Factors influencing the recovery from potentially lethal damage (PLD) in mammalian cells in vitro and in vivo

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

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Cytological changes induced by platinum-thymine in sarcoma-180 cells: Electron microscopy study

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010015900x ◽

1990 ◽

Vol 48 (3) ◽

pp. 290-291

Author(s):

Gustav Ofosu

Keyword(s):

Mammalian Cells ◽

Antitumor Agent ◽

Microscopy Study ◽

Electron Microscopy Study ◽

Limiting Factor ◽

Sarcoma 180 ◽

Electron Microscopic ◽

Cytological Changes

Platinum-thymine has been found to be a potent antitumor agent, which is quite soluble in water, and lack nephrotoxicity as the dose-limiting factor. The drug has been shown to interact with DNA and inhibits DNA, RNA and protein synthesis in mammalian cells in vitro. This investigation was undertaken to elucidate the cytotoxic effects of piatinum-thymine on sarcoma-180 cells in vitro ultrastructurally, Sarcoma-180 tumor bearing mice were treated with intraperitoneal injection of platinum-thymine 40mg/kg. A concentration of 60μg/ml dose of platinum-thymine was used in in vitro experiments. Treatments were at varying time intervals of 3, 7 and 21 days for in vivo experiments, and 30, 60 and 120 min., 6, 12, and 24th in vitro. Controls were not treated with platinum-thymine.Electron microscopic analyses of the treated cells in vivo and in vitro showed drastic cytotoxic effect.

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Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

10.26434/chemrxiv.5844393 ◽

2018 ◽

Author(s):

Noor H. Dashti ◽

Rufika S. Abidin ◽

Frank Sainsbury

Keyword(s):

Self Assembly ◽

Mammalian Cells ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Super Resolution ◽

Cell Engineering ◽

Particle Analysis ◽

Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both in vitro and in vivo cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for in vivo self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by in vitro self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

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The Role of nitro (NO2-), chloro (Cl), and fluoro (F) Substitution in the Design of Antileishmanial and Antichagasic Compounds

Current Drug Targets ◽

10.2174/1389450121666201228122239 ◽

2020 ◽

Vol 21 ◽

Author(s):

Boniface Pone ◽

Ferreira Igne Elizabeth

Keyword(s):

Chagas Disease ◽

Mammalian Cells ◽

Neglected Tropical Diseases ◽

New Drugs ◽

Pharmacokinetic Studies ◽

Scientific Publications ◽

Antitrypanosomal Activity ◽

Chemical Groups

: Neglected tropical diseases (NTDs) are responsible for over 500,000 deaths annually and are characterized by multiple disabilities. Leishmaniasis and Chagas disease are among the most severe NTDs, and are caused by the Leishmania sp, and Trypanosoma cruzi, respectively. Glucantime, pentamidine and miltefosine are commonly used to treat leishmaniasis, whereas nifurtimox, benznidazole are current treatments for Chagas disease. However, these treatments are associated with drug resistance, and severe side effects. Hence, the development of synthetic products, especially those containing N02, F, or Cl, which chemical groups are known to improve the biological activity. The present work summarizes the information on the antileishmanial and antitrypanosomal activity of nitro-, chloro-, and fluoro-synthetic derivatives. Scientific publications referring to halogenated derivatives in relation to antileishmanial and antitrypanosomal activities were hand searched in databases such as SciFinder, Wiley, Science Direct, PubMed, ACS, Springer, Scielo, and so on. According to the literature information, more than 90 compounds were predicted as lead molecules with reference to their IC50/EC50 values in in vitro studies. It is worth to mention that only active compounds with known cytotoxic effects against mammalian cells were considered in the present study. The observed activity was attributed to the presence of nitro-, fluoro- and chloro-groups in the compound backbone. All in all, nitro and h0alogenated derivatives are active antileishmanial and antitrypanosomal compounds and can serve as baseline for the development of new drugs against leishmaniasis and Chagas disease. However, efforts on in vitro and in vivo toxicity studies of the active synthetic compounds is still needed. Pharmacokinetic studies, and the mechanism of action of the promising compounds need to be explored. The use of new catalysts and chemical transformation can afford unexplored halogenated compounds with improved antileishmanial and antitrypanosomal activity.

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Discovery of 3-Cinnamamido-N-Substituted Benzamides as Potential Antimalarial Agents

Medicinal Chemistry ◽

10.2174/1573406416666200817160708 ◽

2020 ◽

Vol 16 ◽

Author(s):

Haicheng Liu ◽

Yushi Futamura ◽

Honghai Wu ◽

Aki Ishiyama ◽

Taotao Zhang ◽

...

Keyword(s):

Mammalian Cells ◽

Antimalarial Activity ◽

In Vitro Assays ◽

Compound Library ◽

Antimalarial Agents ◽

Phenotypic Screen ◽

Ic50 Values ◽

Substituted Benzamides

Background: Malaria is one of the most devastating parasitic diseases, yet the discovery of antimalarial agents remains profoundly challenging. Very few new antimalarials have been developed in the past 50 years, while the emergence of drug-resistance continues to appear. Objective: This study focuses on the discovery, design, synthesis, and antimalarial evaluation of 3-cinnamamido-N-substituted benzamides. Method: In this study, a screening of our compound library was carried out against the multidrug-sensitive Plasmodium falciparum 3D7 strain. Derivatives of the hit were designed, synthesized and tested against P. falciparum 3D7 and the in vivo antimalarial activity of the most active compounds was evaluated using the method of Peters’ 4-day suppressive test. Results: The retrieved hit compound 1 containing a 3-cinnamamido-N-substituted benzamide skeleton showed moderate antimalarial activity (IC50 = 1.20 µM) for the first time. A series of derivatives were then synthesized through a simple four-step workflow, and half of them exhibited slightly better antimalarial effect than the precursor 1 during the subsequent in vitro assays. Additionally, compounds 11, 23, 30 and 31 displayed potent activity with IC50 values of approximately 0.1 µM, and weak cytotoxicity against mammalian cells. However, in vivo antimalarial activity is not effective which might be ascribed to the poor solubility of these compounds. Conclusion: In this study, phenotypic screen of our compound library resulted in the first report of 3-cinnamamide framework with antimalarial activity and 40 derivatives were then designed and synthesized. Subsequent structure-activity studies showed that compounds 11, 23, 30 and 31 exhibited the most potent and selective activity against P. falciparum 3D7 strain with IC50 values around 0.1 µM. Our work herein sets another example of phenotypic screen-based drug discovery, leading to potentially promising candidates of novel antimalarial agents once given further optimization.

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In Silico Predicted Antifungal Peptides: In Vitro and In Vivo Anti-Candida Activity

Journal of Fungi ◽

10.3390/jof7060439 ◽

2021 ◽

Vol 7 (6) ◽

pp. 439

Author(s):

Tecla Ciociola ◽

Walter Magliani ◽

Tiziano De Simone ◽

Thelma A. Pertinhez ◽

Stefania Conti ◽

...

Keyword(s):

In Silico ◽

Mammalian Cells ◽

Galleria Mellonella ◽

Ex Vivo ◽

Consensus Sequence ◽

In Silico Analysis ◽

Significant Activity ◽

Synthetic Antibody

It has been previously demonstrated that synthetic antibody-derived peptides could exert a significant activity in vitro, ex vivo, and/or in vivo against microorganisms and viruses, as well as immunomodulatory effects through the activation of immune cells. Based on the sequence of previously described antibody-derived peptides with recognized antifungal activity, an in silico analysis was conducted to identify novel antifungal candidates. The present study analyzed the candidacidal and structural properties of in silico designed peptides (ISDPs) derived by amino acid substitutions of the parent peptide KKVTMTCSAS. ISDPs proved to be more active in vitro than the parent peptide and all proved to be therapeutic in Galleria mellonella candidal infection, without showing toxic effects on mammalian cells. ISDPs were studied by circular dichroism spectroscopy, demonstrating different structural organization. These results allowed to validate a consensus sequence for the parent peptide KKVTMTCSAS that may be useful in the development of novel antimicrobial molecules.

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Review of T-2307, an Investigational Agent That Causes Collapse of Fungal Mitochondrial Membrane Potential

Journal of Fungi ◽

10.3390/jof7020130 ◽

2021 ◽

Vol 7 (2) ◽

pp. 130

Author(s):

Nathan P. Wiederhold

Keyword(s):

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Candida Species ◽

Mammalian Cells ◽

Mitochondrial Membrane ◽

Fungal Infections ◽

Published Data ◽

Candida Auris

Invasive infections caused by Candida that are resistant to clinically available antifungals are of increasing concern. Increasing rates of fluconazole resistance in non-albicans Candida species have been documented in multiple countries on several continents. This situation has been further exacerbated over the last several years by Candida auris, as isolates of this emerging pathogen that are often resistant to multiple antifungals. T-2307 is an aromatic diamidine currently in development for the treatment of invasive fungal infections. This agent has been shown to selectively cause the collapse of the mitochondrial membrane potential in yeasts when compared to mammalian cells. In vitro activity has been demonstrated against Candida species, including C. albicans, C. glabrata, and C. auris strains, which are resistant to azole and echinocandin antifungals. Activity has also been reported against Cryptococcus species, and this has translated into in vivo efficacy in experimental models of invasive candidiasis and cryptococcosis. However, little is known regarding the clinical efficacy and safety of this agent, as published data from studies involving humans are not currently available.

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Phospholipase D1b and D2a generate structurally identical phosphatidic acid species in mammalian cells

Biochemical Journal ◽

10.1042/bj3600707 ◽

2001 ◽

Vol 360 (3) ◽

pp. 707-715 ◽

Cited By ~ 35

Author(s):

Trevor R. PETTITT ◽

Mark McDERMOTT ◽

Khalid M. SAQIB ◽

Neil SHIMWELL ◽

Michael J. O. WAKELAM

Keyword(s):

Liquid Chromatography ◽

Phosphatidic Acid ◽

Phospholipase D ◽

Mammalian Cells ◽

Inducible Promoter ◽

In Vitro Assays ◽

Protein Levels ◽

Intracellular Messenger

Mammalian cells contain different phospholipase D enzymes (PLDs) whose distinct physiological roles are poorly understood and whose products have not been characterized. The development of porcine aortic endothelial (PAE) cell lines able to overexpress PLD-1b or −2a under the control of an inducible promoter has enabled us to characterize both the substrate specificity and the phosphatidic acid (PtdOH) product of these enzymes under controlled conditions. Liquid chromatography–MS analysis showed that PLD1b- and PLD2a-transfected PAE cells, as well as COS7 and Rat1 cells, generate similar PtdOH and, in the presence of butan-1-ol, phosphatidylbutanol (PtdBut) profiles, enriched in mono- and di-unsaturated species, in particular 16:0/18:1. Although PtdBut mass increased, the species profile did not change in cells stimulated with ATP or PMA. Overexpression of PLD made little difference to basal or stimulated PtdBut formation, indicating that activity is tightly regulated in vivo and that factors other than just PLD protein levels limit hydrolytic function. In vitro assays using PLD-enriched lysates showed that the enzyme could utilize both phosphatidylcholine and, much less efficiently, phosphatidylethanolamine, with slight selectivity towards mono- and di-unsaturated species. Phosphatidylinositol was not a substrate. Thus PLD1b and PLD2a hydrolyse a structurally similar substrate pool to generate an identical PtdOH product enriched in mono- and di-unsaturated species that we propose to function as the intracellular messenger forms of this lipid.

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Preclinical Studies in Anti-Trypanosomatidae Drug Development

Pharmaceuticals ◽

10.3390/ph14070644 ◽

2021 ◽

Vol 14 (7) ◽

pp. 644

Author(s):

Cintya Perdomo ◽

Elena Aguilera ◽

Ileana Corvo ◽

Paula Faral-Tello ◽

Elva Serna ◽

...

Keyword(s):

Rural Communities ◽

Chagas Disease ◽

Murine Model ◽

Mammalian Cells ◽

Drug Candidate ◽

Murine Macrophages ◽

Causative Agents ◽

Trypanosomatid Parasites

The trypanosomatid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania are the causative agents of human African trypanosomiasis, Chagas Disease and Leishmaniasis, respectively. These infections primarily affect poor, rural communities in the developing world, and are responsible for trapping sufferers and their families in a disease/poverty cycle. The development of new chemotherapies is a priority given that existing drug treatments are problematic. In our search for novel anti-trypanosomatid agents, we assess the growth-inhibitory properties of >450 compounds from in-house and/or “Pathogen Box” (PBox) libraries against L. infantum, L. amazonensis, L.braziliensis, T. cruzi and T. brucei and evaluate the toxicities of the most promising agents towards murine macrophages. Screens using the in-house series identified 17 structures with activity against and selective toward Leishmania: Compounds displayed 50% inhibitory concentrations between 0.09 and 25 μM and had selectivity index values >10. For the PBox library, ~20% of chemicals exhibited anti-parasitic properties including five structures whose activity against L. infantum had not been reported before. These five compounds displayed no toxicity towards murine macrophages over the range tested with three being active in an in vivo murine model of the cutaneous disease, with 100% survival of infected animals. Additionally, the oral combination of three of them in the in vivo Chagas disease murine model demonstrated full control of the parasitemia. Interestingly, phenotyping revealed that the reference strain responds differently to the five PBox-derived chemicals relative to parasites isolated from a dog. Together, our data identified one drug candidate that displays activity against Leishmania and other Trypanosomatidae in vitro and in vivo, while exhibiting low toxicity to cultured mammalian cells and low in vivo acute toxicity.

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STRETCHABLE Lab-on-Chip Device With Impedance Spectroscopy Capability for Mammalian Cell Studies

Volume 10: Micro- and Nano-Systems Engineering and Packaging ◽

10.1115/imece2016-66156 ◽

2016 ◽

Cited By ~ 1

Author(s):

Xudong Zhang ◽

Anis Nurashikin Nordin ◽

Fang Li ◽

Ioana Voiculescu

Keyword(s):

Impedance Spectroscopy ◽

Mammalian Cells ◽

Dynamic Environment ◽

Cyclic Strain ◽

Cyclic Stretch ◽

Mechanical Stimuli ◽

Aortic Endothelial Cells ◽

Lab On Chip

This paper presents the fabrication and testing of electric cell-substrate impedance spectroscopy (ECIS) electrodes on a stretchable membrane. This is the first time when ECIS electrodes were fabricated on a stretchable substrate and ECIS measurements on mammalian cells exposed to cyclic strain of 10% were successfully demonstrated. A chemical was used to form strong chemical bond between gold electrodes of ECIS sensor and polymer membrane, which enable the electrodes keep good conductive ability during cyclic stretch. The stretchable membrane integrated with the ECIS sensor can simulate and replicate the dynamic environment of organism and enable the analysis of the cells activity involved in cells attachment and proliferation in vitro. Bovine aortic endothelial cells (BAEC) were used to evaluate the endothelial function influenced by mechanical stimuli in this research because they undergo in vivo cyclic physiologic elongation produced by the blood circulation in the arteries.

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