhibition because of competitive adsorption. Atomic force microscopy revealed distinct variations in film organization, persisting up to 40 mN/m, compared with pure BLES monolayers. Fluorescence confocal microscopy confirmed that albumin remained within the liquid-expanded phase of the monolayer at surface pressures higher than the equilibrium surface pressure of albumin. The remaining albumin mixed with the BLES monolayer so as to increase film compressibility. Such an inhibitory effect could not be relieved by repeated compression-expansion cycles or by adding surfactant protein A. These experimental data indicate a new mechanism of surfactant inhibition by serum proteins, complementing the traditional competitive adsorption mechanism.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1969
I. The relative incorporation of a number of radioactive fatty acids into the different glyceroli... more I. The relative incorporation of a number of radioactive fatty acids into the different glycerolipids of rat liver microsomes has been investigated. 2. Studies on the distribution of the radioactivity incorporated into phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid showed that in all three lipids, the majority of the saturated fatty acids were at the r-position while the polyunsaturated fatty acids were largely confined to the a-position. 3. The phosphatidic acid fraction of rat liver was isolated in a pure form, and its fatty acid distribution agreed with the results of the incorporation studies. 4. The incorporation of radioactive fatty acids into different molecular species of phosphatidic acid, separated as dimethyl phosphatidates was investigated.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1974
1. The properties of a stable glycerophosphate acyltransferase preparation obtained by lyophiliza... more 1. The properties of a stable glycerophosphate acyltransferase preparation obtained by lyophilization of rabbit lung microsomal fractions are described. Phosphatidic acid was the principal radioactive product formed in the presence of either sn-[ * "C] glycerol-3-P or 3H-or ' 4C fatty acids plus rat-glycerol-3-P. 2. The subcellular localization of glycerophosphate acyltransferase was examined by comparing the distribution of this enzyme with that of "marker" enzymes. A similarity was observed between the percentage distribution and the relative specific activities of glycerophosphate acyltransferase and NADPH-cytochrome c reductase in the different fractions, which indicates that endopl~mi~ reticulum is the principal organelle responsible for pulmonary glycerolipid synthesis. The mitochondr~ fraction contained glycerophosphate acyltransferase activity which could not be explained through contamination with endoplasmic reticulum. However, under the assay conditions used, the activity associated with the mitochondria could only account for a small proportion of the total lung glycerophosphate acyltransferase activity. 3. The kinetics of the reaction indicated that considerable free fatty acid was either associated with the preparation or produced during the incubations. Treatment of the fresh microsomal preparations with serum albumin removed some of the endogenous fatty acid. 4. The relative incorporation of radioactive fatty acids into phosphatidic acid was in the following order: linoleate > palmitate, oleate and stearate > myristate > laurate > linolenate and arachidonate. A small acylation of endogenous lyso-derivatives was also indicated by the experimental results. 5. Phospholipase AZ degr%dation of the radioactive phosphatidic acids revealed that laurate, myristate, linolenate and arachidonate were preferenti~ly incorporated to the 2-position. Palm&ate, oleate, stearate and linoleate were randomly incorporated.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1988
Two low-molecular-weight hydrophobic proteins with nominal molecular weights M, = 15 000 and M, =... more Two low-molecular-weight hydrophobic proteins with nominal molecular weights M, = 15 000 and M, = 3 500 have been isolated from the lipid extracts of bovine pulmonary smfactant by several methods, including (a) dialysis plus silicic acid chromatography, (b) elution from Waters SEP-PAK silica cartridges with a variety of solvent mixtures, and (c) ultrafiltration. As detailed in the text, these proteins have been designated surfactant-associated protein-BC (SP-BC) (15 kDa: nonreduced), and SP-C (3.5 kDa). The biophysical activities of reconstituted surfactant containing these proteins and the phospholipids present in lung surfactant have been compared with the biophysical activities of bovine lipid extract surfactant on a pulsating bubble surfactometer using a phospholipid concentration of 10 mg/ml. At this concentration, unmodified lipid extract surfactant reduces the surface tension of the pulsating bubble to near 0 within 10 pulsations at 20 cycles per min. Similar biophysical properties were observed with modified lipid extract surfactant in which the relative concentration of hydrophobic protein had been reduced from 1 to 0.4% (w/w) of the phospholipids by addition of dipalmitoylphosphatidylcholine (DPPC) or DPPC plus phosphatidylglycerol. Reconstituted surfactants, which contained partially delipidated SP-BC (15 kDa: nonreduced) obtained by method (a) at a relative concentration of 0.196, were also capable of reducing the surface tension to near 0 mN/m. Preparations of SP-BC (15 kDa: nonreduced) obtained by method (b), which had been subjected to very low pH levels during isolation and were extensively delipidated, exhibited full biophysical activity only at higher protein concentrations and with prolonged pulsation. Extensively delipidated samples of SP-BC obtained by method (c) exhibited impaired biophysical activities, even when prepared with neutral organic solvents. Reconstituted surfactant samples containing SP-C (3.5 kDa) obtained by any of the methods listed above were only able to reduce the surface tension at minimum bubble radius to approx. 20 mN/m. The biophysical activity of SP-C (3.5 kDa) was not significantly affected by low pH or extensive delipidation. Reconstituted samples containing mixtures of SP-BC (15 kDa: nonreduced) and SP-C (3.5 f&a) were more effective than samples containing either protein alone. Furthermore, with samples containing both hydrophobic proteins the final surface tensions at maximum bubble radius were attained within a few bubble
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1990
The surfactant-associated proteins, SP-A, SP-B and SP-C have been isolated from bovine pulmonary ... more The surfactant-associated proteins, SP-A, SP-B and SP-C have been isolated from bovine pulmonary surfactant. The biophysical roles of SP-B and SP-C in reconstituted surfactants, with various phospholipid mixtures subjected to different thermal treatments, have been examined using a pulsating bubble surfactometer. The phospholipid mixtures were: (A) dipalmitoylphosphatidylcholine (DPPC)/egg phosphatidylcholine (PC)/egg phosphatidylglycerol (PG) (6:2:2, w/w); (B) DPPC/PG (9:1); and (C) DPPC/PG (7:3). Thermal treatments involved mixing SP-B or SP-C, at room temperature, with lipids in chloroform/methanol (9:1, v/v) and removing the solvent under N2 by (1) evaporation at room temperature; (2) evaporation at 45 degrees C; or (3) incubation at 45 degrees C overnight prior to evaporation at 45 degrees C. In all cases, 45 degrees C solvent evaporation was the most effective treatment. DPPC/egg PG (7:3) was the most favourable lipid composition. With either a static or a pulsating bubble, SP-C promoted a rapid decrease in surface tension with little change thereafter. This implies that SP-C is effective in enhancing phospholipid adsorption but does not play an important role in the removal of non-DPPC lipid from the monolayer. While SP-B was not as effective in facilitating phospholipid absorption, samples containing this protein could achieve near zero surface tension upon pulsation. A very low surface tension could also be attained during the initial pulsation of DPPC/PG plus SP-B mixtures which had been allowed to adsorb until equilibrium. This observation indicates that SP-B promotes the removal of PG from the monolayer. SP-A alone had only a slight effect on the surface activity of the DPPC/PG (7:3) mixture, and did not accelerate adsorption of samples containing SP-C. However, SP-A facilitated phospholipid adsorption and may also enhance the removal of PG from monolayers in the presence of SP-B.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1993
We have investigated the ontogeny and hormonal regulation of both synthesis rates and cellular ac... more We have investigated the ontogeny and hormonal regulation of both synthesis rates and cellular accumulation of the mRNA for surfactant-associated protein B (SP-B) in rabbit fetal lung. The developmental pattern for SP-B mRNA synthesis increased as a function of gestational age and paralleled that for SP-B mRNA levels except on days 22-26 of gestation where relatively higher levels of gene transcription were observed. Time-course studies with explants from 26- and 30-day fetal lung maintained in culture revealed a gradual increase in mRNA levels and a much smaller increase in gene transcription relative to adult values. Within 48 h of exposure of 26-day explants to dexamethasone at 10(-8) M there was a rapid increase in SP-B mRNA levels to 7-fold adult levels. A similar overall although somewhat slower and attenuated pattern was observed with 30-day explants. Dexamethasone at 10(-8) M had no effect on SP-B gene transcription with explants of either gestational age. We conclude that the major effect of dexamethasone treatment in vitro on SP-B mRNA levels appears to be post-transcriptional and there are small but distinct differences in the effects of glucocorticoids on SP-B mRNA levels with explant cultures from early and late stages of fetal lung maturation.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1994
The effect of exogenous cholesterol on the stability of surface films at 37°C from various surfac... more The effect of exogenous cholesterol on the stability of surface films at 37°C from various surfactants was stiddled with the pulsating bubble surfactometer. Addition of cholesterol (5%,, w/w) to bovine lipid extract surfactant QbLES) or mixtures of dipalmitoylphosphatidylcholine/l-palmitoyl-Z-oleoyl-pbosphatidylglyceroi/SP-B (7 : 3 : 1%) dispersed in 1.5 r&M CaCIJO.9% NaCl resulted in unstable surface films. Although 10% cholesterol only partially impaired the surface activny of bEESi, it virtuaily abolished that of the reconstituted surfactant. The inhibitory effects of cholesterol were significantly repressed by SPA (ifBG, w/w of lipid) and 3 mM CaCI, or 5 mM CaCl, without SPA. Adsorption of cholesterol from various surfaclants into the air/water interface was examined by measuring the surface radioactivity of [14C]cholasteroi. Cholesterol alone dispersed in I.5 mIt4 CaCl,/O.9% NaCl could not adsorb to the interface, but it adsorbed readily when mixed with bLES. ChoiesteroI adsorption was markedly suppressed by W-A in 3 mM CaC1,/0.9% NaCl or 5 m CaC1,/0.9% NaCI without-A. Eiectron microsco=jy revealed striking ultrastructural differences between bLES/S% ch sterol/lO% %P-A in 3 m CaCl,jO.9% NaC1 and bLES/S% cholesterol in 3 or 5 mM CaC1,/0.9% NaCl. The former exhibited large multilayer and small unilameflar vesicles, while the latter displayed condensed patches of aggregates. Adsorption studies showed aggregated patches adsorbed more rapidly than vesicles but attained lower equilibrium surface pressures. These results indicate SPA and calcium hit he adsorption of surfactant cholesterol to the air-water interface.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1984
Rat lung microsomes washed with increasing conceutrations of NaCl show a displacement of protein ... more Rat lung microsomes washed with increasing conceutrations of NaCl show a displacement of protein from microsomes to the wash supe~at~t. Among the proteins removed from the microsoma~ surface 'was the Mg 2 f-dependent phosphatidate phosphohydrolase, while the Mg ' +-independent activity remained associated with the microsomes. The Mg2+-dependent ac tivity could be quantitatively assayed in the wash supernatant. ~icrosomes washed with increasing concen~ations of MaCl showed a progressive impairment in the synthesis of labelled neutral lipid and phosphatidylcholine from f r4C]glycerol 3-phospbate witb a concomitant increase in the labelling of phosphatidic acid. The impairment was sigmoidal and correlated bigbly with the decrease in Mg2+-dependent phosphatidate phosphohydrolase activity. Wheu ~g~+~de~~dent ~hosp~atidate phosphohydrolase from wash supernatant was incubated with microsomes previously washed witb higb salt concentrations, the labelling of neutral lipid and phosphatidylcholine was returned to control levels. Labelling of neutral lipids and phosphatidylcholine could be restored upon addition of a cytosolic Mg"pendent phosphaiidate phosphohydrol~e isolated by gel filtration. Mg'+-independent ~hos~batidate phosphohydrolase isolated from cytosol was incapable of restoring the labelling of neutral lipids and phosphatidylcholine. These findings confirm that the iMg2+-dependent p hosphatidate phosphohydrolase of sat lenpng is involved in pulmona~ glycerolipid biosyn~esis. The role of the Mg*+-inde~ndent phos~hatidate uhosphohydrolase activity remains unknown.
Under optimal conditions, rat lung microsomal cholinephosphotransferase (EC 2.7.8.2) activity is ... more Under optimal conditions, rat lung microsomal cholinephosphotransferase (EC 2.7.8.2) activity is markedly stimulated by exogenously added 1,2-sn-diacylglycerols containing an unsaturated fatty acid at the 2-position. Diacylglycerols containing long-chain saturated fatty acids at the 1- and 2-positions did not stimulate the incorporation of CDP[14C]choline above the incorporation observed with the endogenous diacylglycerols present in the microsomal preparation. 1-Oleoyl,2-palmitoyl-sn-glycerol was also ineffective in stimulating phosphatidylcholine synthesis. Diacylglycerols containing linoleate or linolenate at the 2-position were not as effective as 1-palmitoyl,2-oleoyl-sn-glycerol. Identical stimulations were observed with the latter diacylglycerol and 'natural' diacylglycerols prepared from egg-yolk phosphatidylcholine or pig liver phosphatidylcholine. Marked selectivities were observed with diacylglycerols containing two unsaturated fatty acids. Only minor amounts of 1,...
Pulmonary surfactant facilitates breathing by reducing the surface tension at the air/liquid inte... more Pulmonary surfactant facilitates breathing by reducing the surface tension at the air/liquid interface. We examined the effect of experimental lung transplantation on the phospholipid pool sizes of alveolar surfactant large and small aggregates, the composition of the large aggregates, the surface tension-reducing ability of lipid extract surfactant, and the leakage of serum proteins into the lung. A double-lung block from the donor animal was stored for 2 or 12 h after perfusion with either Euro-Collins solution or University of Wisconsin solution. The right donor lung was lavaged immediately after the storage period to determine the effects of storage on pulmonary surfactant. The left donor lung was transplanted. The recipient animal, containing its own native right lung and the transplanted left lung, was reperfused for 6 h. After the reperfusion period, the transplanted left lung and the native right lung were lavaged. After an ischemic time of 12 h, impaired gas exchange was observed in the transplanted lung as well as the native lung during the 6 h of reperfusion. This impaired gas exchange was associated with several significant changes in pulmonary surfactant: (1) total serum protein in the lung lavage was increased, (2) the small to large surfactant aggregate ratio was increased, (3) sphingomyelin content was increased and phosphatidylglycerol content was decreased in large aggregates, and (4) the surfactant-associated protein A content was decreased in large aggregates. No significant differences were observed between the results obtained with Euro-Collins and University of Wisconsin solutions.(ABSTRACT TRUNCATED AT 250 WORDS)
The lysophosphatidic acid receptor 1 (LPA1), a G-protein coupled receptor, regulates cell prolife... more The lysophosphatidic acid receptor 1 (LPA1), a G-protein coupled receptor, regulates cell proliferation, migration, and cytokine release. Here, we investigate the molecular signature of LPA1 trafficking to the cell surface. The overexpressed LPA1 with a C-terminal V5 tag (LPA1-V5) is majorly expressed on the cell surface, while two deletion mutants (C320 and ∆84-87) failed to be trafficked to the cell surface. Further, site-directed mutagenesis analysis of the LPA1 revealed that Ile325, Tyr85, and Leu87 within these two fragments regulate LPA1 maturation and trafficking to the cell surface. Over-expression of Sar1, a component of coat protein complex II (COPII), enhances glycosylation of LPA1 wild type, but not these mutants. The mutants of LPA1 are majorly localized in the endoplasmic reticulum (ER) and exhibit a higher binding affinity to heat shock protein 70 (Hsp70), when compared to the LPA1 wild type. Further, we found that all these mutants failed to increase phosphorylation ...
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1989
The potential involvement of the glycerophosphorylcholine (GPC) pathway for the synthesis of phos... more The potential involvement of the glycerophosphorylcholine (GPC) pathway for the synthesis of phosphatidylcholine (PC) has been examined in rat liver and lung and in a human line, the A549 cell which possesses characteristics representative of mature alveolar type |l epithelial cells. Although mitochondrial and microsomai fractions from the above sources readily incorporated radioactive g|ycerophosphate into lipids, the only incorporation observed with radioactive GPC w&s a small variable labelling with the mitochondriai and microsomal fractions from rat lung. Even with these fractions, no radioactivity from GPC was incorporated into PC or |ysoPC. Attempts to increase the incorporation of GPC into lipids by manipulating the incubation conditions were unsuccessful. It was concluded that the occurrence of the GPC pathway in liver and lung is unlikely.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1989
The properties of natural bovine surfactant and its lipid extract have been examined with a pulsa... more The properties of natural bovine surfactant and its lipid extract have been examined with a pulsating bubble surfactometer which assesses the ability of surfactant lipids to adsorb to the air/liquid interface and reduce the surface tension to near 0 dynes/cm during dynamic compression. Studies conducted at 1 mg/ml phospholipid revealed that the surface activity (i.e., the ability to produce low surface tensions) of lipid extracts could be enhanced by incubating the sample at 37 degrees C for 120 min or by addition of CaCl2. In contrast, incubation at 37 degrees C only slightly improved the biophysical activity of natural surfactant and the addition of CaCl2 had a more modest effect than with lipid extracts. With 20 mM CaCl2, the surfactant activity of lipid extract surfactant was similar to that of natural surfactant. Incubation with EDTA reduced the biophysical activity of natural surfactant. Experiments in which increasing amounts of lipid extract were replaced by natural surfactant revealed that small amounts of natural surfactant enhanced the surfactant activity of lipid extract. The biophysical activity of lipid extract surfactant was also increased by the addition of soluble surfactant-associated protein-A (SP-A) (28-36 kDa) purified from natural bovine surfactant. These results indicate that SP-A (28-36 kDa) improves the surfactant activity of lipid extracts by enhancing the rate of adsorption and/or spreading of phospholipid at the air/liquid interface resulting in the formation of a stable lipid monolayer at lower bulk concentrations of either phospholipid or calcium.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1987
The effects of levonorgestrel treatment (4 pg/day per kg body weight o.'5 for 18 days) on rate-li... more The effects of levonorgestrel treatment (4 pg/day per kg body weight o.'5 for 18 days) on rate-limiting enzymes of hepatic triacylglycerol synthesis, namely glycerol-3-phosphate acyltransferase and phosphatidic acid phosphatase were investigated in microsomal, mitochondrial and cytosolic fractions of rat liver. Levonorgestrel treatment resulted in a significant reduction (26%) of hepatic microsomal glycerol-3-phosphate acyltransferase specific activity. Hepatic mitochondrial glycerol-3-phosphate acyltransferase specific activity was unchanged. Levonorgestrel treatment also significantly reduced (by 20%) the specific activity of hepatic microsomal magnesium-independent phosphatidic acid phosphatase. However, magnesium-dependent phosphatic acid phosphatase specific activities in microsomal and cytosolic fractions were unaffected. Cytosolic magnesium-independent phosphatidic acid phosphatase activity was also unchanged. These studies are consistent with the view that levonorgestrel lowers serum triacylglycerol levels, at least in part, by inhibition of the glycerol-3-phosphate acyltransferase (EC 2.3.1.15) step in hepatic triacylglycerol synthesis.
Gold (Au) nanoribbons were synthesized in aqueous phase under ambient conditions by using dimethy... more Gold (Au) nanoribbons were synthesized in aqueous phase under ambient conditions by using dimethylene bis(tetradecyldimethyl-ammonium bromide) (14-2-14) as a capping agent as well as a soft template. A two steps seed-growth (S-G) method was used. The first step of S-G method mainly gave nanorods of high aspect ratio along with nanoparticles of other shapes, but the next step produced mainly fine Au nanoribbons of several micrometers long, 50-150 nm wide, and ≈5 nm thick. They were characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, energy dispersive X-ray, and X-ray diffraction analysis. Isotropic liquid crystalline phase of 14-2-14 provided a soft template effect and derived nanorods of high aspect ratios toward nanoribbon formation at room temperature.
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