Anti-Trypanosoma cruzieffects of cyclosporin A derivatives: possible role of a P-glycoprotein and parasite cyclophilins

SUMMARYCyclophilins are target molecules for cyclosporin A (CsA), an immunosuppressive antimicrobial drug. We have previously reported thein vitroanti-Trypanosoma cruziactivity of H-7-94 and F-7-62 non-immunosuppressive CsA analogues. In this work, we continue the study of the parasiticidal effect of H-7-94 and F-7-62 CsA analoguesin vitroandin vivoand we analyse 3 new CsA derivatives: MeIle-4-CsA (NIM 811), MeVal-4-CsA (MeVal-4) and D-MeAla-3-EtVal-4-CsA, (EtVal-4). The most efficient anti-T. cruzieffect was observed with H-7-94, F-7-62 and MeVal-4 CsA analogues evidenced as inhibition of epimastigote proliferation, trypomastigote penetration, intracellular amastigote development andin vivo T. cruziinfection. This trypanocidal activity could be due to inhibition of the peptidyl prolylcis-transisomerase activity on theT. cruzirecombinant cyclophilins tested. Furthermore, CsA and F-7-62 derivative inhibited the efflux of rhodamine 123 fromT. cruziepimastigotes, suggesting an interference with a P-glycoprotein activity. Moreover, H-7-94 and F-7-62 CsA analogues were not toxic as shown by cell viability and by aminopyrine-N-demethylase activity on mammalian cells. Our results show that H-7-94, F-7-62 and MeVal-4 CsA analogues expressed the highest inhibiting effects onT. cruzi, being promissory parasiticidal drugs worthy of further studies.

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Functional genetic encoding of sulfotyrosine in mammalian cells

Nature Communications ◽

10.1038/s41467-020-18629-9 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Xinyuan He ◽

Yan Chen ◽

Daisy Guiza Beltran ◽

Maia Kelly ◽

Bin Ma ◽

...

Keyword(s):

Mammalian Cells ◽

Biochemical Characterization ◽

Trna Synthetase ◽

Functional Verification ◽

Cellular Processes ◽

Genetic Encoding ◽

Efficient Expression

Abstract Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.

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Streptolysin-induced endoplasmic reticulum stress promotes group A streptococcal in vivo biofilm formation and necrotizing fasciitis

10.1101/183012 ◽

2017 ◽

Author(s):

Anuradha Vajjala ◽

Debabrata Biswas ◽

Kelvin Kian Long Chong ◽

Wei Hong Tay ◽

Emanuel Hanski ◽

...

Keyword(s):

Soft Tissue ◽

Er Stress ◽

Necrotizing Fasciitis ◽

Biofilm Formation ◽

Mammalian Cells ◽

Chronic Infections ◽

Group A

AbstractGroup A Streptococcus (GAS) is a human pathogen that causes infections ranging from mild to fulminant and life-threatening. Biofilms have been implicated in acute GAS soft-tissue infections such as necrotizing fasciitis (NF). However, most in vitro models used to study GAS biofilms have been designed to mimic chronic infections and insufficiently recapitulate in vivo conditions and the host-pathogen interactions that might influence biofilm formation. Here we establish and characterize an in vitro model of GAS biofilm development on mammalian cells that simulates microcolony formation observed in a murine model of human NF. We show that on mammalian cells, GAS forms dense aggregates that display hallmark biofilm characteristics including a three-dimensional architecture and enhanced tolerance to antibiotics. In contrast to abiotic-grown biofilms, host-associated biofilms require the expression of secreted GAS streptolysins O and S (SLO, SLS) resulting in the release of a host-associated biofilm promoting-factor(s). Supernatants from GAS-infected mammalian cells or from cells treated with endoplasmic reticulum (ER) stressors restore biofilm formation to an SLO and SLS null mutant that is otherwise attenuated in biofilm formation on cells, together suggesting a role for streptolysin-induced ER stress in this process. In an in vivo mouse model, the streptolysin-null mutant is attenuated in both microcolony formation and bacterial spread, but pre-treatment of softtissue with an ER-stressor restores the ability of the mutant to form wild type like microcolonies that disseminate throughout the soft tissue. Taken together, we have identified a new role of streptolysin-driven ER stress in GAS biofilm formation and NF disease progression.Significance StatementAlthough it is well-accepted that bacterial biofilms are associated with many chronic infections, little is known about the mechanisms by which group A Streptococcus (GAS) biofilms contribute to acute soft tissue-invasive diseases like necrotizing fasciitis (NF). In this study, we establish a physiologically relevant in vitro model to study GAS biofilm formation on mammalian cells and validate our findings in a mouse model that mimics human NF. This study demonstrates a novel role of GAS streptolysin-mediated ER stress in the development and spread of GAS biofilms in acute softtissue infections. We also show that biofilm formation depends on the release of a host-associated factor that promotes microcolony formation and GAS dissemination in vivo.

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Factors involved in the modulation of cell proliferation in vivo and in vitro: The role of fibroblast and epidermal growth factors in the proliferative response of mammalian cells

In Vitro ◽

10.1007/bf02618177 ◽

1978 ◽

Vol 14 (1) ◽

pp. 85-118 ◽

Cited By ~ 247

Author(s):

D. Gospodarowicz ◽

G. Greenburg ◽

H. Bialecki ◽

B. R. Zetter

Keyword(s):

Cell Proliferation ◽

Growth Factors ◽

Mammalian Cells ◽

Proliferative Response ◽

Epidermal Growth Factors ◽

Epidermal Growth

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Role of proteoglycans and glycosaminoglycans in Duchenne muscular dystrophy

Glycobiology ◽

10.1093/glycob/cwy058 ◽

2018 ◽

Vol 29 (2) ◽

pp. 110-123 ◽

Cited By ~ 8

Author(s):

Laurino Carmen ◽

Vadala’ Maria ◽

Julio Cesar Morales-Medina ◽

Annamaria Vallelunga ◽

Beniamino Palmieri ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Mammalian Cells ◽

Muscle Development ◽

Glycoprotein Complex ◽

Experimental Therapies ◽

Dystrophin Protein

Abstract Duchenne muscular dystrophy (DMD) is an inherited fatal X-linked myogenic disorder with a prevalence of 1 in 3500 male live births. It affects voluntary muscles, and heart and breathing muscles. DMD is characterized by continuous degeneration and regeneration cycles resulting in extensive fibrosis and a progressive reduction in muscle mass. Since the identification of a reduction in dystrophin protein as the cause of this disorder, numerous innovative and experimental therapies, focusing on increasing the levels of dystrophin, have been proposed, but the clinical improvement has been unsatisfactory. Dystrophin forms the dystrophin-associated glycoprotein complex and its proteins have been studied as a promising novel therapeutic target to treat DMD. Among these proteins, cell surface glycosaminoglycans (GAGs) are found almost ubiquitously on the surface and in the extracellular matrix (ECM) of mammalian cells. These macromolecules interact with numerous ligands, including ECM constituents, adhesion molecules and growth factors that play a crucial role in muscle development and maintenance. In this article, we have reviewed in vitro, in vivo and clinical studies focused on the functional role of GAGs in the pathophysiology of DMD with the final aim of summarizing the state of the art of GAG dysregulation within the ECM in DMD and discussing future therapeutic perspectives.

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Relevance of p-glycoprotein for the enteral absorption of cyclosporin A: in vitro-in vivo correlation

British Journal of Pharmacology ◽

10.1111/j.1476-5381.1996.tb15612.x ◽

1996 ◽

Vol 118 (7) ◽

pp. 1841-1847 ◽

Cited By ~ 158

Author(s):

G. Fricker ◽

J. Drewe ◽

J. Huwyler ◽

H. Gutmann ◽

C. Beglinger

Keyword(s):

Cyclosporin A ◽

Enteral Absorption ◽

P Glycoprotein

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Testing of Schefflera vinosa extract in mammalian cells in vitro and in vivo for potential toxicity, genetic damage, and role of oxidation

Journal of Toxicology and Environmental Health Part A ◽

10.1080/15287394.2016.1229238 ◽

2016 ◽

Vol 79 (24) ◽

pp. 1201-1210 ◽

Cited By ~ 7

Author(s):

Natália Helen Ferreira ◽

Kelly Jacqueline Barbosa de Andrade ◽

Luís Fernando Leandro ◽

Nathália Oliveira Acésio ◽

Suzana Amorim Mendes ◽

...

Keyword(s):

Mammalian Cells ◽

Genetic Damage ◽

Potential Toxicity

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NAADP as a second messenger: neither CD38 nor base-exchange reaction are necessary for in vivo generation of NAADP in myometrial cells

AJP Cell Physiology ◽

10.1152/ajpcell.00638.2005 ◽

2007 ◽

Vol 292 (1) ◽

pp. C227-C239 ◽

Cited By ~ 72

Author(s):

Sandra Soares ◽

Michael Thompson ◽

Thomas White ◽

Amir Isbell ◽

Michiko Yamasaki ◽

...

Keyword(s):

Exchange Reaction ◽

Mammalian Cells ◽

Second Messenger ◽

Base Exchange ◽

Myometrial Cells ◽

Intracellular Ca ◽

Messenger System

Nicotinic acid adenine dinucleotide phosphate (NAADP) has recently been shown to act as a second messenger controlling intracellular Ca2+ responses in mammalian cells. Many questions remain regarding this signaling pathway, including the role of the ryanodine receptor (RyR) in NAADP-induced Ca2+ transients. Furthermore, the exact metabolic pathway responsible for the synthesis of NAADP in vivo has not been determined. Here, we demonstrate that the NAADP mediated Ca2+ release system is present in human myometrial cells. We also demonstrate that human myometrial cells use the NAADP second messenger system to generate intracellular Ca2+ transients in response to histamine. It has been proposed in the past that the NAADP system in mammalian cells is dependent on the presence of functional RyRs. Here, we observed that the histamine-induced Ca2+ transients are dependent on both the NAADP and inositol 1,4,5-trisphosphate signaling pathways but are independent of RyRs. The enzyme CD38 has been shown to catalyze the synthesis of NAADP in vitro by the base-exchange reaction. Furthermore, it has been proposed that this enzyme is responsible for the intracellular generation of NAADP in vivo. Using CD38 knockout mice, we observed that both the basal and histamine stimulated levels of NAADP are independent of CD38 and the base-exchange reaction. Our group is the first to demonstrate that NAADP is a second messenger for histamine-elicited Ca2+ transients in human myometrial cells. Furthermore, the NAADP mediated mechanism in mammalian cells can be independent of RyRs and CD38. Our data provides novel insights into the understanding of the mechanism of action and metabolism of this new second messenger system.

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In Vitro and In Vivo Trypanocidal Effects of the Cyclopalladated Compound 7a, a Drug Candidate for Treatment of Chagas' Disease

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00323-10 ◽

2010 ◽

Vol 54 (8) ◽

pp. 3318-3325 ◽

Cited By ~ 32

Author(s):

Alisson L. Matsuo ◽

Luis S. Silva ◽

Ana C. Torrecilhas ◽

Bruno S. Pascoalino ◽

Thiago C. Ramos ◽

...

Keyword(s):

Electron Microscopy ◽

Chagas Disease ◽

Cell Invasion ◽

Mammalian Cells ◽

New Drugs ◽

Drug Candidate ◽

Acute Form ◽

Intracellular Amastigote

ABSTRACT Chagas' disease, a neglected tropical infection, affects about 18 million people, and 100 million are at risk. The only drug available, benznidazole, is effective in the acute form and in the early chronic form, but its efficacy and tolerance are inversely related to the age of the patients. Side effects are frequent in elderly patients. The search for new drugs is thus warranted. In the present study we evaluated the in vitro and in vivo effect of a cyclopalladated compound (7a) against Trypanosoma cruzi, the agent of Chagas' disease. The 7a compound inhibits trypomastigote cell invasion, decreases intracellular amastigote proliferation, and is very effective as a trypanocidal drug in vivo, even at very low dosages. It was 340-fold more cytotoxic to parasites than to mammalian cells and was more effective than benznidazole in all in vitro and in vivo experiments. The 7a cyclopalladate complex exerts an apoptosis-like death in T. cruzi trypomastigote forms and causes mitochondrion disruption seen by electron microscopy.

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Role of P Glycoprotein in the Course and Treatment of Encephalitozoon Microsporidiosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.1.73-78.2001 ◽

2001 ◽

Vol 45 (1) ◽

pp. 73-78 ◽

Cited By ~ 11

Author(s):

Gordon J. Leitch ◽

Mary Scanlon ◽

Andrew Shaw ◽

Govinda S. Visvesvara

Keyword(s):

Multidrug Resistance ◽

Cyclosporin A ◽

Parasitophorous Vacuole ◽

Host Cells ◽

P Glycoprotein ◽

Antiparasitic Drug ◽

Infected Cells ◽

Intracellular Fluorescence

ABSTRACT Encephalitozoon microsporidia are obligate intracellular protozoan parasites that proliferate and differentiate within a parasitophorous vacuole inside host cells that are usually epithelial in nature. Isolates of the three species of theEncephalitozoon microsporidia, E. cuniculi,E. hellem, and E. intestinalis, were obtained from AIDS patients and cultured in green monkey (E6) kidney cells. Anti-P-glycoprotein (anti-Pgp) and anti-multidrug resistance-associated protein (anti-MRP) monoclonal antibodies were used to probe for multidrug resistance (MDR) pump epitopes and verapamil- or cyclosporin A- and probenecid-modulated intracellular calcein fluorescence were used to assess the expression of Pgp and MRP respectively in uninfected and infected cells. Pgp, but not MRP, was detected immunocytochemically and by verapamil- and cyclosporin A-potentiated intracellular fluorescence in both host cells and parasite developing stages. When an in vitro infection assay was employed, verapamil and cyclosporin A acted as chemosensitizing agents for the antiparasitic drug albendazole. These observations suggest that inhibiting host cell and perhaps parasite MDR pumps may increase the efficacy of antiparasitic agents in these and other microsporidia species.

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