Francis H.C. Tsao

PURIFICATION AND CHARACTERIZATION OF TWO RABBIT LUNG CA2+-DEPENDENT PHOSPHOLIPID-BINDING PROTEINS

Two Ca2(+)-dependent phospholipid-binding proteins (PLBPs) in rabbit lung cytosolic fraction have been purified to homogeneity. The apparent molecular weights of these two proteins are 36,000 and 33,000. Both the 36,000 and 33,000 PLBPs aggregated certain negatively charged unilamellar liposomes, but not the neutral phosphatidylcholine (PC) liposomes, in the presence of Ca2+. However, both PLBPs fused PC unilamellar liposomes to membrane acceptors. The 36,000 and 33,000 PLBPs had different specificities for phospholipid head groups, effects of Ca2+ and membrane charges and amino acid compositions. Both the PLBPs aggregated the surfactant membranes (lamellar bodies or from lung lavage) and nonsurfactant membranes (microsomes or mitochondria) to a level similar to that of the synthetic acidic phospholipid vesicles, but the proteins fused [14C]PC liposomes to the surfactant membranes 13- to 16-times more than to the synthetic phospholipid vesicles. The 36,000 PLBP fused [14C]PC liposomes to microsomes or mitochondria only half that of the fusion to the surfactant; the 33,000 PLBP fused [14C]PC liposomes to the surfactant and nonsurfactant alike. The PLBPs aggregate activity was not affected by the depletion of biological membrane proteins and the disruption of the native membrane integrity, but its fusion activity was greatly reduced. These results suggest that: (1) the 36,000 and 33,000 PLBPs are two different proteins; (2) the PLBPs may possess two catalytic reactions, one for the aggregation of small vesicles due to the binding of the protein to phospholipid vesicles and one for the fusion of small vesicles to acceptors; (3) the fusion activity was probably regulated by biological membrane proteins or structures; and (4) lung PLBP(s) might play a role in lung surfactant biogenesis.

Parenteral Nutrition Suppresses the Bactericidal Response of the Small Intestine

BACKGROUND Parenteral nutrition (PN) increases infectious risk in critically ill patients compared with enteral feeding. Previously, we demonstrated that PN feeding suppresses the concentration of the Paneth cell antimicrobial protein secretory phospholipase A2 (sPLA2) in the gut lumen. sPLA2 and other Paneth cell proteins are released in response to bacterial components, such as lipopolysaccharide (LPS), and they modulate the intestinal microbiome. Because the Paneth cell protein sPLA2 was suppressed with PN feeding, we hypothesized PN would diminish the responsiveness of the small bowel to LPS through reduced secretions and as a result exhibit less bactericidal activity. METHODS The distal ileum was harvested from Institute of Cancer Research mice, washed, and randomized for incubation with LPS (0, 1, or 10 μg/mL). Culture supernatant was collected and sPLA2 activity was measured. Bactericidal activity of the ileum segment secretions was assessed against Pseudomonas aeruginosa with and without an sPLA2 inhibitor at 2 concentrations, 100 nmol/L and 1 μmol/L. Institute of Cancer Research mice were randomized to chow or PN for 5 days. Tissue was collected for immunohistochemistry (IHC) and ileal segments were incubated with LPS (0 or 10 μg/mL). sPLA2 activity and bactericidal activity were measured in secretions from ileal segments. RESULTS Ileal segments responded to 10 μg/mL LPS with significantly greater sPLA2 activity and bactericidal activity. The bactericidal activity of secretions from LPS stimulated tissue was suppressed 50% and 70%, respectively, with the addition of the sPLA2-inhibitor. Chow displayed greater sPLA2 in the Paneth cell granules and secreted higher levels of sPLA2 than PN before and after LPS. Accordingly, media collected from chow was more bactericidal than PN. IHC confirmed a reduction in Paneth cell granules after PN. CONCLUSION This work demonstrates that ileal segments secrete bactericidal secretions after LPS exposure and the inhibition of the Paneth cell antimicrobial protein sPLA2 significantly diminishes this. PN feeding resulted in suppressed secretion of the sPLA2 and resulted in increased bacterial survival. This demonstrates that PN significantly impairs the innate immune response by suppressing Paneth cell function.

Bacteria causing false positive test for phosphatidylglycerol in amniotic fluid

A case is reported in which phosphatidylglycerol was detected in a sample of vaginal pool amniotic fluid but absent in amniotic fluid obtained at amniocentesis. Bacteria contaminating the fluid are shown to be capable of phosphatidylglycerol production.

Specific transfer of dipalmitoyl phosphatidylcholine in rabbit lung

Liposomes were used as a tool to probe the specific transfer of dipalmitoyl phosphatidylcholine in rabbit lung. After incubation of liposomes with lung slices, lamellar bodies took up 30-50% more radioactively labeled dipalmitoyl phosphatidylcholine from egg yolk phosphatidylcholine liposomes than the labeled dioleoyl phosphatidylcholine, whereas mitochondria and microsomes took up both species at relatively equal rates. Furthermore, when 50% egg yolk phosphatidylcholine was replaced by dipalmitoyl phosphatidylcholine, the uptake of [1-14C]dipalmitoyl phosphatidylcholine by lamellar bodies was greatly reduced due to the isotope-dilution effect of the unlabeled dipalmitoyl species. This competitive uptake was not observed in mitochondria and microsomes. Similar results were also observed when liposomes were incubated with the cell-free homogenate. On the other hand, distearoyl or dioleoyl phosphatidylcholine did not compete with the uptake of [1-14C]dipalmitoyl phosphatidylcholine. These results suggest an intracellular specific transfer of dipalmitoyl phosphatidylcholine from liposomes to lamellar bodies, presumably by a specific transfer protein. When liposomes were labeled with [1-14C]dioleoyl phosphatidylcholine, the addition of unlabeled dioleoyl phosphatidylcholine up to 50% of total phosphatidylcholine concentration did not compete with the uptake of [1-14C]dioleoyl phosphatidylcholine in all fractions including lamellar bodies. This indicates that the intracellular uptake of liposomal dioleoyl phosphatidylcholine by lamellar bodies and other subcellular organelles was probably dominated by the fusion mechanism.

Lung calcium-dependent phospholipid-binding proteins: structure and function

Distinct peptide maps of two rabbit lung Ca2(+)-dependent phospholipid-binding proteins (PLBPs), 36,000 and 33,000, were generated by cyanogen bromide (CNBr) cleavage, trypsin or Staphylococcus aureus V8 proteinase digestion. The amino acid sequence of a CNBr-cleaved peptide of the 36,000 PLBP was aligned to the amino terminus of human lipocortin I with more than 77% identity, but had no identity with the known amino terminal sequence of other known annexins. Partial amino acid sequence of a 33,000 PLBP peptide demonstrated a close (56%) relationship to endonexin II, human placental anticoagulant protein, and porcine intestine protein II, but shared only 32% identity with lipocortin I, 30% with lipocortin II. Antiserum generated against purified 36,000 PLBP reacted strongly with the 33,000 PLBP, but did not react with any other rabbit lung cytosolic proteins. Both PLBPs inhibited the phospholipase A2 reaction when dioleoyl phosphatidylcholine and phosphatidylglycerol vesicles or monolayers were used as substrates. In the vesicle assay, the phospholipase A2 reaction was inhibited at lower substrate phospholipid concentrations but not at nearly saturating substrate concentrations. In the monolayer assay, the phospholipid-binding proteins did not inhibit phospholipase A2 at a low phospholipid surface concentration of 3.8.10(-3) molecules/A2, but they did at higher surface concentrations between 1.1 x 10(-2) and 3.8 x 10(-2) molecules/A2. The inhibition of phospholipase A2 by rabbit lung phospholipid-binding proteins is most likely due to the prevention of penetration by phospholipase A2 into the interface, a requirement for the enzyme to act on the substrate.

Identification of calcium-dependent phospholipid-binding proteins (annexins) from guinea pig alveolar type II cells

A new group of calcium-regulating proteins, called annexins or Ca++-dependent phospholipid-binding proteins (PLBP), have been detected in different species, organs and cell types. In the present study, we have identified and quantitated PLBP from guinea pig lung, lavage fluid and alveolar type II cells to elucidate the possible role of PLBP in lung surfactant biogenesis and secretion. Lungs were lavaged and type II cells from lavaged lung were isolated by elastase digestion and purified by centrifugal elutriation. For the quantitative identification of PLBP, we performed ELISA assays and Western blot analysis by using an antiserum raised in guinea pigs against a pure rabbit lung 36 kDa PLBP. The lavage fluid, cytosol from lung and type II cells contained 784,167 and 435 ng per mg protein, respectively, of PLBP. The SDS-PAGE electrophoretic pattern and Western blot confirmed that all lung samples have band corresponding to a 36 kDa protein. This indicates that both alveolar type II cells and lavage fluid have higher levels of PLBP than whole lung cytosol.

Immunocharacterization and developmental regulation of rabbit lung calcium-dependent phospholipid-binding proteins

The purpose of this work was to use the immunoblotting methods to study the 36 kDa calcium-dependent phospholipid-binding protein (PLBP) in the adult and fetal rabbit lungs to gain insight into the significance of this protein in lung development. The identity of the 36 kDa PLBP and the antigen specificity of the antiserum raised against this protein in the guinea pig were tested against known annexins and antibodies to the annexins. Our results showed that the rabbit lung 36 kDa PLBP contained only one protein which cross-reacted with antibodies against annexin 1. However, the 36 kDa PLBP was slightly smaller (36 vs. 37 kDa) and more acidic (pI 6.0 vs. 6.9) than the recombinant human annexin 1. The guinea pig antiserum only reacted with annexin 1, not with any of the other annexins tested. In the cytosolic fractions of the lung and the alveolar epithelial type II cells, and in the lung lavage fluid, the 36 kDa PLBP was by far the most prominent protein with minor presence of a 33 kDa protein recognized by the guinea pig antiserum. The amount of the 36 kDa PLBP of type II cells was 55% higher than that in the lung tissue and 2.6-times higher than that in the lung lavage (9.3 +/- 0.62, 6.0 +/- 0.31 and 3.6 +/- 0.04 micrograms/mg protein, respectively). The 36 kDa PLBP appeared in the fetal rabbit lungs as early as at 21 days gestation and increased 2-fold to reach the adult level at 27 days gestation (term 31 days). The high content of PLBP in type II cells and the rapid increase in this protein in the fetal lungs at late gestations suggest an important role of the 36 kDa PLBP in lung development and surfactant biogenesis.

Route and Type of Nutrition and Surgical Stress Influence Secretory Phospholipase A2 Secretion of the Murine Small Intestine

BACKGROUND The function of secretory phospholipase A2 (sPLA2) is site dependent. In tissue, sPLA2 regulates eicosanoid production; in circulation, sPLA2 primes neutrophils; and in the intestinal lumen, sPLA2 provides innate bactericidal immunity as a defensin-related protein. Since parenteral nutrition (PN) primes leukocytes while suppressing intraluminal mucosal immunity, the authors hypothesized that (1) PN would diminish luminal sPLA2 activity but increase activity in intestinal tissue and serum and (2) stress would accentuate these changes. METHODS Mice received chow, a complex enteral diet (CED), intragastric PN (IG-PN), or PN in experiment 1 and chow, chow+stress, PN, or PN+stress in experiment 2. RESULTS In experiment 1, luminal sPLA2 activity was greatest in chow and decreased in CED, IG-PN, and PN, with PN lower than CED and IG-PN. Compared to that after chow, serum sPLA2 activity dropped after CED, IG-PN, and PN. Serum sPLA2 was higher in portal than systemic serum. In experiment 2, PN lowered luminal sPLA2 activity vs chow. Stress lowered luminal sPLA2 activity in chow, without change in PN. Following stress, luminal immunoglobulin A increased in chow but not PN. Serum sPLA2 activity increased in PN. CONCLUSIONS PN attenuates sPLA2 activity in intestinal fluid, consistent with suppressed innate mucosal defense. Stress suppresses luminal fluid sPLA2 activity in chow but not the immunoglobulin A response; PN impairs both. Stress significantly elevates serum sPLA2 in PN-fed mice, consistent with known increased neutrophil priming with PN. PN reduces innate bactericidal immunity of the gut but upregulates serum proinflammatory products poststress.

Annexin I in female rabbit reproductive organs: Varying levels in relation to maturity and pregnancy

The level of annexin I, a 36 kDa calcium-dependent phospholipid-binding protein (36 kDa PLBP) in the reproductive organs of young, mature, and pregnant rabbits was determined immunologically with antibodies raised against purified rabbit lung annexin I. In the cytosolic fractions of the ovary, fallopian tube, uterus, and placenta, annexin I was the only major immunoreactive protein. The reproductive organs appeared to have higher annexin I levels than most nonreproductive organ tissues, except the lung and the spleen which were also rich in annexin I. A small amount of annexin I and a nearly equal amount of its hydrolytic product, a 33 kDa polypeptide, were detected in the amniotic fluid between 21 and 27 days gestation. Structural similarity of annexin I in the reproductive organs and in the lung was suggested by their identical isoelectric point values. Annexin I in the ovary of adult rabbits was 70% higher than that in the respective organ of immature rabbits. The uterus of pregnant rabbits had about 84% higher annexin I contents than that of the nonpregnant rabbits., The placenta had more annexin I per mg cytosolic protein than either the ovary or the uterus during pregnancy. The high concentration of annexin I in the reproductive organs may reflect specific functions of these organs in the reproductive years and during the reproductive cycle.

Effect of Hydrocortisone on the Metabolism of Phosphatidylcholine in Maternal and Fetal Rabbit Lungs and Livers

Summary: It has been observed that glucocorticoids stimulate fetal lung maturation. This study examined the effects of glucocorticoids on the phosphatidylcholine (PC) metabolism in maternal and fetal lungs and livers. At 24 days of gestation, pregnant does were injected im with either hydrocortisone or an equal volume of saline. At 27 days of gestation, the maternal and fetal lungs and livers were removed for study. Maternal hydrocortisone treatment significantly decreased the fetal body weight and lung weight, but had no effect on the weights of fetal liver or the ratios of maternal lung/body or liver/body weights. Concentrations of protein, total phospholipids, or PC of fetal lung and liver and of maternal liver were not affected by hydrocortisone. However, the amounts of maternal lung protein, total phospholipids, and PC were significantly increased. No stimulation by hydrocortisone was seen in the specific activities of the enzymes: choline kinase, phosphocholine cytidyltransferase, and choline phosphotransferase of cytidine 5′-diphosphocholine (CDP-choline) pathway and lysoPC-lysoPC acyltransferase, lysophospholipase, and acyl-CoA lysoPC acyl-transferase of PC-lysoPC cycle pathway.Maternal administration of hydrocortisone stimulated the incorporation of [methyl-14C] choline into fetal lung PC. Additionally, hydrocortisone also accelerated the secretion of PC from maternal lung tissue. The acceleration of the secretion of PC was not observed in the fetal lung tissue, possibly because of a low basal secretory rate. The acceleration of PC secretion by hydrocortisone in maternal lung may suggest a relation of this mechanism to the fetal lung maturation affected by steroids.Speculation: One biochemical effect of antenatal maternal hydrocortisone is the acceleration of choline incorporation into fetal lung PC. Maternally administered glucocorticoids, in addition to affecting fetal lung, might also affect maternal lung and liver PC metabolism by alteration of enzyme activity or changing turnover or transfer rates. Though studied indirectly, hydrocortisone might stimulate the secretion of lung PC onto the alveolar surface.