scholarly journals De Novo Sphingolipid Synthesis Is Essential for Viability, but Not for Transport of Glycosylphosphatidylinositol-Anchored Proteins, in African Trypanosomes

Eukaryotic Cell â—½  
10.1128/ec.00283-06 â—½  
2007 â—½  
Vol 6 (3) â—½  
pp. 454-464 â—½  
Author(s):  
Shaheen S. Sutterwala â—½  
Caleb H. Creswell â—½  
Sumana Sanyal â—½  
Anant K. Menon â—½  
James D. Bangs
Keyword(s):  
Adverse Effect â—½  
Hela Cells â—½  
Leishmania Major â—½  
De Novo â—½  
Eukaryotic Cells â—½  
De Novo Synthesis â—½  
General Requirement â—½  
Prolonged Incubation â—½  
African Trypanosomes â—½  
Biochemical Analyses

ABSTRACT De novo sphingolipid synthesis is required for the exit of glycosylphosphatidylinositol (GPI)-anchored membrane proteins from the endoplasmic reticulum in yeast. Using a pharmacological approach, we test the generality of this phenomenon by analyzing the transport of GPI-anchored cargo in widely divergent eukaryotic systems represented by African trypanosomes and HeLa cells. Myriocin, which blocks the first step of sphingolipid synthesis (serine + palmitate → 3-ketodihydrosphingosine), inhibited the growth of cultured bloodstream parasites, and growth was rescued with exogenous 3-ketodihydrosphingosine. Myriocin also blocked metabolic incorporation of [3H]serine into base-resistant sphingolipids. Biochemical analyses indicate that the radiolabeled lipids are not sphingomyelin or inositol phosphorylceramide, suggesting that bloodstream trypanosomes synthesize novel sphingolipids. Inhibition of de novo sphingolipid synthesis with myriocin had no adverse effect on either general secretory trafficking or GPI-dependent trafficking in trypanosomes, and similar results were obtained with HeLa cells. A mild effect on endocytosis was seen for bloodstream trypanosomes after prolonged incubation with myriocin. These results indicate that de novo synthesis of sphingolipids is not a general requirement for secretory trafficking in eukaryotic cells. However, in contrast to the closely related kinetoplastid Leishmania major, de novo sphingolipid synthesis is essential for the viability of bloodstream-stage African trypanosomes.

scholarly journals Cyclooxygenase-2 Glycosylation Is Affected by Peroxynitrite in Endothelial Cells: Impact on Enzyme Activity and Degradation

Antioxidants â—½  
2021 â—½  
Vol 10 (3) â—½  
pp. 496
Author(s):  
Sonia Eligini â—½  
Susanna Colli â—½  
Aida Habib â—½  
Giancarlo Aldini â—½  
Alessandra Altomare â—½  
...  
Keyword(s):  
Endothelial Cells â—½  
De Novo â—½  
Cyclooxygenase 2 â—½  
Nitric Oxide Donors â—½  
Metabolic Labeling â—½  
Dependent Manner â—½  
De Novo Synthesis â—½  
Human Endothelial Cells â—½  
Prolonged Incubation â—½  
Cox 2

The exposure of human endothelial cells to 3-morpholinosydnonimine (SIN-1) induced the expression of cyclooxygenase-2 (COX-2) in a dose- and time-dependent manner. Interestingly, after a prolonged incubation (>8 h) several proteoforms were visualized by Western blot, corresponding to different states of glycosylation of the protein. This effect was specific for SIN-1 that generates peroxynitrite and it was not detected with other nitric oxide-donors. Metabolic labeling experiments using 35S or cycloheximide suggested that the formation of hypoglycosylated COX-2 was dependent on de novo synthesis of the protein rather than the deglycosylation of the native protein. Moreover, SIN-1 reduced the activity of the hexokinase, the enzyme responsible for the first step of glycolysis. The hypoglycosylated COX-2 induced by SIN-1 showed a reduced capacity to generate prostaglandins and the activity was only partially recovered after immunoprecipitation. Finally, hypoglycosylated COX-2 showed a more rapid rate of degradation compared to COX-2 induced by IL-1α and an alteration in the localization with an accumulation mainly detected in the nuclear membrane. Our results have important implication to understand the effect of peroxynitrite on COX-2 expression and activity, and they may help to identify new pharmacological tools direct to increase COX-2 degradation or to inhibit its activity.

scholarly journals De novo synthesis of phosphatidylcholine is essential for the promastigote but not amastigote stage in Leishmania major

2020 â—½  
Author(s):  
Samrat Moitra â—½  
Somrita Basu â—½  
Mattie Pawlowic â—½  
Fong-fu Hsu â—½  
Kai Zhang
Keyword(s):  
Culture Medium â—½  
Leishmania Major â—½  
De Novo â—½  
Total Lipids â—½  
De Novo Synthesis â—½  
Targeted Deletion â—½  
Amastigote Stage â—½  
Kennedy Pathway â—½  
Choline Phosphate

ABSTRACTPhosphatidylcholine (PC) is the most abundant type of phospholipids in eukaryotes constituting ~30% of total lipids in Leishmania. PC synthesis mainly occurs via the choline branch of the Kennedy pathway (choline ⇒ choline-phosphate ⇒ CDP-choline ⇒ PC) and the N-methylation of phosphatidylethanolamine (PE). In addition, Leishmania parasites can acquire PC and other lipids from the host or culture medium. In this study, we assessed the function and essentiality of choline ethanolamine phosphotransferase (CEPT) in Leishmania major which is responsible for the final step of the de novo synthesis of PC and PE. Our data indicate that CEPT is localized in the endoplasmic reticulum and possesses the activity to generate PC from CDP-choline and diacylglycerol. Targeted deletion of CEPT is only possible in the presence of an episomal CEPT gene in the promastigote stage of L. major. These chromosomal null parasites require the episomal expression of CEPT to survive in culture, confirming its essentiality during the promastigote stage. In contrast, during in vivo infection of BALB/c mice, these chromosomal null parasites appeared to lose the episomal copy of CEPT while maintaining normal levels of virulence, replication and cellular PC. Therefore, while the de novo synthesis of PC/PE is indispensable for the proliferation of promastigotes, intracellular amastigotes appear to acquire most of their lipids through salvage and remodeling.

scholarly journals Two pathways for cysteine biosynthesis in Leishmania major

Biochemical Journal â—½  
10.1042/bj20082441 â—½  
2009 â—½  
Vol 420 (3) â—½  
pp. 451-462 â—½  
Author(s):  
Roderick A. M. Williams â—½  
Gareth D. Westrop â—½  
Graham H. Coombs
Keyword(s):  
Drug Target â—½  
Leishmania Major â—½  
De Novo â—½  
Genome Mining â—½  
Sulfur Amino Acids â—½  
Cysteine Synthase â—½  
De Novo Biosynthesis â—½  
Cysteine Synthesis â—½  
Mercaptopyruvate Sulfurtransferase â—½  
Biochemical Analyses

Genome mining and biochemical analyses have shown that Leishmania major possesses two pathways for cysteine synthesis – the de novo biosynthesis pathway comprising SAT (serine acetyltransferase) and CS (cysteine synthase) and the RTS (reverse trans-sulfuration) pathway comprising CBS (cystathionine β-synthase) and CGL (cystathionine γ-lyase). The LmjCS (L. major CS) is similar to the type A CSs of bacteria and catalyses the synthesis of cysteine using O-acetylserine and sulfide with Kms of 17.5 and 0.13 mM respectively. LmjCS can use sulfide provided by the action of MST (mercaptopyruvate sulfurtransferase) on 3-MP (3-mercaptopyruvate). LmjCS forms a bi-enzyme complex with Leishmania SAT (and Arabidopsis SAT), with residues Lys222, His226 and Lys227 of LmjCS being involved in the complex formation. LmjCBS (L. major CBS) catalyses the synthesis of cystathionine from homocysteine, but, unlike mammalian CBS, also has high cysteine synthase activity (but with the Km for sulfide being 10.7 mM). In contrast, LmjCS does not have CBS activity. CS was up-regulated when promastigotes were grown in medium with limited availability of sulfur amino acids. Exogenous methionine stimulated growth under these conditions and also the levels of intracellular cysteine, glutathione and trypanothione, whereas cysteine had no effect on growth or the intracellular cysteine levels, correlating with the low rate of transport of cysteine into the cell. These results suggest that cysteine is generated endogenously by promastigotes of Leishmania. The absence of CS from mammals and the clear differences between CBS of mammals and Leishmania suggest that each of the parasite enzymes could be a viable drug target.

scholarly journals De Novo Synthesis of Phosphatidylcholine Is Essential for the Promastigote But Not Amastigote Stage in Leishmania major

2021 â—½  
Vol 11 â—½  
Author(s):  
Samrat Moitra â—½  
Somrita Basu â—½  
Mattie Pawlowic â—½  
Fong-fu Hsu â—½  
Kai Zhang
Keyword(s):  
Culture Medium â—½  
Leishmania Major â—½  
De Novo â—½  
Total Lipids â—½  
De Novo Synthesis â—½  
Targeted Deletion â—½  
Amastigote Stage â—½  
Kennedy Pathway â—½  
Choline Phosphate

Phosphatidylcholine (PC) is the most abundant type of phospholipids in eukaryotes constituting ~30% of total lipids in Leishmania. PC synthesis mainly occurs via the choline branch of the Kennedy pathway (choline ⇒ choline-phosphate ⇒ CDP-choline ⇒ PC) and the N-methylation of phosphatidylethanolamine (PE). In addition, Leishmania parasites can acquire PC and other lipids from the host or culture medium. In this study, we assessed the function and essentiality of choline ethanolamine phosphotransferase (CEPT) in Leishmania major which is responsible for the final step of the de novo synthesis of PC and PE. Our data indicate that CEPT is localized in the endoplasmic reticulum and possesses the activity to generate PC from CDP-choline and diacylglycerol. Targeted deletion of CEPT is only possible in the presence of an episomal CEPT gene in the promastigote stage of L. major. These chromosomal null parasites require the episomal expression of CEPT to survive in culture, confirming its essentiality during the promastigote stage. In contrast, during in vivo infection of BALB/c mice, these chromosomal null parasites appeared to lose the episomal copy of CEPT while maintaining normal levels of virulence, replication and cellular PC. Therefore, while the de novo synthesis of PC/PE is indispensable for the proliferation of promastigotes, intracellular amastigotes appear to acquire most of their lipids through salvage and remodeling.

Characterization of Monocyte-Associated Factor V

1993 â—½  
Vol 70 (02) â—½  
pp. 273-280 â—½  
Author(s):  
Janos Kappelmayer â—½  
Satya P Kunapuli â—½  
Edward G Wyshock â—½  
Robert W Colman
Keyword(s):  
Monoclonal Antibody â—½  
Light Chain â—½  
Peripheral Blood â—½  
De Novo â—½  
Factor V â—½  
Blood Monocytes â—½  
De Novo Synthesis â—½  
Hep G2 â—½  
Hep G2 Cells â—½  
Peripheral Blood Monocytes

SummaryWe demonstrate that in addition to possessing binding sites for intact factor V (FV), unstimulated peripheral blood monocytes also express activated factor V (FVa) on their surfaces. FVa was identified on the monocyte surface by monoclonal antibody B38 recognizing FVa light chain and by human oligoclonal antibodies H1 (to FVa light chain) and H2 (to FVa heavy chain) using immunofluorescence microscopy and flow cytometry. On Western blots, partially cleaved FV could be identified as a 220 kDa band in lysates of monocytes. In addition to surface expression of FVa, monocytes also contain intracellular FV as detected only after permeabilization by Triton X-100 by monoclonal antibody B10 directed specifically to the Cl domain not present in FVa. We sought to determine whether the presence of FV in peripheral blood monocytes is a result of de novo synthesis.Using in situ hybridization, no FV mRNA could be detected in monocytes, while in parallel control studies, factor V mRNA was detectable in Hep G2 cells and CD18 mRNA in monocytes. In addition, using reverse transcriptase and the polymerase chain reaction, no FV mRNA was detected in mononuclear cells or in U937 cells, but mRNA for factor V was present in Hep G2 cells using the same techniques. These data suggest that FV is present in human monocytes, presumably acquired by binding of plasma FV, and that the presence of this critical coagulation factor is not due to de novo synthesis.

Platelets Stimulate Thromboplastin Synthesis in Human Endothelial Cells

1983 â—½  
Vol 49 (02) â—½  
pp. 069-072 â—½  
Author(s):  
U L H Johnsen â—½  
T Lyberg â—½  
K S Galdal â—½  
H Prydz
Keyword(s):  
Endothelial Cells â—½  
De Novo â—½  
Umbilical Vein â—½  
Time Dependent â—½  
De Novo Synthesis â—½  
Human Endothelial Cells â—½  
Tumor Promotor â—½  
Coagulation Assay â—½  
Platelet Aggregates â—½  
Umbilical Vein Endothelial Cells

SummaryHuman umbilical vein endothelial cells in culture synthesize thromboplastin upon stimulation with phytohaemagglutinin (PHA) or the tumor promotor 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The thromboplastin activity is further strongly enhanced in a time dependent reaction by the presence of gel-filtered platelets or platelet aggregates. This effect was demonstrable at platelet concentrations lower than those normally found in plasma, it may thus be of pathophysiological relevance. The thromboplastin activity increased with increasing number of platelets added. Cycloheximide inhibited the increase, suggesting that de novo synthesis of the protein component of thromboplastin, apoprotein III, is necessary.When care was taken to remove monocytes no thromboplastin activity and no apoprotein HI antigen could be demonstrated in suspensions of gel-filtered platelets, platelets aggregated with thrombin or homogenized platelets when studied with a coagulation assay and an antibody neutralization technique.

DE NOVO SYNTHESIS OF STEROIDS BY THE TESTES OF THE HUMAN FOETUS AT MIDGESTATION

1971 â—½  
Vol 68 (1_Supplb) â—½  
pp. S135 â—½  
Author(s):  
R. S. Mathur â—½  
N. Wiqvist â—½  
E. Diczfalusy
Keyword(s):  
De Novo â—½  
De Novo Synthesis â—½  
Human Foetus
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