Lipidomics of cellular and secreted phospholipids from differentiated human fetal type II alveolar epithelial cells

Maturation of fetal alveolar type II epithelial cells in utero is characterized by specific changes to lung surfactant phospholipids. Here, we quantified the effects of hormonal differentiation in vitro on the molecular specificity of cellular and secreted phospholipids from human fetal type II epithelial cells using electrospray ionization mass spectrometry. Differentiation, assessed by morphology and changes in gene expression, was accompanied by restricted and specific modifications to cell phospholipids, principally enrichments of shorter chain species of phosphatidylcholine (PC) and phosphatidylinositol, that were not observed in fetal lung fibroblasts. Treatment of differentiated epithelial cells with secretagogues stimulated the secretion of functional surfactant-containing surfactant proteins B and C (SP-B and SP-C). Secreted material was further enriched in this same set of phospholipid species but was characterized by increased contents of short-chain monounsaturated and disaturated species other than dipalmitoyl PC (PC16:0/16:0), principally palmitoylmyristoyl PC (PC16:0/14:0) and palmitoylpalmitoleoyl PC (PC16:0/16:1). Mixtures of these PC molecular species, phosphatidylglycerol, and SP-B and SP-C were functionally active and rapidly generated low surface tension on compression in a pulsating bubble surfactometer. These results suggest that hormonally differentiated human fetal type II cells do not select the molecular composition of surfactant phospholipid on the basis of saturation but, more likely, on the basis of acyl chain length.     

Variations in lethal toxin and cholesterol-dependent cytolysin production correspond to differences in cytotoxicity among strains of Clostridium sordellii

Clostridium sordellii is an emerging human pathogen and frequent contaminant of cadaver-derived tissue transplant material. Herein, we provide data suggesting the potential for severe C. sordellii-associated disease may be dictated by whether the specific strain produces lethal toxin (TcsL) or sordellilysin (SDL), a cholesterol-dependent cytolysin. The virulence factor profiles of 14 C. sordellii isolates were determined, and culture supernatant from six of the isolates was found to be cytotoxic to mammalian cells; yet, only one of these strains conferred cytotoxicity via production of TcsL. Cytotoxicity of TcsL- strains correlated with the production of sordellilysin, which was also recognized by an antiperfringolysin O antibody. However, supernatant from TcsL+, SDL- strains demonstrated a lower LD50 relative to TcsL-, SDL+ strains, suggesting the potential for severe C. sordellii-associated disease may be determined by the particular strain colonizing the host.     

HTII-280, a Biomarker Specific to the Apical Plasma Membrane of Human Lung Alveolar Type II Cells

The pulmonary alveolar epithelium is composed of two morphologically distinct cell types, type I (TI) and type II (TII) cells. Alveolar TII cells synthesize, secrete, and recycle surfactant components; contain ion transporters; and secrete immune effector molecules. In response to alveolar injury, TII cells have the capacity to act as progenitor cells, proliferating and transdifferentiating into TI cells. Although various proteins are associated with TII cells, a plasma membrane marker specific to human TII cells that would be useful for identification in tissue and for isolating this cell type has not been described previously. We devised a strategy to produce a monoclonal antibody (MAb) specific to the apical surface of human TII cells and developed an MAb that appears to be specific for human TII cells. The antibody recognizes a 280- to 300-kDa protein, HTII-280, which has the biochemical characteristics of an integral membrane protein. HTII-280 is detected by week 11 of gestation and is developmentally regulated. HTII-280 is useful for isolating human TII cells with purities and viabilities >95%. HTII-280 is likely to be a useful morphological and biochemical marker of human TII cells that may help to advance our understanding of various lung pathological conditions, including the origin and development of various lung tumors. (J Histochem Cytochem 58:891–901, 2010)     

Multivalent nanobodies targeting death receptor 5 elicit superior tumor cell killing through efficient caspase induction

Multiple therapeutic agonists of death receptor 5 (DR5) have been developed and are under clinical evaluation. Although these agonists demonstrate significant anti-tumor activity in preclinical models, the clinical efficacy in human cancer patients has been notably disappointing. One possible explanation might be that the current classes of therapeutic molecules are not sufficiently potent to elicit significant response in patients, particularly for dimeric antibody agonists that require secondary cross-linking via Fcγ receptors expressed on immune cells to achieve optimal clustering of DR5. To overcome this limitation, a novel multivalent Nanobody approach was taken with the goal of generating a significantly more potent DR5 agonist. In the present study, we show that trivalent DR5 targeting Nanobodies mimic the activity of natural ligand, and furthermore, increasing the valency of domains to tetramer and pentamer markedly increased potency of cell killing on tumor cells, with pentamers being more potent than tetramers in vitro. Increased potency was attributed to faster kinetics of death-inducing signaling complex assembly and caspase-8 and caspase-3 activation. In vivo, multivalent Nanobody molecules elicited superior anti-tumor activity compared to a conventional DR5 agonist antibody, including the ability to induce tumor regression in an insensitive patient-derived primary pancreatic tumor model. Furthermore, complete responses to Nanobody treatment were obtained in up to 50% of patient-derived primary pancreatic and colon tumor models, suggesting that multivalent DR5 Nanobodies may represent a significant new therapeutic modality for targeting death receptor signaling.     

A rapid method for the analysis of N tau-methylhistidine in human urine

A novel, automated method is described for the determination of N^τ-methylhistidine in human urine. The method uses a modification (H. Nakamura and J. J. Pisano (1976) Arch. Biochem. Biophys.177, 334–335) of the reaction of fluorescamine with amines, which renders it specific for certain imidazoles. Interference due to histidine and histamine is selectively removed by prior reaction with aldehydes. The fluorescence yield for N^τ-methylhistamine is 280, 440, 50, and 1.7, respectively. The concentrations of N^τ-methylhistidine in human urine as determined by this technique correlate well (r = 0.99) with those determined by ion-exchange chromatography. Furthermore, the technique is rapid (6–7 samples/h net throughout), is reproducible (coefficient of variation 1.8%), requires no prior treatment of the sample, and is implemented with widely available equipment.     

Genomic concatemerization/deletion in rotaviruses: a new mechanism for generating rapid genetic change of potential epidemiological importance.

Three variants of group A rotavirus with large changes in their gene 5 structures have been analyzed at the molecular level. The first of these, P9 delta 5, was obtained during plaque purification undertaken as part of the biological cloning of a field isolate of virus. The gene 5 homolog in this isolate migrated just ahead of the normal segment 6 RNA, giving an estimated size of 1,300 bp. Molecular cloning and sequencing of this homolog revealed it to have a single 308-bp deletion in the center of the normal gene 5 sequence extending between nucleotides 460 and 768 of the normal gene sequence. This deletion caused a frameshift in the gene such that a stop codon was encountered 8 amino acids downstream of the deletion point, giving a predicted size for the protein product of this gene of 150 amino acids compared with the 490 amino acids of its normal-size counterpart. Attempts to detect this shortened protein in virus-infected cells were not successful, indicating that it was much less stable than the full-length protein and/or had suffered a large change in its antigenicity. The second two variants, brvA and brvE, were generated in an earlier study following the high-multiplicity passage of the UKtc strain of bovine rotavirus. Polyacrylamide gel electrophoresis analysis of these nondefective variants showed that brvA had a gene 5 homolog approximately equal in size to the normal RNA segment 2 (approximately 2,700 bp) and that brvE had a size of approximately 2,300 bp. Both variants showed changes in their gene 5 protein products, with brvA mimicking P9 delta 5 in failing to produce a detectable product whereas brvE produced a new virus-specific protein approximately 80 kDa in size. Full-length cDNA clones of the brvE gene 5 homolog were isolated, and analysis of their structure revealed a head-to-tail concatemerization of the normal gene 5 sequence with the first copy of the concatemer covering nucleotides 1 to 808 and the second covering nucleotides 92 to 1579, giving a total length of 2,296 bp. Sequencing across the junction region of the two copies of the gene showed that they were joined in frame to give a predicted combined open reading frame of 728 amino acids with the amino-terminal region consisting of amino acids 1 to 258 fused at the carboxy terminus to amino acids 21 to 490.(ABSTRACT TRUNCATED AT 400 WORDS)     

Home range,activity and sociality of a top predator,the dingo: a test of the Resource Dispersion Hypothesis

The idea that groups of individuals may develop around resource patches led to the formulation of the Resource Dispersion Hypothesis (RDH). We tested the predictions of the RDH, within a quasi‐experimental framework, using Australia’s largest terrestrial predator, the dingo Canis lupus dingo. Average dingo group sizes were higher in areas with abundant focal food sources around two mine sites compared with those in more distant areas. This supports the notion that resource richness favours larger group size, consistent with the RDH. Irrespective of season or sex, average home range estimates and daily activity for dingoes around the mine sites were significantly less than for dingoes that lived well away. Assuming that a territory is the defended part of the home range and that territory size is correlated with home range size, consistent with the RDH, the spatial dispersion of food patches therefore determined territory size for dingoes in our study. However, although sample size was small, some dingoes that accessed the supplementary food resource at the mines also spent a large proportion of their time away, suggesting a breakdown of territorial defence around the focal food resource. This, in combination with the large variation in home range size among dingoes that accessed the same supplementary food resource, limits the predictive capabilities of the RDH for this species. We hypothesize that constraints on exclusive home range occupancy will arise if a surfeit of food resources (in excess of requirements for homeostasis) is available in a small area, and that this will have further effects on access to mates and social structure. We present a conceptual model of facultative territorial defence where focal resources are available to demonstrate our findings.     

Production and utilization of acetate in mammals

1. In an attempt to define the importance of acetate as a metabolic precursor, the activities of acetyl-CoA synthetase (EC 6.2.1.1) and acetyl-CoA hydrolase (Ec 3.1.2.1) were assayed in tissues from rats and sheep. In addition, the concentrations of acetate in blood and liver were measured, as well as the rates of acetate production by tissue slices and mitochondrial fractions of these tissues. 2. Acetyl-CoA synthetase occurs at high activities in heart and kidney cortex of both species as well as in rat liver and the sheep masseter muscle. The enzyme is mostly in the cytosol fraction of liver, whereas it is associated with the mitochondrial fraction in heart tissue. Both mitochondrial and cytosol activities have a K(m) for acetate of 0.3mm. Acetyl-CoA synthetase activity in liver was not altered by changes in diet, age or alloxan-diabetes. 3. Acetyl-CoA hydrolase is widely distributed in rat and sheep tissues, the highest activity being found in liver. Essentially all of the activity in liver and heart is localized in the mitochondrial fraction. Hepatic acetyl-CoA hydrolase activity is increased by starvation in rats and sheep and during the suckling period in young rats. 4. The concentrations of acetate in blood are decreased by starvation and increased by alloxan-diabetes in both species. The uptake of acetate by the sheep hind limb is proportional to the arterial concentration of acetate, except in alloxan-treated animals, where uptake is impaired. 5. Acetate is produced by liver and heart slices and also by heart mitochondrial fractions that are incubated with either pyruvate or palmitoyl-(-)-carnitine. Liver mitochondrial fractions do not form acetate from either substrate but instead convert acetate into acetoacetate. 6. We propose that acetate in the blood of rats or starved sheep is derived from the hydrolysis of acetyl-CoA. Release of acetate from tissues would occur under conditions when the function of the tricarboxylic acid cycle is restricted, so that the circulating acetate serves to redistribute oxidizable substrate throughout the body. This function is analogous to that served by ketone bodies.