scholarly journals Characterization of the inositol phosphorylceramide synthase activity from Trypanosoma cruzi

2005 ◽  
Vol 387 (2) ◽  
pp. 519-529 ◽  
Author(s):  
Juliana M. FIGUEIREDO ◽  
Wagner B. DIAS ◽  
Lucia MENDONÇA-PREVIATO ◽  
José O. PREVIATO ◽  
Norton HEISE
Keyword(s):  
Trypanosoma Cruzi ◽  
Inositol Phosphate ◽  
Protozoan Parasite ◽  
Peritoneal Macrophages ◽  
Dependent Manner ◽  
Life Cycle Stages ◽  
Aureobasidin A ◽  
Triton X ◽  
First Time

IPC (inositol phosphorylceramide) synthase is an enzyme essential for fungal viability, and it is the target of potent antifungal compounds such as rustmicin and aureobasidin A. Similar to fungi and some other lower eukaryotes, the protozoan parasite Trypanosoma cruzi is capable of synthesizing free or protein-linked glycoinositolphospholipids containing IPC. As a first step towards understanding the importance and mechanism of IPC synthesis in T. cruzi, we investigated the effects of rustmicin and aureobasidin A on the proliferation of different life-cycle stages of the parasite. The compounds did not interfere with the axenic growth of epimastigotes, but aureobasidin A decreased the release of trypomastigotes from infected murine peritoneal macrophages and the number of intracellular amastigotes in a dose-dependent manner. We have demonstrated for the first time that all forms of T. cruzi express an IPC synthase activity that is capable of transferring inositol phosphate from phosphatidylinositol to the C-1 hydroxy group of C6-NBD-cer {6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-amino]hexanoylceramide} to form inositol phosphoryl-C6-NBD-cer, which was purified and characterized by its chromatographic behaviour on TLC and HPLC, sensitivity to phosphatidylinositol-specific phospholipase C and resistance to mild alkaline hydrolysis. Unlike the Saccharomyces cerevisiae IPC synthase, the T. cruzi enzyme is stimulated by Triton X-100 but not by bivalent cations, CHAPS or fatty-acid-free BSA, and it is not inhibited by rustmicin or aureobasidin A, or the two in combination. Further studies showed that aureobasidin A has effects on macrophages independent of the infecting T. cruzi cells. These results suggest that T. cruzi synthesizes its own IPC, but by a mechanism that is not affected by rustmicin and aureobasidin A.

scholarly journals Anti-Trypanosoma cruzi activity, cytotoxicity and, chemical characterization of extracts from seeds of Lonchocarpus cultratus

2021 ◽  
Vol 15 (02) ◽  
pp. 270-279
Author(s):  
Aline Griebler ◽  
Fernanda Weyand Banhuk ◽  
Izabela Virginia Staffen ◽  
Aline Antunes Maciel Bortoluzzi ◽  
Thaís Soprani Ayala ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Methanolic Extract ◽  
Chemical Characterization ◽  
Peritoneal Macrophages ◽  
Chemical Profile ◽  
Murine Macrophages ◽  
Human Red Blood Cells

Introduction: Trypanosoma cruzi is the agent of Chagas’ disease and affects approximately 6-8 million people worldwide. The search for new anti-T. cruzi drugs are relevant because only two drugs exist actually. The objective of this study was to investigate the effect of the extracts from the seeds of Lonchocarpus cultratus on T. cruzi, its cytotoxicity as well as to elucidate its chemical profile. Methodology: The characterization of the extracts was done using 1H-RMN. T. cruzi forms were treated with increasing concentrations of the extracts and after, the percentage of inhibition and IC50 or LC50 were calculated. Murine peritoneal macrophages were treated with different concentrations of the extracts to evaluate the cellular viability. The hemotoxicity was accessed by verifying the levels of hemolysis caused by the extracts on human red blood cells. Results: Chalcones isocordoin and lonchocarpin were detected in the dichloromethane extract, and chalcone lonchocarpin was detected in the hexane extract. The dichloromethane extract showed higher activity against all the forms of T. cruzi compared to the other two extracts, but the hexane showed the best selectivity index. The cytotoxicity observed in murine macrophages was confirmed in human erythrocytes, with dichloromethane extract having the highest toxicity. The methanolic extract showed the lowest anti-T. cruzi activity but was nontoxic to peritoneal murine macrophages and red blood cells. Conclusions: L. cultratus extracts have the potential to be explored for the development of new anti-trypanosomal drugs. This study was the first to demonstrate the action of extracts from the genus Lonchocarpus on infecting forms of T. cruzi.

scholarly journals Gene and cDNA cloning and characterization of the mouse V3/V1b pituitary vasopressin receptor

1999 ◽  
Vol 22 (3) ◽  
pp. 251-260 ◽  
Author(s):  
MA Ventura ◽  
P Rene ◽  
Y de Keyzer ◽  
X Bertagna ◽  
E Clauser
Keyword(s):  
Inositol Phosphate ◽  
Rank Order ◽  
Single Gene ◽  
Dependent Manner ◽  
Binding Studies ◽  
V1b Receptor ◽  
Competition Binding ◽  
Receptor Cdna ◽  
Dose Dependent

The gene of the mouse V3/V1b receptor was identified by homology cloning. One of the genomic clones contained the entire coding sequence. The cDNA presented high identity with rat (92%) and human (84%) sequences. Southern blot analysis indicated the existence of a single gene. Tissue distribution was studied by RT-PCR. The major site of expression was the pituitary. A faint signal was also present in hypothalamus, brain, adrenal, pancreas and colon. The mouse corticotroph cell line, AtT20, did not express the transcript. In order to confirm the identity of the sequence, the V3/V1b receptor cDNA was cloned and stably expressed in CHO-AA8 Tet-Off cells under the control of tetracycline. When transfected cells were treated with arginine vasopressin (AVP), inositol phosphate production increased in a dose-dependent manner, indicating that the V3/V1b receptor couples to phospholipase C. Moreover, AVP did not stimulate cAMP production. Binding studies with [3H]AVP indicated that the affinity of the mouse V3/V1b receptor (Kd=0.5 nM) is similar to that reported for rat and human receptors. The rank order of potency established in competition binding experiments with different analogues was representative of a V3/V1b profile, distinct from V1a and V2. However, significant differences were found between human and mouse receptors tested in parallel. Thus the pharmacology of V3/V1b receptors can not be transposed among different species.

scholarly journals Characterization of the Novel Mitochondrial Genome Segregation Factor TAP110 in Trypanosoma brucei

2021 ◽  
pp. jcs.254300
Author(s):  
Simona Amodeo ◽  
Ana Kalichava ◽  
Albert Fradera-Sola ◽  
Eloïse Bertiaux-Lequoy ◽  
Paul Guichard ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Trypanosoma Brucei ◽  
Proteome Analysis ◽  
Protozoan Parasite ◽  
Eukaryotic Cell ◽  
The Novel ◽  
Precise Position ◽  
Associated Proteins ◽  
First Time

Proper mitochondrial genome inheritance is important for eukaryotic cell survival. Trypanosoma brucei, a protozoan parasite, contains a singular mitochondrial genome, the kDNA. The kDNA is anchored to the basal body via the tripartite attachment complex (TAC) to ensure proper segregation. Several components of the TAC have been described. However, the connection of the TAC to the kDNA remains elusive. Here, we characterize the TAC associated protein TAP110. Depletion as well as overexpression of TAP110 leads to a delay in the separation of the replicated kDNA networks. Proteome analysis after TAP110 overexpression identified several kDNA associated proteins including a TEX-like protein that dually localizes to the nucleus and the kDNA potentially linking replication/segregation in the two compartments. The assembly of TAP110 into the TAC region seems to require the TAC but not the kDNA itself, however once TAP110 has been assembled it also interacts with the kDNA. Finally, for the first time we use ultrastructure expansion microscopy in trypanosomes to reveal the precise position of TAP110 between TAC102 and the kDNA, showcasing the potential of this approach.

scholarly journals Characterization of the Novel Mitochondrial Genome Segregation Factor TAP110 in Trypanosoma brucei

2020 ◽  
Author(s):  
Simona Amodeo ◽  
Ana Kalichava ◽  
Albert Fradera-Sola ◽  
Eloïse Bertiaux-Lequoy ◽  
Paul Guichard ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Trypanosoma Brucei ◽  
Protozoan Parasite ◽  
Eukaryotic Cell ◽  
The Novel ◽  
Detergent Treatment ◽  
Summary Statement ◽  
First Time

AbstractProper mitochondrial genome inheritance is key for eukaryotic cell survival, however little is known about the molecular mechanism controlling this process. Trypanosoma brucei, a protozoan parasite, contains a singular mitochondrial genome aka kinetoplast DNA (kDNA). kDNA segregation requires anchoring of the genome to the basal body via the tripartite attachment complex (TAC). Several components of the TAC as well as their assembly have been described, it however remains elusive how the TAC connects to the kDNA. Here, we characterize the TAC associated protein TAP110 and for the first time use ultrastructure expansion microscopy in trypanosomes to reveal that TAP110 is the currently most proximal kDNA segregation factor. The kDNA proximal positioning is also supported by RNAi depletion of TAC102, which leads to loss of TAP110 at the TAC. Overexpression of TAP110 leads to expression level changes of several mitochondrial proteins and a delay in the separation of the replicated kDNA networks. In contrast to other kDNA segregation factors TAP110 remains only partially attached to the flagellum after DNAse and detergent treatment and can only be solubilized in dyskinetoplastic cells, suggesting that interaction with the kDNA might be important for stability of the TAC association. Furthermore, we demonstrate that the TAC, but not the kDNA, is required for correct TAP110 localization in vivo and suggest that TAP110 might interact with other proteins to form a >669 kDa complex.Summary StatementTAP110 is a novel mitochondrial genome segregation factor in Trypanosoma brucei that associates with the previously described TAC component TAC102. Ultrastructure expansion microscopy reveals its proximal position to the kDNA.

scholarly journals A new model for Trypanosoma cruzi heme homeostasis depends on modulation of TcHTE protein expression

2020 ◽  
Vol 295 (38) ◽  
pp. 13202-13212
Author(s):  
Lucas Pagura ◽  
Evelyn Tevere ◽  
Marcelo L. Merli ◽  
Julia A. Cricco
Keyword(s):  
Trypanosoma Cruzi ◽  
De Novo ◽  
Mrna Levels ◽  
Biological Processes ◽  
Transport Activity ◽  
Critical Pathway ◽  
Heme Transport ◽  
Life Cycle Stages ◽  
Insight Into

Heme is an essential cofactor for many biological processes in aerobic organisms, which can synthesize it de novo through a conserved pathway. Trypanosoma cruzi, the etiological agent of Chagas disease, as well as other trypanosomatids relevant to human health, are heme auxotrophs, meaning they must import it from their mammalian hosts or insect vectors. However, how these species import and regulate heme levels is not fully defined yet. It is known that the membrane protein TcHTE is involved in T. cruzi heme transport, although its specific role remains unclear. In the present work, we studied endogenous TcHTE in the different life cycle stages of the parasite to gain insight into its function in heme transport and homeostasis. We have confirmed that TcHTE is predominantly detected in replicative stages (epimastigote and amastigote), in which heme transport activity was previously validated. We also showed that in epimastigotes, TcHTE protein and mRNA levels decrease in response to increments in heme concentration, confirming it as a member of the heme response gene family. Finally, we demonstrated that T. cruzi epimastigotes can sense intracellular heme by an unknown mechanism and regulate heme transport to adapt to changing conditions. Based on these results, we propose a model in which T. cruzi senses intracellular heme and regulates heme transport activity by adjusting the expression of TcHTE. The elucidation and characterization of heme transport and homeostasis will contribute to a better understanding of a critical pathway for T. cruzi biology allowing the identification of novel and essential proteins.

scholarly journals Purification and Characterization of Lipase Produced by Leuconostoc mesenteroides Subsp. mesenteroides ATCC 8293 Using an Aqueous Two-Phase System (ATPS) Composed of Triton X-100 and Maltitol

Molecules ◽  
2018 ◽  
Vol 23 (7) ◽  
pp. 1800 ◽  
Author(s):  
Nurfadhilah Eko Sukohidayat ◽  
Mohammad Zarei ◽  
Badlishah Baharin ◽  
Mohd Manap
Keyword(s):  
Phase System ◽  
Two Phase ◽  
Aqueous Two Phase System ◽  
Process Characteristics ◽  
Sds Page ◽  
Ph Range ◽  
Lipase Purification ◽  
Triton X ◽  
First Time

Purification of lipase produced by L. mesenteroides subsp. mesenteroides ATCC 8293 was conducted for the first time using a novel aqueous two-phase system (ATPS) composed of Triton X-100 and maltitol. The partitioning of lipase was optimized according to several parameters including pH, temperature, and crude load. Results showed that lipase preferentially migrated to the Triton X-100 rich phase and optimum lipase partitioning was achieved in ATPS at TLL of 46.4% and crude load of 20% at 30 °C and pH 8, resulting in high lipase purification factor of 17.28 and yield of 94.7%. The purified lipase showed a prominent band on SDS-PAGE with an estimated molecular weight of 50 kDa. The lipase was stable at the temperature range of 30–60 °C and pH range of 6–11, however, it revealed its optimum activity at the temperature of 37 °C and pH 8. Moreover, lipase exhibited enhanced activity in the presence of non-ionic surfactants with increased activity up to 40%. Furthermore, results exhibited that metals ions such as Na+, Mg2+, K+ and Ca2+ stimulated lipase activity. This study demonstrated that this novel system could be potentially used as an alternative to traditional ATPS for the purification and recovery of enzymes since the purified lipase still possesses good process characteristics after undergoing the purification process.

scholarly journals Amastigotes of Trypanosoma cruzi escape destruction by the terminal complement components.

1989 ◽  
Vol 169 (3) ◽  
pp. 881-891 ◽  
Author(s):  
K Iida ◽  
M B Whitlow ◽  
V Nussenzweig
Keyword(s):  
Trypanosoma Cruzi ◽  
Alternative Pathway ◽  
Surface Protein ◽  
Protozoan Parasite ◽  
Mammalian Host ◽  
Insect Vector ◽  
Complement Components ◽  
Life Cycle Stages ◽  
Sds Page ◽  
Terminal Complement

We studied the effect of complement on two life cycle stages of the protozoan parasite Trypanosoma cruzi: epimastigotes, found in the insect vector, and amastigotes, found in the mammalian host. We found that while both stages activate vigorously the alternative pathway, only epimastigotes are destroyed. The amounts of C3 and C5b-7 deposited on the amastigotes were similar to those bound to the much larger epimastigotes. Binding of C9 to amastigotes was four to six times less than binding to epimastigotes, resulting in a lower C9/C5b-7 ratio. Although a fairly large amount of C9 bound stably to amastigotes, no functional channels were formed as measured by release of incorporated 86Rb. The bound C9 had the characteristic properties of poly-C9, that is, it expressed a neo-antigen unique to poly-C9, and migrated in SDS-PAGE with an apparent Mr greater than 10(5). The poly-C9 was removed from the surface of amastigotes by treatment with trypsin, indicating that it was not inserted in the lipid bilayer. Modification of amastigote surface by pronase treatment rendered the parasites susceptible to complement attack. These results suggest that amastigotes have a surface protein that binds to the C5b-9 complex and inhibits membrane insertion, thus protecting the parasites from complement-mediated lysis.

Characterization of an ABCA-like transporter involved in vesicular trafficking in the protozoan parasite Trypanosoma cruzi

2004 ◽  
Vol 54 (3) ◽  
pp. 632-646 ◽  
Author(s):  
Cristina Torres ◽  
F. Javier Pérez-Victoria ◽  
Adriana Parodi-Talice ◽  
Santiago Castanys ◽  
Francisco Gamarro
Keyword(s):  
Trypanosoma Cruzi ◽  
Protozoan Parasite ◽  
Vesicular Trafficking

scholarly journals Molecular Characterization of Sarcocystis Species Isolated from Sheep and Goats in Riyadh, Saudi Arabia

Animals ◽  
2019 ◽  
Vol 9 (5) ◽  
pp. 256 ◽  
Author(s):  
Dina M. Metwally ◽  
Mashael A. Al-Damigh ◽  
Isra M. Al-Turaiki ◽  
Manal F. El-Khadragy
Keyword(s):  
Saudi Arabia ◽  
Protozoan Parasite ◽  
Final Host ◽  
Molecular Testing ◽  
Sarcocystis Species ◽  
Sheep And Goats ◽  
Transmission Electron ◽  
First Time ◽  
Intracellular Protozoan Parasite

Sarcocystosis is induced by species of Sarcocystis, which is an intracellular protozoan parasite in the phylum Apicomplexa. The diversity and importance of Sarcocystis species in sheep and goats in Saudi Arabia are poorly understood. In this study, the tongue, esophagus, heart, diaphragm, and skeletal muscles were collected from 230 sheep and 84 goats, and the tissues were examined for the presence of Sarcocystis species by macroscopic examination and light microscopy. Microscopic Sarcocystis species cysts were found in both sheep and goats. Transmission electron microscopy (TEM) revealed S. tenella in sheep and S. capracanis in goats. Sarcocystis species were confirmed for the first time in Saudi Arabian sheep and goats by molecular testing. S. capracanis was most closely related to S. tenella, with the COX1 sequences sharing 91.7% identity. A phylogenetic analysis produced similar results and indicated that the Sarcocystis isolates were within a group of Sarcocystis species in which dogs were the final host. Finally, the Sarcocystis species cysts from sheep and goats could be grouped together, indicating that they were strongly related.

Heterogeneity of metalloprotease expression in Trypanosoma cruzi

Parasitology ◽  
1996 ◽  
Vol 112 (4) ◽  
pp. 393-399 ◽  
Author(s):  
C. M. Lowndes ◽  
M. C. Bonaldo ◽  
N. Thomaz ◽  
S. Goldenberg
Keyword(s):  
Trypanosoma Cruzi ◽  
Developmental Regulation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cysteine Proteases ◽  
Published Data ◽  
Regulation Of Expression ◽  
Life Cycle Stages ◽  
Different Strains ◽  
Triton X ◽  
High Degree

SUMMARYExpression of metalloprotease activities during metacyclogenesis of a series of strains and clones of Trypanosoma cruzi was investigated using SDS-polyacrylamide gel electrophoresis with a range of different co-polymerized proteolytic substrates. A complex pattern of metalloprotease expression, with considerable quantitative and qualitative variability between strains and life-cycle stages, was apparent. This is in contrast to previously published data, and data presented in this study, which demonstrate a high degree of conservation of expression of cysteine proteases in different strains and clones of T. cruzi. All the metalloprotease activities identified partitioned into the detergent phase of a Triton X-114 extract, suggesting that they are membrane-bound. Developmental regulation of expression during metacyclogenesis, either in terms of quantity, or in terms of difference in relative amounts of different isoforms, was apparent for all isolates studied except CL14. However, a clearly metacyclic-specific/metacyclic-enriched metalloprotease was detected only in T. cruzi Dm28c and 383, and our results demonstrate that a metacyclic-specific metalloprotease common to all isolates of the parasite could not be detected, at least at this level of analysis.

Sign in / Sign up

Export Citation Format

Share Document