A Bioassay Using a Pentadecanal Derivative to Measure S1P Lyase Activity

Sphingosine-1-phosphate (S1P) is a unique lipid ligand binding to S1P receptors to transduce various cell survival or proliferation signals via small G proteins. S1P lyase (S1PL) is the specific enzyme that degrades S1P to phosphoethanolamine and (2E)-hexadecenal and therefore regulates S1P levels. S1PL also degrades dihydrosphingosine-1-phosphate (Sa1P), with a higher affinity to produce hexadecanal. Here, we developed a newly designed assay using a C17-Sa1P substrate that degrades into pentadecanal and phosphoethanolamine. For higher sensitivity in pentadecanal analysis, we developed a quantitative protocol as well as a 5,5-dimethyl cyclohexanedione (5,5-dimethyl CHD) derivatization method. The derivatization conditions were optimized for the reaction time, temperature, and concentrations of the 5,5-dimethyl CHD reagent, acetic acid, and ammonium acetate. The S1PL reaction in the cell lysate after spiking 20 µM of C17-Sa1P for 20 min was linear to the total protein concentrations of 50 µg. The S1PL levels (4 pmol/mg/min) were readily detected in this HPLC with fluorescence detection (λex = 366 nm, λem = 455 nm). The S1PL-catalyzed reaction was linear over 30 min and yielded a Km value of 2.68 μM for C17-Sa1P. This new method was validated to measure the S1PL activity of mouse embryonal carcinoma cell lines of the standard cell (F9-0), S1PL knockdown cells (F9-2), and S1PL-overexpressed cells (F9-4). Furthermore, we treated F9-4 cells with different S1PL inhibitors such as FTY720, 4-deoxypyridoxine (DOP), and the deletion of pyridoxal-5-phosphate (P5P), an essential cofactor for S1PL activity, and observed a significant decrease in pentadecanal relative to the untreated cells. In conclusion, we developed a highly sensitive S1PL assay using a C17-Sa1P substrate for pentadecanal quantification for application in the characterization of S1PL activity in vitro.

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In Vitro Identification and Characterization of CD133pos Cancer Stem-Like Cells in Anaplastic Thyroid Carcinoma Cell Lines

PLoS ONE ◽

10.1371/journal.pone.0003544 ◽

2008 ◽

Vol 3 (10) ◽

pp. e3544 ◽

Cited By ~ 69

Author(s):

Giovanni Zito ◽

Pierina Richiusa ◽

Alessandra Bommarito ◽

Elvira Carissimi ◽

Leonardo Russo ◽

...

Keyword(s):

Thyroid Carcinoma ◽

Cell Lines ◽

Carcinoma Cell ◽

Anaplastic Thyroid Carcinoma ◽

Carcinoma Cell Lines ◽

Identification And Characterization ◽

Thyroid Carcinoma Cell

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SipA, SopA, SopB, SopD, and SopE2 Contribute to Salmonella enterica Serotype Typhimurium Invasion of Epithelial Cells

Infection and Immunity ◽

10.1128/iai.73.1.146-154.2005 ◽

2005 ◽

Vol 73 (1) ◽

pp. 146-154 ◽

Cited By ~ 169

Author(s):

Manuela Raffatellu ◽

R. Paul Wilson ◽

Daniela Chessa ◽

Helene Andrews-Polymenis ◽

Quynh T. Tran ◽

...

Keyword(s):

Epithelial Cells ◽

Type Iii Secretion ◽

Salmonella Enterica ◽

T84 Cells ◽

Carcinoma Cell Lines ◽

Effector Genes ◽

Ht 29 ◽

Invasion Assays

ABSTRACT The centisome 63 type III secretion system (T3SS-1) encoded by Salmonella pathogenicity island 1 (SPI1) mediates invasion of epithelial cells by Salmonella enterica serotype Typhimurium. Characterization of mutants lacking individual genes has revealed that T3SS-1 secreted proteins (effectors) SopE2 and SopB are required for invasion while the SipA protein accelerates entry into cells. Here we have revisited the question of which T3SS-1 effectors contribute to the invasion of epithelial cells by complementing a strain lacking all of the effector genes that are required to cause diarrhea in a calf (a sipA sopABDE2 mutant). Introduction of either the cloned sipA, the cloned sopB, or the cloned sopE2 gene increased the invasiveness of the sipA sopABDE2 mutant for nonpolarized HT-29 cells. However, a contribution of sopA or sopD to invasion was not apparent when invasion assays were performed with the nonpolarized colon carcinoma cell lines T84 and HT-29. In contrast, introduction of either the sopA, the sopB, the sopD, or the sopE2 gene increased the invasiveness of the sipA sopABDE2 mutant for polarized T84 cells. Furthermore, introduction of a plasmid carrying sipA and sopB increased the invasiveness of the sipA sopABDE2 mutant for polarized T84 cells significantly compared to the introduction of plasmids carrying only sipA or sopB. We conclude that SipA, SopA, SopB, SopD, and SopE2 contribute to S. enterica serotype Typhimurium invasion of epithelial cells in vitro.

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Molecular Characterization of Extrachromosomal Circular DNAs from an Embryonal Carcinoma Cell Line Induced to Differentiate into Neuron-like Cells in vitro.

Cell Structure and Function ◽

10.1247/csf.18.261 ◽

1993 ◽

Vol 18 (4) ◽

pp. 261-266 ◽

Cited By ~ 4

Author(s):

Takuji Iwasato ◽

Takeyuki Shimizu ◽

Yasuyoshi Kanari ◽

Hideo Yamagishi

Keyword(s):

Cell Line ◽

Molecular Characterization ◽

Embryonal Carcinoma ◽

Carcinoma Cell ◽

Carcinoma Cell Line ◽

Embryonal Carcinoma Cell ◽

Embryonal Carcinoma Cell Line ◽

Circular Dnas

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Higher sensitivity to patupilone versus paclitaxel chemotherapy in primary uterine serous papillary carcinoma cell lines with high versus low HER-2/neu expression in vitro

Gynecologic Oncology ◽

10.1016/j.ygyno.2010.06.024 ◽

2010 ◽

Vol 119 (1) ◽

pp. 140-145 ◽

Cited By ~ 12

Author(s):

Daniel Paik ◽

Emiliano Cocco ◽

Stefania Bellone ◽

Francesca Casagrande ◽

Marta Bellone ◽

...

Keyword(s):

Papillary Carcinoma ◽

Cell Lines ◽

Carcinoma Cell ◽

Carcinoma Cell Lines ◽

Her 2 ◽

Serous Papillary Carcinoma ◽

Higher Sensitivity

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Characterization of Streptococcus gordonii SecA2 as a Paralogue of SecA

Journal of Bacteriology ◽

10.1128/jb.00365-09 ◽

2009 ◽

Vol 191 (11) ◽

pp. 3482-3491 ◽

Cited By ~ 23

Author(s):

Barbara A. Bensing ◽

Paul M. Sullam

Keyword(s):

Protein Transport ◽

Maximal Activity ◽

Biochemical Properties ◽

Streptococcus Gordonii ◽

Lower Solubility ◽

Highly Sensitive ◽

Sec System ◽

High Degree

ABSTRACT The accessory Sec system of Streptococcus gordonii is essential for transport of the glycoprotein GspB to the bacterial cell surface. A key component of this dedicated transport system is SecA2. The SecA2 proteins of streptococci and staphylococci are paralogues of SecA and are presumed to have an analogous role in protein transport, but they may be specifically adapted for the transport of large, serine-rich glycoproteins. We used a combination of genetic and biochemical methods to assess whether the S. gordonii SecA2 functions similarly to SecA. Although mutational analyses demonstrated that conserved amino acids are essential for the function of SecA2, replacing such residues in one of two nucleotide binding folds had only minor effects on SecA2 function. SecA2-mediated transport is highly sensitive to azide, as is SecA-mediated transport. Comparison of the S. gordonii SecA and SecA2 proteins in vitro revealed that SecA2 can hydrolyze ATP at a rate similar to that of SecA and is comparably sensitive to azide but that the biochemical properties of these enzymes are subtly different. That is, SecA2 has a lower solubility in aqueous solutions and requires higher Mg2+ concentrations for maximal activity. In spite of the high degree of similarity between the S. gordonii paralogues, analysis of SecA-SecA2 chimeras indicates that the domains are not readily interchangeable. This suggests that specific, unique contacts between SecA2 and other components of the accessory Sec system may preclude cross-functioning with the canonical Sec system.

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A2B Adenosine Receptors and Sphingosine 1-Phophate Signaling Cross-Talk in Oligodendrogliogenesis

Frontiers in Neuroscience ◽

10.3389/fnins.2021.677988 ◽

2021 ◽

Vol 15 ◽

Author(s):

Elisabetta Coppi ◽

Francesca Cencetti ◽

Federica Cherchi ◽

Martina Venturini ◽

Chiara Donati ◽

...

Keyword(s):

Adenosine Receptors ◽

Adult Life ◽

Oligodendrocyte Progenitor Cells ◽

Voltage Dependent ◽

Sphingosine 1 Phosphate ◽

Demyelinating Disorders ◽

S1p Lyase ◽

Stimulation Of

Oligodendrocyte-formed myelin sheaths allow fast synaptic transmission in the brain. Impairments in the process of myelination, or demyelinating insults, might cause chronic diseases such as multiple sclerosis (MS). Under physiological conditions, remyelination is an ongoing process throughout adult life consisting in the differentiation of oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes (OLs). During pathological events, this process fails due to unfavorable environment. Adenosine and sphingosine kinase/sphingosine 1-phosphate signaling axes (SphK/S1P) play important roles in remyelination processes. Remarkably, fingolimod (FTY720), a sphingosine analog recently approved for MS treatment, plays important roles in OPC maturation. We recently demonstrated that the selective stimulation of A2B adenosine receptors (A2BRs) inhibit OPC differentiation in vitro and reduce voltage-dependent outward K+ currents (IK) necessary to OPC maturation, whereas specific SphK1 or SphK2 inhibition exerts the opposite effect. During OPC differentiation A2BR expression increases, this effect being prevented by SphK1/2 blockade. Furthermore, selective silencing of A2BR in OPC cultures prompts maturation and, intriguingly, enhances the expression of S1P lyase, the enzyme responsible for irreversible S1P catabolism. Finally, the existence of an interplay between SphK1/S1P pathway and A2BRs in OPCs was confirmed since acute stimulation of A2BRs activates SphK1 by increasing its phosphorylation. Here the role of A2BR and SphK/S1P signaling during oligodendrogenesis is reviewed in detail, with the purpose to shed new light on the interaction between A2BRs and S1P signaling, as eventual innovative targets for the treatment of demyelinating disorders.

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Generation of CD1 tetramers as a tool to monitor glycolipid–specific T cells

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2003.1267 ◽

2003 ◽

Vol 358 (1433) ◽

pp. 875-877 ◽

Cited By ~ 12

Author(s):

Stephan D. Gadola ◽

Anastasios Karadimitris ◽

Nathan R. Zaccai ◽

Mariolina Salio ◽

Nicolas Dulphy ◽

...

Keyword(s):

Ex Vivo ◽

Functional Characterization ◽

Hla Class I ◽

Lipid Complex ◽

Highly Sensitive ◽

M Proteins ◽

Human T Lymphocyte

CD1 molecules are β 2 m–associated HLA class–I–like glycoproteins which have the unique ability to present glycolipid and phospholipid antigens to specific T lymphocytes. To study the biology of CD1 and its role in human disease we developed novel techniques for generation of recombinant CD1/lipid complexes by in vitro refolding. Fluorescent tetrameric complexes made from soluble recombinant CD1d/α–galactosylceramide complexes allowed highly sensitive and specific ex vivo and in vitro detection and functional characterization of novel human T–lymphocyte populations. Furthermore, protein crystals were obtained from soluble recombinant CD1b/β 2 m–proteins loaded either with phosphatidylinositol or ganglioside GM2, which led to the first atomic structure determination of a CD1/lipid complex. The analysis of these crystal structures clarified how CD1b molecules can bind lipid ligands of different size, and revealed a broader spectrum of potential CD1b ligands than previously predicted.

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