Fatty acids of pulmonary surfactant phosphatidylcholine from fetal rabbit lung tissue in culture. Biosynthesis of n-10 monoenoic fatty acids

We have previously reported that fetal rabbit lung tissue in organ culture produces a lamellar body material (pulmonary surfactant) with a lower percentage of disaturated phosphatidylcholine than is typically found in rabbit lung in vivo (Longmuir, K.J., C. Resele-Tiden, and L. Sykes. 1985. Biochim. Biophys. Acta. 833: 135-143). This investigation was conducted to identify all fatty acids present in the lamellar body phosphatidylcholine, and to determine whether the low level of disaturated phosphatidylcholine is due to excessive unsaturated fatty acid at position sn-1, sn-2, or both. Fetal rabbit lung tissue, 23 days gestation, was maintained in culture for 7 days in defined (serum-free) medium. Phospholipids were labeled in culture with [1-14C]acetate or [U-14C]glycerol (to follow de novo fatty acid biosynthesis), or with [1-14C]palmitic acid (to follow incorporation of exogenously supplied fatty acid). Radiolabeled fatty acid methyl esters obtained from lamellar body phosphatidylcholine were first separated by reverse-phase thin-layer chromatography (TLC) into two fractions of 1) 14:0 + 16:1 and 2) 16:0 + 18:1. Complete separation of the individual saturated and monoenoic fatty acids was achieved by silver nitrate TLC of the two fractions. Monoenoic fatty acid double bond position was determined by permanganate-periodate oxidation followed by HPLC of the carboxylic acid phenacyl esters. Lamellar body phosphatidylcholine contained four monoenoic fatty acids: 1) palmitoleic acid, 16:1 cis-9; 2) oleic acid, 18:1 cis-9; 3) cis-vaccenic acid, 18:1 cis-11; and 4) 6-hexadecenoic acid, 16:1 cis-6. In addition, 8-octadecenoic acid, 18:1 cis-8, was found in the fatty acids of the tissue homogenate. The abnormally low disaturated phosphatidylcholine content in lamellar body material was the result of abnormally high levels of monoenoic fatty acid (principally 16:1 cis-9) found at position sn-2. Position sn-1 contained normal levels of saturated fatty acid. The biosynthesis of the unusual n-10 fatty acids was observed from the start of culture throughout the entire 7-day culture period, and was observed in incubations of tissue slices of day 23 fetal rabbit lung. This is the first report of the biosynthesis of n-10 fatty acids (16:1 cis-6 and 18:1 cis-8) in a mammalian tissue other than skin, where these fatty acids are found in the secretory product (sebum) of sebaceous glands.     

The phospholipids of lamellar body material from fetal rabbit lung tissue in culture. Unusual phosphatidylcholine fatty acid profile

Tissue pieces of rabbit fetal lung, 23 days gestation, were cultured for 7 days in serum-free medium to obtain lamellar body material for phospholipid analysis. Cultures were maintained in culture medium without serum and (1) with no added hormones (control cultures), (2) with thyroxine (1 x 10(-7) M), (3) with cortisol (1 x 10(-7) M) and (4) with thyroxine plus cortisol (1 x 10(-7) M each). The hormonal response was evaluated by measuring the quantity of lamellar body material isolated from the tissue pieces after the 7-day culture period. Compared to control cultures, more lamellar body material was recovered from cultures treated with cortisol (180% of control) and with thyroxine plus cortisol (250% of control). Cultures treated with thyroxine alone yielded the same amount of lamellar body material as the controls. Hormone treatment produced only minor changes in the glycerophospholipid profile of the lamellar body material. A small but significant increase in the percentage of phosphatidylglycerol and a small but significant decrease in phosphatidylinositol were found in lamellar body material from cultures treated with thyroxine and thyroxine plus cortisol. The disaturated phosphatidylcholine content of the lamellar body material from culture was 28% of the total lamellar body phospholipid and was not affected by hormone treatment. This disaturated phosphatidylcholine content was low compared to the disaturated phosphatidylcholine of lamellar body material from adult lung (46%). The low proportion of disaturated phosphatidylcholine was due to the unusual presence of palmitoleic acid (16:1(cis-9)), which was more than one-fourth of the total fatty acid of the lamellar body phosphatidylcholine. It is possible that an abnormal delta 9 fatty acid desaturation activity was expressed in the lung tissue in vitro, which resulted in the high incorporation of the 16:1 fatty acid into lamellar body phosphatidylcholine.     

Cytokine- and calcium ionophore A23187-mediated arachidonic acid metabolism in neutrophils

Arachidonic acid (AA) metabolism is implicated as an intracellular and/or intercellular second messenger system for the transmission of cytokine-initiated signals that affect neutrophils and mediate systemic toxicity. The purpose of the present study is to ascertain if cytokines that are known to affect neutrophil function in vivo and in vitro directly stimulate neutrophil AA metabolism in vitro. The recombinant human cytokines multi-colony stimulating factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 1, tumor necrosis factor (TNF), and interleukin 6 and the calcium ionophore A23187 were incubated with purified 14C-AA radiolabeled human peripheral blood neutrophils and the effects were assayed by one- and two-dimensional thin layer lipid chromatography. None of the cytokines appeared to induce the release of cell-incorporated AA or to increase the level of radiolabeled phosphatidic acid. TNF induces severe systemic toxicity that is inhibited by cyclooxygenase inhibitors, which suggests a role for AA metabolites in the pathophysiologic effects of TNF; we have confirmed that TNF and endotoxin act synergistically to induce indomethacin-inhibitable fatal shock in rats. However, when in 3H-AA radiolabeled human neutrophils were incubated with TNF in kinetic, cold-chase, and TNF preincubation experiments, TNF was not found to increase AA metabolism, although changes in the intracellular neutral lipid content were noted. GM-CSF, which has been reported by previous investigators to directly induce the release of AA, did not release neutrophil-associated 3H-AA. In conclusion, the direct release of AA from membrane-associated phospholipids does not appear to be a major second messenger pathway for cytokine-initiated activation of neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)     

Considerations for operational space definition and optimization of a no-salt flowthrough hydrophobic interaction chromatography purification step

No-salt flowthrough hydrophobic interaction chromatography (HIC) has been shown to effectively remove process and product-related impurities from bioprocess streams. In this publication, a panel of six antibodies has been used to demonstrate operating principles for the application of no-salt flowthrough HIC in antibody purification processes. The results indicate that no-salt flowthrough HIC provides robust aggregate clearance across operating conditions including flow rate, and variations in resin ligand density. Additionally, HMW reduction has an optimal pH range relative to the isoelectric point of each molecule and high molecular weight (HMW) reduction can be improved by altering the total protein load and/or HMW concentration to drive binding of high molecular weight species to the resin.     

Investigation of inanimate objects by the greater bushbaby (Otolemur garnettii)

Investigatory behavior with novel, inanimate objects by two groups of four juvenile greater bushbabies (Otolemur garnettii) was examined in the laboratory. Substantial investigatory behavior was shown by all subjects. In the first study, subjects showed interest in a wide variety of nonfood stimulus objects. In the second, subjects displayed sustained interest in and investigation of non-food stimulus objects over three sessions. Bushbabies showed preferences for larger, more manipulable objects and variations in total contact over days. Individual differences were observed in the duration and types of contact with objects. These observations contradict earlier reports that prosimians show little interest in inanimate, non-food objects.     

Duration of Maternal Antibodies against Canine Distemper Virus and Hendra Virus in Pteropid Bats

Old World frugivorous bats have been identified as natural hosts for emerging zoonotic viruses of significant public health concern, including henipaviruses (Nipah and Hendra virus), Ebola virus, and Marburg virus. Epidemiological studies of these viruses in bats often utilize serology to describe viral dynamics, with particular attention paid to juveniles, whose birth increases the overall susceptibility of the population to a viral outbreak once maternal immunity wanes. However, little is understood about bat immunology, including the duration of maternal antibodies in neonates. Understanding duration of maternally derived immunity is critical for characterizing viral dynamics in bat populations, which may help assess the risk of spillover to humans. We conducted two separate studies of pregnant Pteropus bat species and their offspring to measure the half-life and duration of antibodies to 1) canine distemper virus antigen in vaccinated captive Pteropus hypomelanus; and 2) Hendra virus in wild-caught, naturally infected Pteropus alecto. Both of these pteropid bat species are known reservoirs for henipaviruses. We found that in both species, antibodies were transferred from dam to pup. In P. hypomelanus pups, titers against CDV waned over a mean period of 228.6 days (95% CI: 185.4–271.8) and had a mean terminal phase half-life of 96.0 days (CI 95%: 30.7–299.7). In P. alecto pups, antibodies waned over 255.13 days (95% CI: 221.0–289.3) and had a mean terminal phase half-life of 52.24 days (CI 95%: 33.76–80.83). Each species showed a duration of transferred maternal immunity of between 7.5 and 8.5 months, which was longer than has been previously estimated. These data will allow for more accurate interpretation of age-related Henipavirus serological data collected from wild pteropid bats.     

The Escherichia coli CydX Protein Is a Member of the CydAB Cytochrome bd Oxidase Complex and Is Required for Cytochrome bd Oxidase Activity

Cytochrome bd oxidase operons from more than 50 species of bacteria contain a short gene encoding a small protein that ranges from ∼30 to 50 amino acids and is predicted to localize to the cell membrane. Although cytochrome bd oxidases have been studied for more than 70 years, little is known about the role of this small protein, denoted CydX, in oxidase activity. Here we report that Escherichia coli mutants lacking CydX exhibit phenotypes associated with reduced oxidase activity. In addition, cell membrane extracts from ΔcydX mutant strains have reduced oxidase activity in vitro. Consistent with data showing that CydX is required for cytochrome bd oxidase activity, copurification experiments indicate that CydX interacts with the CydAB cytochrome bd oxidase complex. Together, these data support the hypothesis that CydX is a subunit of the CydAB cytochrome bd oxidase complex that is required for complex activity. The results of mutation analysis of CydX suggest that few individual amino acids in the small protein are essential for function, at least in the context of protein overexpression. In addition, the results of analysis of the paralogous small transmembrane protein AppX show that the two proteins could have some overlapping functionality in the cell and that both have the potential to interact with the CydAB complex.     

Reduction of product-related species during the fermentation and purification of a recombinant IL-1 receptor antagonist at the laboratory and pilot scale

Through a parallel approach of tracking product quality through fermentation and purification development, a robust process was designed to reduce the levels of product-related species. Three biochemically similar product-related species were identified as byproducts of host-cell enzymatic activity. To modulate intracellular proteolytic activity, key fermentation parameters (temperature, pH, trace metals, EDTA levels, and carbon source) were evaluated through bioreactor optimization, while balancing negative effects on growth, productivity, and oxygen demand. The purification process was based on three non-affinity steps and resolved product-related species by exploiting small charge differences. Using statistical design of experiments for elution conditions, a high-resolution cation exchange capture column was optimized for resolution and recovery. Further reduction of product-related species was achieved by evaluating a matrix of conditions for a ceramic hydroxyapatite column. The optimized fermentation process was transferred from the 2-L laboratory scale to the 100-L pilot scale and the purification process was scaled accordingly to process the fermentation harvest. The laboratory- and pilot-scale processes resulted in similar process recoveries of 60 and 65%, respectively, and in a product that was of equal quality and purity to that of small-scale development preparations. The parallel approach for up- and downstream development was paramount in achieving a robust and scalable clinical process.