1,721,113 research outputs found
Alan Hunt
No full textAlan R Hunt, Alan Hunt, Alan, Hunthttps://digitalcommons.montclair.edu/sust-seminar-headshots/1096/thumbnail.jp
CTP: Choline Phosphate Cytidylyltransferase in human lung
No full textHuman lung cytidylyltransferase was found associated with both 'soluble' (S100) and membrane-rich particulate (P100) fractions of Tris-buffered saline homogenates. S100 enzyme activities in 15 - 16 week fetal and adult human lungs represented a constant proportion of overall recovery, (66.8 4.8% vs 66.1 7.5%, means standard error). A lack of support for the regulatory translocation of human lung cytidylyltransferase at these extremes of development was unable to rule out a transient change in distribution near term. Rat lung P100 enzyme increased from 31% to 40% between d18 gestation and term, d22, but a concomitant increase in total.S100 cytidylyltransferase, measured in the presence of the lipid activator PG, questioned the physiological significance of the apparent translocation.Cytidylyltransferase from human and rat lung S100 were resolved into a high molecular weight H form (> 106 daltons) and a lower molecular weight L form (~200,000 daltons). Incubation of S100 at 37oC for 2 hours yielded insoluble, protein-rich aggregates which were strongly associated with rat H form cytidylyltransferase, while less strongly with the human H form. The principal 43,000 dalton, protein in these aggregates was identified as a cytoplasmic actin on the basis of its properties, amino acid composition and western blot analysis. The association of H form cytidylyltransferase with cytoskeletal F-actin containing fractions in vitro was disrupted by the detergent CHAPS, which was also able to release a portion of P100 enzyme. Separation of human S100 H and L form enzyme, by gel filtration or ultracentrifugation, revealed the presence of latent cytidylyltransferase, often as high as 3 fold, which questioned activity determinations in fresh S100. Within the framework of an emerging concept of a highly ordered aqueous cytoplasm, the incorporation of these results suggested that a portion of human lung cytidylyltransferase might by cytoskeletally bound in vivo, as has been described for many enzymes or enzyme systems.The use of conventional purification techniques, including affinity chromatography, with a view to testing these ideas in defined systems, met with little success. Low yields or highly unstable enzyme characterised many individual steps, especially where cytidylyltransferase was separated from F-actin enriched fractions. A number of triazine dyes screened as pseudoaffinity ligands revealed a rapid inhibition with Procion Green H-4G and a partial protection with MgCTP. Sepharose CL4B-immobilised Green H-4G bound cytidylyltransferase, but MgCTP was unable to effect elution. Increasing ionic strength eluted some activity but also inhibited enzyme irreversibly, while CHAPS at 1% released a maximum of only 18% bound enzyme and SDS PAGE revealed a relatively non-specific binding. The use of dye-affinity matrix offered the potential of a useful purification step with partially purified enzyme if suitable elution conditions could be devised. <br/
Dynamic lipidomic insights into phosphatidylcholine synthesis from organelle to organism
No full textRecent technical improvement and technological innovation in small molecule mass spectrometry have provided powerful tools for the intensive metabolomic biochemical investigations that will necessarily characterise the "post-genomic" era of biomedical research. For membrane phospholipids, use of tandem electrospray ionisation mass spectrometry (ESI-MS/MS) exploiting precursor scanning of class-specific diagnostic fragments, can provide detailed quantitative profiles at the level of individual molecular species for many hundreds of unique lipids. Such "snapshot" measurements provide little information concerning metabolic flux. However, recent use of metabolic labelling with stable isotope derivatives of phospholipid headgroups combined with precursor scans of unlabelled and labelled fragments have yielded considerable insight into phosphatidylcholine metabolism in vivo. Here, we briefly review some of the recent work on pathways of phosphatidylcholine metabolism ranging from studies at subcellular organelle level through to whole organism. The sensitivity, specificity and suitability of this powerful methodological approach to numerous questions of phospholipid metabolism place ESI-MS/MS at the very heart of dynamic lipidomics in the foreseeable future
Mass spectrometry determination of endonuclear phospholipid composition and dynamics
No full textMammalian cell lipid analyses using tandem electrospray ionization mass spectrometry, in conjunction with stable isotope labeling, permit unparalleled access to membrane phospholipid molecular species compositions and turnover. Lipidomic data from isolable compartments of lipid second messenger generation, such as membrane-free nuclei, can provide dynamic insights into the topology of phospholipid turnover. For example, ESI-MS/MS precursor scans of characteristic phosphocholine m/z 184+ fragments reveal a highly saturated endonuclear phosphatidylcholine pool with homeostatic maintenance properties. A spatially distinct CDPcholine pathway yields, within minutes of choline-d9 labeling, unsaturated endonuclear phosphatidylcholines progressively remodeled to more saturated species evidenced by tracking the deuteriated headgroup through precursor scans of phosphocholine-d9 (m/z 193+ fragment). Among the other endonuclear phospholipids, diacyl phosphatidylethanolamines (neutral loss of m/z 141+) are also highly saturated compared with those of whole cell whereas, phophatidylinositols (precursor scans of m/z 241? fragment) are essentially identical in nuclei and whole cells. Moreover, the pattern of myo-inositol-d6 acquisition into endonuclear phosphatidylinositol (precursor scans of m/z 247? fragment) is inconsistent with compartment-specific synthesis. Endonuclear sphingomyelins (seen in precursor scans of m/z 184+ and confirmed from precursor scans of m/z 168? fragments) are enriched but similar in composition to whole cell species whereas endonuclear phosphatidylserines (neutral loss of m/z 87?) are more saturated than their whole cell counterparts. The focus of described methodologies emphasize their value in probing the compositions and dynamics of endonuclear phospholipids, but in principle may be extended to exploration of other isolable compartments including ER or plasma membranes
Phosphatidylcholine biosynthesis inside the nucleus: is it involved in regulating cell proliferation?
No full textDynamic lipidomics with stable isotope labeling
No full textIncorporation of stable isotope labelled precursors enables estimation of the kinetics of lipid synthesis and turnover (dynamic lipidomics) in the clinical as well the experimental setting. Recent advances in tandem mass spectrometry extend the analytical possibilities from measurements of isotope enrichments to determinations of intact substrates. Incorporations of deuteriated choline, ethanolamine and inositol can be determined by precursor and neutral loss scans of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, respectively. This experimental approach provides information on the kinetics of individual phospholipid molecular species and has considerable potential to probe diseases of lipid metabolism in vivo
Regulation of lung surfactant phospholipid synthesis and metabolism
No full textThe alveolar type II epithelial (ATII) cell is highly specialised for the synthesis and storage, in intracellular lamellar bodies, of phospholipid destined for secretion as pulmonary surfactant into the alveolus. Regulation of the enzymology of surfactant phospholipid synthesis and metabolism has been extensively characterised at both molecular and functional levels, but understanding of surfactant phospholipid metabolism in vivo in either healthy or, especially, diseased lungs is still relatively poorly understood. This review will integrate recent advances in the enzymology of surfactant phospholipid metabolism with metabolic studies in vivo in both experimental animals and human subjects. It will highlight developments in the application of stable isotope-labelled precursor substrates and mass spectrometry to probe lung phospholipid metabolism in terms of individual molecular lipid species and identify areas where a more comprehensive metabolic model would have considerable potential for direct application to disease states
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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