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

2020 ◽  
Author(s):  
Samrat Moitra ◽  
Somrita Basu ◽  
Mattie Pawlowic ◽  
Fong-fu Hsu ◽  
Kai Zhang
Keyword(s):  
Culture Medium ◽  
Leishmania Major ◽  
De Novo ◽  
Total Lipids ◽  
De Novo Synthesis ◽  
Targeted Deletion ◽  
Amastigote Stage ◽  
Kennedy Pathway ◽  
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 De Novo Synthesis of Phosphatidylcholine Is Essential for the Promastigote But Not Amastigote Stage in Leishmania major

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

Divergent effects of intravenous GSH and cysteine on renal and hepatic GSH

1992 ◽  
Vol 263 (2) ◽  
pp. R348-R352 ◽  
Author(s):  
S. Aebi ◽  
B. H. Lauterburg
Keyword(s):  
Steady State ◽  
De Novo ◽  
Feedback Inhibition ◽  
Therapeutic Use ◽  
Steady State Concentration ◽  
De Novo Synthesis ◽  
State Concentration ◽  
Cellular Thiol

There is a growing interest in the therapeutic use of sulfhydryls. To assess the effect of glutathione (GSH) and cysteine on the cellular thiol status, thiols were administered intravenously to rats in doses ranging from 1.67 to 8.35 mmol/kg with and without pretreatment with 4 mmol/kg buthionine-[S,R]-sulfoximine (BSO), an inhibitor of GSH synthesis. One hour after administration of 1.67 mmol/kg GSH, the concentration of GSH rose from 5.2 +/- 1.0 to 8.4 +/- 0.9 mumol/g and from 2.5 +/- 0.5 to 3.7 +/- 0.7 mumol/g in liver and kidneys, respectively. After 8.35 mmol/kg, hepatic GSH did not increase further, but renal GSH rose to 6.7 +/- 1.8 mumol/g. Infusion of cysteine increased hepatic GSH to the same extent as intravenous GSH, but renal GSH did not increase after 1.67 mmol/kg and even significantly decreased to 0.6 +/- 0.2 mumol/g after 8.35 mmol/kg. In the presence of BSO, GSH resulted in a significant increase in renal but not hepatic GSH, suggesting that the kidneys take up intact GSH and indicating that the increment in hepatic GSH was due to de novo synthesis. The present data show that hepatic GSH can be markedly increased in vivo by increasing the supply of cysteine. Measurements of hepatic cysteine indicate that up to a concentration of approximately 0.5 mumol/g cysteine is a key determinant of hepatic GSH, such that the physiological steady-state concentration of GSH in the liver appears to be mainly determined by the availability of cysteine. At higher concentrations GSH does not increase further, possibly due to feedback inhibition of GSH synthesis or increased efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

LB Broth-lyophilized Rabbit Anti-sheep Cell Haemolysin as a Simple Culture Medium for Cultivation of Leishmania Major Promastigotes

2019 ◽  
Author(s):  
Vahid Nasiri ◽  
Farnoosh Jameie ◽  
Habibollah Paykari
Keyword(s):  
Blood Cell ◽  
Culture Medium ◽  
Leishmania Major ◽  
Low Cost ◽  
Protozoan Parasites ◽  
Causative Agents ◽  
Genus Leishmania ◽  
Leishmania Parasites ◽  
Growth Ability

Background and Aims: The protozoan parasites of the genus Leishmania are the causative agents of various clinical diseases. Different methods of cultivation of Leishmania parasites are available. In the present study, the efficacy of the LB broth with rabbit lyophilized anti-sheep red blood cell haemolysin was evaluated in the cultivation of promastigotes of Leishmania major. Materials and Methods: Conventional LB broth medium was prepared and autoclaved for 15 min at 121°C and then lyophilized rabbit anti-sheep cell haemolysin was added at 1-10% final concentrations. The efficacy of the medium was evaluated by assessing the growth ability and replication patterns of the promastigotes of Leishmania major. Results: Medium with 1-10% lyophilized rabbit haemolysin supported the growth of the parasites and can be used for cultivation of Leishmania parasites with acceptable In vivo infectivity for research purpose. Conclusions: The ability of the parasites to survive and proliferate in the presence of lyophilized rabbit haemolysin indicates that this material is a good nutritional source. This study opens a new way to make low-cost medium that can be used in cultivation of Leishmania parasites

scholarly journals An Acid-activated Nucleobase Transporter from Leishmania major

2009 ◽  
Vol 284 (24) ◽  
pp. 16164-16169 ◽  
Author(s):  
Diana Ortiz ◽  
Marco A. Sanchez ◽  
Hans P. Koch ◽  
H. Peter Larsson ◽  
Scott M. Landfear
Keyword(s):  
Leishmania Major ◽  
De Novo ◽  
Low Ph ◽  
Acidic Ph ◽  
Physiological Conditions ◽  
Neutral Ph ◽  
Amastigote Stage ◽  
Electrode Voltage ◽  
Ph Conditions ◽  
Micromolar Range

Parasitic protozoa are unable to synthesize purines de novo and must import preformed purine nucleobases or nucleosides from their hosts. Leishmania major expresses two purine nucleobase transporters, LmaNT3 and LmaNT4. Previous studies revealed that at neutral pH, LmaNT3 is a broad specificity, high affinity nucleobase transporter, whereas LmaNT4 mediates the uptake of only adenine. Because LmaNT4 is required for optimal viability of the amastigote stage of the parasite that lives within acidified phagolysomal vesicles of mammalian macrophages, the function of this permease was examined under acidic pH conditions. At acidic pH, LmaNT4 acquires the ability to transport adenine, hypoxanthine, guanine, and xanthine with Km values in the micromolar range, indicating that this transporter is activated at low pH. Thus, LmaNT4 is an acid-activated purine nucleobase transporter that functions optimally under the physiological conditions the parasite is exposed to in the macrophage phagolysosome. In contrast, LmaNT3 functions optimally at neutral pH. Two-electrode voltage clamp experiments performed on LmaNT3 and LmaNT4 expressed in Xenopus oocytes revealed substrate-induced inward directed currents at acidic pH, and application of substrates induced acidification of the oocyte cytosol. These observations imply that LmaNT3 and LmaNT4 are nucleobase/proton symporters.

scholarly journals Cloning and expression of a human choline/ethanolaminephosphotransferase: synthesis of phosphatidylcholine and phosphatidylethanolamine

1999 ◽  
Vol 339 (2) ◽  
pp. 291-298 ◽  
Author(s):  
Annette L. HENNEBERRY ◽  
Christopher R. McMASTER
Keyword(s):  
De Novo ◽  
Active Form ◽  
Fatty Acyl ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Kennedy Pathway ◽  
Membrane Spanning

Cholinephosphotransferase catalyses the final step in the synthesis of phosphatidylcholine (PtdCho) via the Kennedy pathway by the transfer of phosphocholine from CDP-choline to diacylglycerol. Ethanolaminephosphotransferase catalyses an analogous reaction with CDP-ethanolamine as the phosphobase donor for the synthesis of phosphatidylethanolamine (PtdEtn). Together these two enzyme activities determine both the site of synthesis and the fatty acyl composition of PtdCho and PtdEtn synthesized de novo. A human choline/ethanolaminephosphotransferase cDNA (hCEPT1) was cloned, expressed and characterized. Northern blot analysis revealed one hCEPT1 2.3 kb transcript that was ubiquitous and not enriched, with respect to actin, in any particular cell type. The open reading frame predicts a protein (hCEPT1p) of 416 amino acid residues with a molecular mass of 46550 Da containing seven membrane-spanning domains. A predicted amphipathic helix resides within the active site of the enzyme with the final two aspartic residues of the CDP-alcohol phosphotransferase motif, DG(X)2AR(X)8G(X)3D(X)3D, positioned within this helix. hCEPT1p was successfully expressed in a full-length, active form in Saccharomyces cerevisiae cells devoid of endogenous cholinephosphotransferase or ethanolaminephosphotransferase activities (HJ091, cpt1::LEU2 ept1-). In vitro, hCEPT1p displayed broad substrate specificity, utilizing both CDP-choline and CDP-ethanolamine as phosphobase donors to a broad range of diacylglycerols, resulting in the synthesis of both PtdCho and PtdEtn. In vivo, S. cerevisiae cells (HJ091, cpt1::LEU2 ept1-) expressing hCEPT1 efficiently incorporated both radiolabelled choline and ethanolamine into phospholipids, demonstrating that hCEPT1p has the ability to synthesize both choline- and ethanolamine- containing phospholipids in vitro and in vivo.

scholarly journals De Novo Synthesis of VP16 Coordinates the Exit from HSV Latency In Vivo

2009 ◽  
Vol 5 (3) ◽  
pp. e1000352 ◽  
Author(s):  
Richard L. Thompson ◽  
Chris M. Preston ◽  
Nancy M. Sawtell
Keyword(s):  
De Novo ◽  
De Novo Synthesis

scholarly journals Gene expression induced by Toll-like receptors in macrophages requires the transcription factor NFAT5

2012 ◽  
Vol 209 (2) ◽  
pp. 379-393 ◽  
Author(s):  
Maria Buxadé ◽  
Giulia Lunazzi ◽  
Jordi Minguillón ◽  
Salvador Iborra ◽  
Rosa Berga-Bolaños ◽  
...  
Keyword(s):  
Histone Deacetylases ◽  
Target Genes ◽  
Leishmania Major ◽  
De Novo ◽  
Specific Gene ◽  
Toll Like Receptors ◽  
Tlr Agonists ◽  
High Doses ◽  
Pathogen Burden

Toll-like receptors (TLRs) engage networks of transcriptional regulators to induce genes essential for antimicrobial immunity. We report that NFAT5, previously characterized as an osmostress responsive factor, regulates the expression of multiple TLR-induced genes in macrophages independently of osmotic stress. NFAT5 was essential for the induction of the key antimicrobial gene Nos2 (inducible nitric oxide synthase [iNOS]) in response to low and high doses of TLR agonists but is required for Tnf and Il6 mainly under mild stimulatory conditions, indicating that NFAT5 could regulate specific gene patterns depending on pathogen burden intensity. NFAT5 exhibited two modes of association with target genes, as it was constitutively bound to Tnf and other genes regardless of TLR stimulation, whereas its recruitment to Nos2 or Il6 required TLR activation. Further analysis revealed that TLR-induced recruitment of NFAT5 to Nos2 was dependent on inhibitor of κB kinase (IKK) β activity and de novo protein synthesis, and was sensitive to histone deacetylases. In vivo, NFAT5 was necessary for effective immunity against Leishmania major, a parasite whose clearance requires TLRs and iNOS expression in macrophages. These findings identify NFAT5 as a novel regulator of mammalian anti-pathogen responses.

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