Mass spectroscopic analysis of phosphatidylinositol synthesis using 6-deuteriated-myo-inositol: comparison of the molecular specificities and acyl remodelling mechanisms in mouse tissues and cultured cells

2004 ◽  
Vol 32 (6) ◽  
pp. 1057-1059 ◽  
Author(s):  
A.D. Postle ◽  
H. Dombrowsky ◽  
H. Clarke ◽  
C.J. Pynn ◽  
G. Koster ◽  
...  
Keyword(s):  
Molecular Species ◽  
Cultured Cells ◽  
Ionization Mass ◽  
Membrane Composition ◽  
Mouse Tissues ◽  
Wide Range ◽  
Kinetics Of ◽  
Hydrolysis Of

Mammalian cell PtdIns (phosphatidylinositol) in vivo is enriched in the sn-1-stearoyl 2-arachidonoyl species, the physiological precursor of phosphatidylinositol 4,5-bisphosphate. Mechanisms regulating this specificity are unclear but are typically lost for cells in culture. We used ESI-MS (tandem electrospray ionization-mass spectrometry) to determine the molecular species of PtdIns synthesized by mouse tissues in vivo compared with cultured cells in vitro. After incorporation of deuteriated myo-d6-inositol over 3 h, endogenous and newly synthesized PtdIns and lysoPtdIns species were quantified from precursor scans of m/z 241− and m/z 247− respectively. PtdIns was synthesized as a wide range of species irrespective of the final membrane composition. Analyses of isotope enrichments argued against acyl remodelling as the major regulatory mechanism: composition of the lysoPtdIns pool under all conditions reflected that of either endogenous or newly synthesized PtdIns and was always at equilibrium. The kinetics of PtdIns synthesis, together with the prolonged time scale required for achieving final equilibrium compositions suggest that selective transport between membranes and/or hydrolysis of selected molecular species are the most probable mechanisms regulating compositions of PtdIns and, ultimately, phosphatidylinositol 4,5-bisphosphate.

Lipid characterization of in vitro-produced bovine embryos with distinct kinetics of development

Zygote ◽  
2019 ◽  
Vol 27 (6) ◽  
pp. 413-422 ◽  
Author(s):  
Kelly Annes ◽  
Mateus José Sudano ◽  
Katia Roberta A. Belaz ◽  
Alessandra Tata ◽  
Vanessa Gonçalves Santos ◽  
...  
Keyword(s):  
Lipid Profile ◽  
Lipid Composition ◽  
Cell Signalling ◽  
Lipid Profiles ◽  
Structural Function ◽  
Ionization Mass ◽  
Bovine Embryos ◽  
Kinetics Of

SummaryHuman embryo studies have proposed the use of additional morphological evaluations related to the moment of the first cell divisions as relevant to embryo viability. Nevertheless, there are still not enough data available related to morphokinetic analysis and its relationship with lipid composition in embryos. Therefore, the aim of this study was to address the lipid profile of bovine embryos with different developmental kinetics: fast (four or more cells) and slow (two or three cells) at 40 h post-insemination (hpi), at three time points of in vitro culture (40, 112 and 186 hpi) and compare these to profiles of in vivo embryos. The lipid profiles of embryos were analyzed by matrix-assisted laser desorption ionization mass spectrometry, which mainly detected pools of membrane lipids such as phosphatidylcholine and sphingomyelin. In addition to their structural function, these lipid classes have an important role in cell signalling, particularly regarding events such as stress and pregnancy. Different patterns of lipids in the fast and slow groups were revealed in all the analyzed stages. Also, differences between in vitro embryos were more pronounced at 112 hpi, a critical moment due to embryonic genome activation. At the blastocyst stage, in vitro-produced embryos, despite the kinetics, had a closer lipid profile when compared with in vivo blastocysts. In conclusion, the kinetics of development had a greater effect on the membrane lipid profiles throughout the embryo culture, especially at the 8–16-cell stage. The in vitro environment affects lipid composition and may compromise cell signalling and function in blastocysts.

scholarly journals Characterization of the small molecule ARC39, a direct and specific inhibitor of acid sphingomyelinase in vitro

2020 ◽  
Vol 61 (6) ◽  
pp. 896-910 ◽  
Author(s):  
Eyad Naser ◽  
Stephanie Kadow ◽  
Fabian Schumacher ◽  
Zainelabdeen H. Mohamed ◽  
Christian Kappe ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Specific Inhibitor ◽  
Acid Sphingomyelinase ◽  
Intact Cells ◽  
Protein Levels ◽  
High Doses ◽  
Hydrolysis Of

Inhibition of acid sphingomyelinase (ASM), a lysosomal enzyme that catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine, may serve as an investigational tool or a therapeutic intervention to control many diseases. Specific ASM inhibitors are currently not sufficiently characterized. Here, we found that 1-aminodecylidene bis-phosphonic acid (ARC39) specifically and efficiently (>90%) inhibits both lysosomal and secretory ASM in vitro. Results from investigating sphingomyelin phosphodiesterase 1 (SMPD1/Smpd1) mRNA and ASM protein levels suggested that ARC39 directly inhibits ASM’s catalytic activity in cultured cells, a mechanism that differs from that of functional inhibitors of ASM. We further provide evidence that ARC39 dose- and time-dependently inhibits lysosomal ASM in intact cells, and we show that ARC39 also reduces platelet- and ASM-promoted adhesion of tumor cells. The observed toxicity of ARC39 is low at concentrations relevant for ASM inhibition in vitro, and it does not strongly alter the lysosomal compartment or induce phospholipidosis in vitro. When applied intraperitoneally in vivo, even subtoxic high doses administered short-term induced sphingomyelin accumulation only locally in the peritoneal lavage without significant accumulation in plasma, liver, spleen, or brain. These findings require further investigation with other possible chemical modifications. In conclusion, our results indicate that ARC39 potently and selectively inhibits ASM in vitro and highlight the need for developing compounds that can reach tissue concentrations sufficient for ASM inhibition in vivo.

scholarly journals Sequence, infectivity and replication kinetics of SARS-CoV-2 isolated from COVID-19 patients in Canada

2020 ◽  
Author(s):  
Arinjay Banerjee ◽  
Jalees A. Nasir ◽  
Patrick Budylowski ◽  
Lily Yip ◽  
Patryk Aftanas ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Productive Infection ◽  
Nucleotide Polymorphisms ◽  
Peripheral Blood Mononuclear ◽  
Single Nucleotide ◽  
Infection Models ◽  
Wide Range ◽  
Kinetics Of

ABSTRACTSARS-CoV-2 emerged in December 2019 in Wuhan, China and has since infected over 1.5 million people, of which over 107,000 have died. As SARS-CoV-2 spreads across the planet, speculations remain about the range of human cells that can be infected by SARS-CoV-2. In this study, we report the isolation of SARS-CoV-2 from two COVID-19 patients in Toronto, Canada. We determined the genomic sequences of the two isolates and identified single nucleotide changes in representative populations of our virus stocks. More importantly, we tested a wide range of human immune cells for productive infection with SARS-CoV-2. Here we confirm that human primary peripheral blood mononuclear cells (PBMCs) are not permissive to SARS-CoV-2. As SARS-CoV-2 continues to spread globally, it is essential to monitor small nucleotide polymorphisms in the virus and to continue to isolate circulating viruses to determine cell susceptibility and pathogenicity using in vitro and in vivo infection models.

scholarly journals SIMILARITY OF THE KINETICS OF INVERTASE ACTION IN VIVO AND IN VITRO

1932 ◽  
Vol 15 (5) ◽  
pp. 491-495 ◽  
Author(s):  
J. M. Nelson ◽  
Elizabeth T. Palmer ◽  
B. G. Wilkes
Keyword(s):  
Yeast Cells ◽  
Enzymic Hydrolysis ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Live Yeast

1. A method is given whereby the course of hydrolysis of sucrose by live yeast cells may be followed with precision equal to that found when invertase solutions prepared from autolyzed yeast are used to cause inversion. 2. The practical value of the equation of Nelson and Hitchcock as a means of following the course of enzymic hydrolysis of sucrose is hereby extended. 3. The inversion of sucrose by live yeast cells and by extracted invertase has been quantitatively compared. 4. The course of hydrolysis of sucrose by the invertase of Fleischmann's yeast has been found to be identical in vivo and in vitro.

scholarly journals Differential regulation of the retinoblastoma family of proteins during cell proliferation and differentiation

1998 ◽  
Vol 333 (3) ◽  
pp. 645-654 ◽  
Author(s):  
Judit GARRIGA ◽  
Ana LIMÓN ◽  
Xavier MAYOL ◽  
Sushil G. RANE ◽  
Jeffrey H. ALBRECHT ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Protein Expression ◽  
Cultured Cells ◽  
Protein Levels ◽  
Pocket Protein ◽  
Cell Proliferation And Differentiation ◽  
Mouse Tissues ◽  
Proliferation And Differentiation

In the present study we have analysed the regulation of pocket protein expression and post-transcriptional modifications on cell proliferation and differentiation, both in vivo and in vitro. There are marked changes in pocket protein levels during these transitions, the most striking differences being observed between p130 and p107. The mechanisms responsible for regulating pocket protein levels seem to be dependent on both cell type and pocket protein, in addition to their dependence on the cell growth status. Changes in retinoblastoma protein and p107 levels are independent of their state of phosphorylation. However, whereas p130 phosphorylation to forms characteristic of quiescent/differentiated cells results in the accumulation of p130 protein, phosphorylation of p130 to one or more forms characteristic of cycling cells is accompanied by down-regulation of its protein levels. We also show here that the phosphorylation status and protein levels of p130 and p107 are regulated in vivo as in cultured cells. In vivo, changes in p130 forms are correlated with changes in E2F complexes. Moreover, the modulation of p130 and p107 status during cell differentiation in vitro is consistent with the patterns of protein expression and phosphorylation status found in mouse tissues. Thus in addition to the direct disruption of pocket protein/E2F complexes induced by cyclin/cyclin-dependent kinase, the results we report here indicate that the differential modulation of pocket protein levels constitutes a major mechanism that regulates the pool of each pocket protein that is accessible to E2F and/or other transcription factors.

scholarly journals ATP13A2 facilitates HDAC6 recruitment to lysosome to promote autophagosome–lysosome fusion

2018 ◽  
Vol 218 (1) ◽  
pp. 267-284 ◽  
Author(s):  
Ruoxi Wang ◽  
Jieqiong Tan ◽  
Tingting Chen ◽  
Hailong Han ◽  
Runyi Tian ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Cultured Cells ◽  
Cellular Functions ◽  
Autophagy Flux ◽  
Mouse Tissues ◽  
In Vitro Reconstitution ◽  
Recessive Form ◽  
Atypical Parkinson’S Disease

Mutations in ATP13A2 cause Kufor-Rakeb syndrome, an autosomal recessive form of juvenile-onset atypical Parkinson’s disease (PD). Recent work tied ATP13A2 to autophagy and other cellular features of neurodegeneration, but how ATP13A2 governs numerous cellular functions in PD pathogenesis is not understood. In this study, the ATP13A2-deficient mouse developed into aging-dependent phenotypes resembling those of autophagy impairment. ATP13A2 deficiency impaired autophagosome–lysosome fusion in cultured cells and in in vitro reconstitution assays. In ATP13A2-deficient cells or Drosophila melanogaster or mouse tissues, lysosomal localization and activity of HDAC6 were reduced, with increased acetylation of tubulin and cortactin. Wild-type HDAC6, but not a deacetylase-inactive mutant, restored autophagosome–lysosome fusion, antagonized cortactin hyperacetylation, and promoted lysosomal localization of cortactin in ATP13A2-deficient cells. Mechanistically, ATP13A2 facilitated recruitment of HDAC6 and cortactin to lysosomes. Cortactin overexpression in cultured cells reversed ATP13A2 deficiency–associated impairment of autophagosome–lysosome fusion. PD-causing ATP13A2 mutants failed to rescue autophagosome–lysosome fusion or to promote degradation of protein aggregates and damaged mitochondria. These results suggest that ATP13A2 recruits HDAC6 to lysosomes to deacetylate cortactin and promotes autophagosome–lysosome fusion and autophagy. This study identifies ATP13A2 as an essential molecular component for normal autophagy flux in vivo and implies potential treatments targeting HDAC6-mediated autophagy for PD.

Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

1982 ◽  
Vol 40 ◽  
pp. 210-211
Author(s):  
M.J. Murphy ◽  
R.R. Price ◽  
J.C. Sloman
Keyword(s):  
Cultured Cells ◽  
Human Tumor ◽  
Cell Type ◽  
Tumor Mass ◽  
Initial Cell ◽  
Cloning Assay ◽  
Human Tumor Cloning ◽  
The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

Preparation of cultured astrocytes for x-ray microanalysis

1989 ◽  
Vol 47 ◽  
pp. 988-989
Author(s):  
N.K.R. Smith ◽  
K.E. Hunter ◽  
P. Mobley ◽  
L.P. Felpel
Keyword(s):  
Cultured Cells ◽  
Elemental Content ◽  
Elemental Distribution ◽  
Sprague Dawley ◽  
X Ray ◽  
Cultured Astrocytes ◽  
Freeze Dried ◽  
Ultimate Objective

Electron probe energy dispersive x-ray microanalysis (XRMA) offers a powerful tool for the determination of intracellular elemental content of biological tissue. However, preparation of the tissue specimen , particularly excitable central nervous system (CNS) tissue , for XRMA is rather difficult, as dissection of a sample from the intact organism frequently results in artefacts in elemental distribution. To circumvent the problems inherent in the in vivo preparation, we turned to an in vitro preparation of astrocytes grown in tissue culture. However, preparations of in vitro samples offer a new and unique set of problems. Generally, cultured cells, growing in monolayer, must be harvested by either mechanical or enzymatic procedures, resulting in variable degrees of damage to the cells and compromised intracel1ular elemental distribution. The ultimate objective is to process and analyze unperturbed cells. With the objective of sparing others from some of the same efforts, we are reporting the considerable difficulties we have encountered in attempting to prepare astrocytes for XRMA.Tissue cultures of astrocytes from newborn C57 mice or Sprague Dawley rats were prepared and cultured by standard techniques, usually in T25 flasks, except as noted differently on Cytodex beads or on gelatin. After different preparative procedures, all samples were frozen on brass pins in liquid propane, stored in liquid nitrogen, cryosectioned (0.1 μm), freeze dried, and microanalyzed as previously reported.

Effect of pH and inhibitors on beta-amyloid fibril assembly

1996 ◽  
Vol 54 ◽  
pp. 752-753
Author(s):  
Beverly E. Maleeff ◽  
Timothy K. Hart ◽  
Stephen J. Wood ◽  
Ronald Wetzel
Keyword(s):  
Amyloid Fibril ◽  
Peptide Fragment ◽  
Hydrophobic Peptides ◽  
Amyloid Fibril Formation ◽  
Effects Of Ph ◽  
Severity Of The Disease ◽  
Kinetics Of ◽  
The Brain

Alzheimer's disease is characterized post-mortem in part by abnormal extracellular neuritic plaques found in brain tissue. There appears to be a correlation between the severity of Alzheimer's dementia in vivo and the number of plaques found in particular areas of the brain. These plaques are known to be the deposition sites of fibrils of the protein β-amyloid. It is thought that if the assembly of these plaques could be inhibited, the severity of the disease would be decreased. The peptide fragment Aβ, a precursor of the p-amyloid protein, has a 40 amino acid sequence, and has been shown to be toxic to neuronal cells in culture after an aging process of several days. This toxicity corresponds to the kinetics of in vitro amyloid fibril formation. In this study, we report the biochemical and ultrastructural effects of pH and the inhibitory agent hexadecyl-N-methylpiperidinium (HMP) bromide, one of a class of ionic micellar detergents known to be capable of solubilizing hydrophobic peptides, on the in vitro assembly of the peptide fragment Aβ.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

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