The incorporation of14C‐glycerol into different species of diglycerides and triglycerides in rat liver slices

The relative rates of de novo synthesis of species of diglycerides and triglycerides from14C‐glycerol were examined in rat liver slices. Diglycerides containing one or two double bonds per molecule and triglycerides containing four or more double bonds per molecule represented 70% and 60% respectively of the newly synthesized diglycerides and triglycerides. The newly synthesized triglycerides were more unsaturated than the endogenous triglycerides. Our results suggest that a nonrandom synthesis of species of diglycerides occurred followed by an almost random utilization of the various diglyceride species for the biosynthesis of triglycerides.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142207/1/lipd0411.pd

Smoking reduces surfactant protein D and phospholipids in patients with and without chronic obstructive pulmonary disease

<p>Abstract</p> <p>Background</p> <p>Pulmonary surfactant D (SP-D) has important regulatory functions for innate immunity and has been implicated as a biomarker for chronic obstructive pulmonary disease (COPD). We hypothesized that COPD patients would have reduced bronchoalveolar lavage (BAL) fluid SP-D levels compared to healthy smoking and non-smoking controls.</p> <p>Methods</p> <p>BAL SP-D and phospholipids were quantified and corrected for dilution in 110 subjects (65 healthy never smokers, 23 smokers with normal spirometry, and 22 smokers with COPD).</p> <p>Results</p> <p>BAL SP-D was highest in never smokers (mean 51.9 μg/mL ± 7.1 μg/mL standard error) compared to both smokers with normal spirometry (16.0 μg/mL ± 11.8 μg/mL) and subjects with COPD (19.1 μg/mL ± 12.9 μg/mL; P < 0.0001). Among smokers with COPD, BAL SP-D correlated significantly with FEV₁% predicted (R = 0.43; P < 0.05); however, the strongest predictor of BAL SP-D was smoking status. BAL SP-D levels were lowest in current smokers (12.8 μg/mL ± 11.0 μg/mL), intermediate in former smokers (25.2 μg/mL ± 14.2 μg/mL; P < 0.008), and highest in never smokers. BAL phospholipids were also lowest in current smokers (6.5 nmol ± 1.5 nmol), intermediate in former smokers (13.1 nmol ± 2.1 nmol), and highest in never smokers (14.8 nmol ± 1.1 nmol; P < 0.0001).</p> <p>Conclusions</p> <p>These data suggest that smokers, and especially current smokers, exhibit significantly reduced BAL SP-D and phospholipids compared to nonsmokers. Our findings may help better explain the mechanism that leads to the rapid progression of disease and increased incidence of infection in smokers.</p

Surfactant replacement and open lung concept – Comparison of two treatment strategies in an experimental model of neonatal ARDS

Background: Several concepts of treatment in neonatal ARDS have been proposed in the last years. The present study compared the effects of open lung concept positive pressure ventilation (PPVOLC) with a conventional ventilation strategy combined with administration of two different surfactant preparations on lung function and surfactant homoeostasis. Methods: After repeated whole-lung saline lavage, 16 newborn piglets were assigned to either PPVOLC(n = 5) or surfactant treatment under conventional PPV using a natural bovine (n = 5) or a monomeric protein B based surfactant (n = 6). Results: Comprehensive monitoring showed each treatment strategy to improve gas exchange and lung function, although the effect on PaO2and pulmonary compliance declined over the study period in the surfactant groups. The overall improvement of the ventilation efficiency index (VEI) was significantly greater in the PPVOLCgroup. Phospholipid and protein analyses of the bronchoalveolar lavage fluid showed significant alterations to surfactant homoeostasis in the PPVOLCgroup, whereas IL-10 and SP-C mRNA expression was tendentially increased in the surfactant groups. Conclusion: The different treatment strategies applied could be shown to improve gas exchange and lung function in neonatal ARDS. To which extent differences in maintenance of lung function and surfactant homeostasis may lead to long-term consequences needs to be studied further

Investigation into the Role of Tumor-Associated Macrophages in the Antitumor Activity of Doxil

Purpose. Our recent studies show specific localization of long-circulating liposomes (LCL) within the endosomal/lysosomal compartment of tumor-associated macrophages (TAM). Based on this finding, the present study aims to investigate whether clinically applied LCL formulations such as Doxil (LCLencapsulated doxorubicin), have alternative mechanisms of action additionally to direct drug-mediated cytotoxicity towards tumor cells. Methods. The antitumor activity of Doxil was evaluated in B16.F10 melanoma-bearing mice, in the presence and in the absence of TAM. To suppress TAM functions, liposomal clodronate (Lip-CLOD) was injected 24 h before the actual treatment. The effect of Doxil on the levels of angiogenic factors was determined using an angiogenic protein array. As positive control, the same experiments were conducted with LCL-encapsulated prednisolone phosphate (LCL-PLP), a tumor-targeted formulation with known strong anti-angiogenic/anti-inflammatory effects on TAM. Results. Our results show that the antitumor efficacy of Doxil was only partially attributed to the inhibition of TAM-mediated angiogenesis whereas LCL-PLP inhibited tumor growth through strong suppressive effects on pro-angiogenic functions of TAM. As described previously, the main mechanism of Doxil might be a cytotoxic effect on tumor cells. Conclusions. Our findings suggest that the antitumor activity of Doxil does not depend mainly on the presence of functional TAM in tumors

Purification and Activity Testing of the Full-Length YycFGHI Proteins of Staphylococcus aureus

Background: The YycFG two-component regulatory system (TCS) of Staphylococcus aureus represents the only essential TCS that is almost ubiquitously distributed in Gram-positive bacteria with a low G+C-content. YycG (WalK/VicK) is a sensor histidine-kinase and YycF (WalR/VicR) is the cognate response regulator. Both proteins play an important role in the biosynthesis of the cell envelope and mutations in these proteins have been involved in development of vancomycin and daptomycin resistance. Methodology/Principal Findings: Here we present high yield expression and purification of the full-length YycG and YycF proteins as well as of the auxiliary proteins YycH and YycI of Staphylococcus aureus. Activity tests of the YycG kinase and a mutated version, that harbours an Y306N exchange in its cytoplasmic PAS domain, in a detergent-micelle-model and a phosholipid-liposome-model showed kinase activity (autophosphorylation and phosphoryl group transfer to YycF) only in the presence of elevated concentrations of alkali salts. A direct comparison of the activity of the kinases in the liposomemodel indicated a higher activity of the mutated YycG kinase. Further experiments indicated that YycG responds to fluidity changes in its microenvironment. Conclusions/Significance: The combination of high yield expression, purification and activity testing of membrane and membrane-associated proteins provides an excellent experimental basis for further protein-protein interaction studies an

Effect of Liposome Characteristics and Dose on the Pharmacokinetics of Liposomes Coated with Poly(amino acid)s

Long-circulating liposomes, such as PEG-liposomes, are frequently studied for drug delivery and diagnostic purposes. In our group, poly(amino acid) (PAA)-based coatings for long-circulating liposomes have been developed. These coatings provide liposomes with similar circulation times as compared to PEG-liposomes, but have the advantage of being enzymatically degradable. For PEG-liposomes it has been reported that circulation times are relatively independent of their physicochemical characteristics. In this study, the influence of factors such as PAA grafting density, cholesterol inclusion, surface charge, particle size, and lipid dose on the circulation kinetics of PAA-liposomes was evaluated after intravenous administration in rats. Prolonged circulation kinetics of PAA-liposomes can be maintained upon variation of liposome characteristics and the lipid dose given. However, the use of relatively high amounts of strongly charge-inducing lipids and a too large mean size is to be avoided. In conclusion, PAA-liposomes represent a versatile drug carrier system for a wide variety of applications

Induction of effective and antigen-specific antitumour immunity by a liposomal ErbB2/HER2 peptide-based vaccination construct

Efficient delivery of tumour-associated antigens to appropriate cellular compartments of antigen-presenting cells is of prime importance for the induction of potent, cell-mediated antitumour immune responses. We have designed novel multivalent liposomal constructs that co-deliver the p63–71 cytotoxic T Lymphocyte epitope derived from human ErbB2 (HER2), and HA307–319, a T-helper (Th) epitope derived from influenza haemagglutinin. Both peptides were conjugated to the surface of liposomes via a Pam3CSS anchor, a synthetic lipopeptide with potent adjuvant activity. In a murine model system, vaccination with these constructs completely protected BALB/c mice from subsequent s.c. challenge with ErbB2-expressing, but not ErbB2-negative, murine renal carcinoma (Renca) cells, indicating the induction of potent, antigen-specific immune responses. I.v. re-challenge of tumour-free animals 2 months after the first tumour cell inoculation did not result in the formation of lung tumour nodules, suggesting that long-lasting, systemic immunity had been induced. While still protecting the majority of vaccinated mice, a liposomal construct lacking the Th epitope was less effective than the diepitope construct, also correlating with a lower number of CD8+ IFN-γ+ T-cells identified upon ex vivo peptide restimulation of splenocytes from vaccinated animals. Importantly, in a therapeutic setting treatment with the liposomal vaccines resulted in cures in the majority of tumour-bearing mice and delayed tumour growth in the remaining ones. Our results demonstrate that liposomal constructs which combine Tc and Th peptide antigens and lipopeptide adjuvants can induce efficient, antigen-specific antitumour immunity, and represent promising synthetic delivery systems for the design of specific antitumour vaccines

Metabolic Regulation of Invadopodia and Invasion by Acetyl-CoA Carboxylase 1 and De novo Lipogenesis

Invadopodia are membrane protrusions that facilitate matrix degradation and cellular invasion. Although lipids have been implicated in several aspects of invadopodia formation, the contributions of de novo fatty acid synthesis and lipogenesis have not been defined. Inhibition of acetyl-CoA carboxylase 1 (ACC1), the committed step of fatty acid synthesis, reduced invadopodia formation in Src-transformed 3T3 (3T3-Src) cells, and also decreased the ability to degrade gelatin. Inhibition of fatty acid synthesis through AMP-activated kinase (AMPK) activation and ACC phosphorylation also decreased invadopodia incidence. The addition of exogenous 16∶0 and 18∶1 fatty acid, products of de novo fatty acid synthesis, restored invadopodia and gelatin degradation to cells with decreased ACC1 activity. Pharmacological inhibition of ACC also altered the phospholipid profile of 3T3-Src cells, with the majority of changes occurring in the phosphatidylcholine (PC) species. Exogenous supplementation with the most abundant PC species, 34∶1 PC, restored invadopodia incidence, the ability to degrade gelatin and the ability to invade through matrigel to cells deficient in ACC1 activity. On the other hand, 30∶0 PC did not restore invadopodia and 36∶2 PC only restored invadopodia incidence and gelatin degradation, but not cellular invasion through matrigel. Pharmacological inhibition of ACC also reduced the ability of MDA-MB-231 breast, Snb19 glioblastoma, and PC-3 prostate cancer cells to invade through matrigel. Invasion of PC-3 cells through matrigel was also restored by 34∶1 PC supplementation. Collectively, the data elucidate the novel metabolic regulation of invadopodia and the invasive process by de novo fatty acid synthesis and lipogenesis