scholarly journals Direct Observation of Sophorolipid Micelle Docking in Model Membranes and Cells by Single Particle Studies Reveals Optimal Fusion Conditions

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1291 ◽  
Author(s):  
Pradeep Kumar Singh ◽  
Søren S.-R. Bohr ◽  
Nikos S. Hatzakis
Keyword(s):  
Cancer Cells ◽  
Hela Cells ◽  
Single Particle ◽  
Mammalian Cells ◽  
Model Membranes ◽  
Live Cells ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Sophorolipids (SLs) are naturally produced glycolipids that acts as drug delivery for a spectrum of biomedical applications, including as an antibacterial antifungal and anticancer agent, where they induce apoptosis selectively in cancerous cells. Despite their utility, the mechanisms underlying their membrane interactions, and consequently cell entry, remains unknown. Here, we combined a single liposome assay to observe directly and quantify the kinetics of interaction of SL micelles with model membrane systems, and single particle studies on live cells to record their interaction with cell membranes and their cytotoxicity. Our single particle readouts revealed several repetitive docking events on individual liposomes and quantified how pH and membrane charges, which are known to vary in cancer cells, affect the docking of SL micelles on model membranes. Docking of sophorolipids micelles was found to be optimal at pH 6.5 and for membranes with −5% negatively charge lipids. Single particle studies on mammalian cells reveled a two-fold increased interaction on Hela cells as compared to HEK-293 cells. This is in line with our cell viability readouts recording an approximate two-fold increased cytotoxicity by SLs interactions for Hela cells as compared to HEK-293 cells. The combined in vitro and cell assays thus support the increased cytotoxicity of SLs on cancer cells to originate from optimal charge and pH interactions between membranes and SL assemblies. We anticipate studies combining quantitative single particle studies on model membranes and live cell may reveal hitherto unknown molecular insights on the interactions of sophorolipid and additional nanocarriers mechanism.

Effect of human acetylcholinesterase subunit assembly on its circulatory residence

2001 ◽  
Vol 354 (3) ◽  
pp. 613-625 ◽  
Author(s):  
Theodor CHITLARU ◽  
Chanoch KRONMAN ◽  
Baruch VELAN ◽  
Avigdor SHAFFERMAN
Keyword(s):  
Laser Desorption Ionization ◽  
Wild Type ◽  
Ionization Time ◽  
Hek 293 ◽  
Enzyme Preparations ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Differential Behaviour ◽  
Oligomeric Forms

Sialylated recombinant human acetylcholinesterase (rHuAChE), produced by stably transfected cells, is composed of a mixed population of monomers, dimers and tetramers and manifests a time-dependent circulatory enrichment of the higher-order oligomeric forms. To investigate this phenomenon further, homogeneous preparations of rHuAChE differing in their oligomerization statuses were generated: (1) monomers, represented by the oligomerization-impaired C580A-rHuAChE mutant, (2) wild-type (WT) dimers and (3) tetramers of WT-rHuAChE generated in vitro by complexation with a synthetic ColQ-derived proline-rich attachment domain (‘PRAD’) peptide. Three different series of each of these three oligoform preparations were produced: (1) partly sialylated, derived from HEK-293 cells; (2) fully sialylated, derived from engineered HEK-293 cells expressing high levels of sialyltransferase; and (3) desialylated, after treatment with sialidase to remove sialic acid termini quantitatively. The oligosaccharides associated with each of the various preparations were extensively analysed by matrix-assisted laser desorption ionization–time-of-flight MS. With the enzyme preparations comprising the fully sialylated series, a clear linear relationship between oligomerization and circulatory mean residence time (MRT) was observed. Thus monomers, dimers and tetramers exhibited MRTs of 110, 195 and 740min respectively. As the level of sialylation decreased, this differential behaviour became less pronounced; eventually, after desialylation all oligoforms had the same MRT (5min). These observations suggest that multiple removal systems contribute to the elimination of AChE from the circulation. Here we also demonstrate that by the combined modulation of sialylation and tetramerization it is possible to generate a rHuAChE displaying a circulatory residence exceeding that of all other known forms of native or recombinant human AChE.

The effects of zolpidem treatment and withdrawal on the in vitro expression of recombinant α1β2γ2s GABAA receptors expressed in HEK 293 cells

2010 ◽  
Vol 382 (3) ◽  
pp. 201-212 ◽  
Author(s):  
Josipa Vlainić ◽  
Maja Jazvinšćak Jembrek ◽  
Dubravka Švob Štrac ◽  
Danka Peričić
Keyword(s):  
Gabaa Receptors ◽  
Hek 293 ◽  
In Vitro Expression ◽  
Hek 293 Cells ◽  
293 Cells

scholarly journals Critical roles for p22phox in the structural maturation and subcellular targeting of Nox3

2007 ◽  
Vol 403 (1) ◽  
pp. 97-108 ◽  
Author(s):  
Yoko Nakano ◽  
Botond Banfi ◽  
Algirdas J. Jesaitis ◽  
Mary C. Dinauer ◽  
Lee-Ann H. Allen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Subcellular Targeting ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Regulatory Subunits ◽  
Hek 293 Cells ◽  
293 Cells ◽  
A Subunit ◽  
And Function

Otoconia are small biominerals in the inner ear that are indispensable for the normal perception of gravity and motion. Normal otoconia biogenesis requires Nox3, a Nox (NADPH oxidase) highly expressed in the vestibular system. In HEK-293 cells (human embryonic kidney cells) transfected with the Nox regulatory subunits NoxO1 (Nox organizer 1) and NoxA1 (Nox activator 1), functional murine Nox3 was expressed in the plasma membrane and exhibited a haem spectrum identical with that of Nox2, the electron transferase of the phagocyte Nox. In vitro Nox3 cDNA expressed an ∼50 kDa primary translation product that underwent N-linked glycosylation in the presence of canine microsomes. RNAi (RNA interference)-mediated reduction of endogenous p22phox, a subunit essential for stabilization of Nox2 in phagocytes, decreased Nox3 activity in reconstituted HEK-293 cells. p22phox co-precipitated not only with Nox3 and NoxO1 from transfectants expressing all three proteins, but also with NoxO1 in the absence of Nox3, indicating that p22phox physically associated with both Nox3 and with NoxO1. The plasma membrane localization of Nox3 but not of NoxO1 required p22phox. Moreover, the glycosylation and maturation of Nox3 required p22phox expression, suggesting that p22phox was required for the proper biosynthesis and function of Nox3. Taken together, these studies demonstrate critical roles for p22phox at several distinct points in the maturation and assembly of a functionally competent Nox3 in the plasma membrane.

Antibodies against the melanocortin-4 receptor act as inverse agonists in vitro and in vivo

2007 ◽  
Vol 292 (6) ◽  
pp. R2151-R2158 ◽  
Author(s):  
Jean-Christophe Peter ◽  
Janet R. Nicholson ◽  
Déborah Heydet ◽  
Anne-Catherine Lecourt ◽  
Johan Hoebeke ◽  
...  
Keyword(s):  
Food Intake ◽  
Third Ventricle ◽  
Separate Experiment ◽  
Inverse Agonists ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Melanocortin 4 Receptor ◽  
293 Cells ◽  
Immunofluorescence Labeling

Functionally active antibodies (Abs) against central G-protein-coupled receptors have not yet been reported. We selected the hypothalamic melanocortin-4 receptor (MC4-R) as a target because of its crucial role in the regulation of energy homeostasis. A 15 amino acid sequence of the N-terminal (NT) domain was used as an antigen. This peptide showed functional activity in surface plasmon resonance experiments and in studies on HEK-293 cells overexpressing the human MC4-R (hMC4-R). Rats immunized against the NT peptide produced specific antibodies, which were purified and characterized in vitro. In HEK-293 cells, rat anti-NT Abs showed specific immunofluorescence labeling of hMC4-R. They reduced the production of cAMP under basal conditions and after stimulation with a synthetic MC4-R agonist. Rats immunized against the NT peptide developed a phenotype consistent with MC4-R blockade, that is, increased food intake and body weight, increased liver and fat pad weight, and elevated plasma triglycerides. In a separate experiment in rats, an increase in food intake could be produced after injection of purified Abs into the third ventricle. Similar results were obtained in rats injected with anti-NT Abs raised in rabbits. Our data show for the first time that active immunization of rats against the NT sequence of the MC4-R results in specific Abs, which appear to stimulate food intake by acting as inverse agonists in the hypothalamus.

scholarly journals Characterization of the reversible phosphorylation and activation of ERK8

2006 ◽  
Vol 394 (1) ◽  
pp. 365-373 ◽  
Author(s):  
Iva V. Klevernic ◽  
Margaret J. Stafford ◽  
Nicholas Morrice ◽  
Mark Peggie ◽  
Simon Morton ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Growth Factor ◽  
Protein Kinase ◽  
Insect Cells ◽  
Osmotic Shock ◽  
Tyrosine Phosphatase ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

ERK8 (extracellular-signal-regulated protein kinase 8) expressed in Escherichia coli or insect cells was catalytically active and phosphorylated at both residues of the Thr-Glu-Tyr motif. Dephosphorylation of the threonine residue by PP2A (protein serine/threonine phosphatase 2A) decreased ERK8 activity by over 95% in vitro, whereas complete dephosphorylation of the tyrosine residue by PTP1B (protein tyrosine phosphatase 1B) decreased activity by only 15–20%. Wild-type ERK8 expressed in HEK-293 cells was over 100-fold less active than the enzyme expressed in bacteria or insect cells, but activity could be increased by exposure to hydrogen peroxide, by incubation with the protein serine/threonine phosphatase inhibitor okadaic acid, or more weakly by osmotic shock. In unstimulated cells, ERK8 was monophosphorylated at Tyr-177, and exposure to hydrogen peroxide induced the appearance of ERK8 that was dually phosphorylated at both Thr-175 and Tyr-177. IGF-1 (insulin-like growth factor 1), EGF (epidermal growth factor), PMA or anisomycin had little effect on activity. In HEK-293 cells, phosphorylation of the Thr-Glu-Tyr motif of ERK8 was prevented by Ro 318220, a potent inhibitor of ERK8 in vitro. The catalytically inactive mutants ERK8[D154A] and ERK8[K42A] were not phosphorylated in HEK-293 cells or E. coli, whether or not the cells had been incubated with protein phosphatase inhibitors or exposed to hydrogen peroxide. Our results suggest that the activity of ERK8 in transfected HEK-293 cells depends on the relative rates of ERK8 autophosphorylation and dephosphorylation by one or more members of the PPP family of protein serine/threonine phosphatases. The major residue in myelin basic protein phosphorylated by ERK8 (Ser-126) was distinct from that phosphorylated by ERK2 (Thr-97), demonstrating that, although ERK8 is a proline-directed protein kinase, its specificity is distinct from ERK1/ERK2.

scholarly journals Nephroprotective activity of Annona Squamosa leaves against paracetamol-induced nephrotoxicity in rats: in vitro and in vivo experiments

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
S. Neelima ◽  
P. Dwarakanadha Reddy ◽  
Chandra Sekhar Kothapalli Bannoth
Keyword(s):  
Kidney Tissue ◽  
Ethanolic Extract ◽  
Tissue Homogenate ◽  
Annona Squamosa ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
In Vivo Experiments ◽  
293 Cells

Abstract Background Paracetamol (PCM), being extensively adapted analgesic and anti-inflammatory drug all over the world, beyond therapeutic dosages, the oxidative stress-involved nephrotoxicity has been evidenced. However, herbal plants are the windfall for the humankind providing solution for most of the wellness breakdowns. Annona squamosa (AS) is one of such plants with enormous therapeutic and nutraceutical potencies. The main aspiration of the current investigation is to evaluate the nephroprotective ability of ethanolic extract of Annona squamosa (EEAS) leaves against paracetamol-induced nephrotoxicity using in vitro human embryonic kidney (HEK)-293 cells and in vivo experiments in Wistar rats through biochemical parameters, oxidative parameters, and histopathological findings. Results When HEK-293 cells were incubated with PCM, an increased cell death associated with alterations in the morphology of normal cells was observed. At variable concentrations, HEK-293 cells co-treated with PCM and EEAS extracts gave a significant improvement in cell growth on comparing with PCM treatment showing cytoprotective feature of EEAS with an IC50 28.75 μg/mL. In vivo nephroprotective property was assessed from the amount of blood urea nitrogen (BUN) along with creatinine and uric acid which were reduced (P < 0.001) within serum and compact levels of glutathione, catalase, and superoxide dismutase which were termed as GSH, CAT, and SOD, respectively, were increased (P < 0.001) in kidney tissue homogenate in the treated groups than the PCM alone group. Results were additionally supported by histopathological observations. Conclusion The results exhibited that EEAS has impending benefits against PCM-induced nephrotoxicity through in vitro and in vivo experiments.

scholarly journals In vitro 3D Spheroid Culture Developed on the Parafilm Surface Using HEK-293 Cells

2020 ◽  
Vol 3 (1) ◽  
pp. 220-227
Author(s):  
Erdal Eroğlu
Keyword(s):  
Three Dimensional ◽  
Culture Method ◽  
In Vivo Studies ◽  
Preclinical Research ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Hanging Drop Culture

Preclinical research to predict the effects of drugs and chemicals on humans is commonly carried out either by cell culture studies in vitro condition or on animals in vivo condition. While drug studies tested on cells cultured as a monolayer in plastic flasks are incompatible with realistic results, falsifying findings can also be achieved from in vivo studies performed on different species. In recent years, research on drug tests using spheroid cultures formed by growing cells in three-dimensional (3D) in vitro has attracted great interest. 3D spheroid structures are formed by growing the cells in a drop suspended on superhydrophobic surfaces. In this study, HEK-293 cells were investigated on parafilm surfaces displaying superhydrophobic properties by growing in 2 &amp;micro;l volume using hanging drop culture method in terms of spheroid formation. Light microscopy images from spheroid structures were taken on different incubation days and the area of spheroids was measured using the ImageJ program. Our study holds important findings for a chip platform that can be developed for use in vitro drug tests.

334 ADENOVIRAL VECTOR-MEDIATED EXPRESSION OF RECOMBINANT HUMAN GLUCOCEREBROSIDASE IN THE MAMMARY GLAND OF RATS

2013 ◽  
Vol 25 (1) ◽  
pp. 314
Author(s):  
K. C. S. Tavares ◽  
C. Feltrin ◽  
I. S. Carneiro ◽  
A. S. Morais ◽  
C. D. Medeiros ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Mammary Glands ◽  
Santa Cruz ◽  
Cmv Promoter ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Control Milk

Glucocerebrosidase is a lysosomal enzyme that plays a key role in sphingolipid cleavage, an intermediate in glycolipid metabolism. A recessive mutation in the glucocerebrosidase gene leads to the accumulation of glucosylceramide in macrophages (sphingolipidosis), a lysosomal storage disease known in humans as the Gaucher disease. The enzyme replacement treatment with recombinant human glucocerebrosidase (hGCase) dramatically reduces and reverses symptoms, with the need of lifelong treatment for patients to attain a normal life. Currently, hGCase is very costly, being produced through in vitro expression in Chinese hamster ovary cells or in vivo, in plants. The aim of this study was to develop a model for the production of hGCase in the mammary gland of rats transiently transduced with recombinant adenovirus. A replication-defective adenovirus carrying hGCase was generated using the AdEasy™ adenoviral vector system (Stratagene, La Jolla, CA, USA). The hGCase cDNA (NM_001005741) was in vitro-synthesized and ligated in the XhoI site of the pAdTrack-CMV vector (pAdT-hGCase). The resulting plasmid was recombined with the pAdEasy™ vector in BJ5183 electro-competent cells. The purified pAdE-pAdT-hGCase vector was linearized and transfected into HEK-293 cells for the production of a primary viral stock. Further amplifications and the titration assay were done in HEK-293 cells, monitoring the transduction by the qualitative evaluation of green fluorescent protein (GFP) expression. Following transfection, the HEK-293 cells increasingly expressed the GFP reporter, regulated by a CMV promoter, in tandem with the hGCase cDNA, under another CMV promoter. On Day 18 of gestation, a female rat (Rattus norvegicus) was anesthetized and the 2 left caudal mammary glands were infused with 109 GTU mL–1 of the pAdE-pAdT-hGCase in PBS solution supplemented with 36 mM EGTA. The 2 right caudal mammary glands were infused only with PBS-EGTA (control milk). Milk samples collected from Days 2 through 9 post-partum were mixed with separation buffer (10 mM Tris-HCl, pH 8.0; 10 mM CaCl2) and centrifuged, with the supernatant assayed for hGCase by Western blot using a monoclonal anti-human glucocerebrosidase antibody (sc-166407, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Relative quantification of the hGCase expression was done using the FluorChem FC2 system (Alpha Innotech, San Leandro, CA, USA), with hGCase band intensity being normalized against GAPDH expression. The in vivo expression assay confirmed the production of hGCase in the secreted portion of the rat milk, with a specific band between 50 to 60 kDa observed on the Western blot, and no detection of the protein in the control milk. The hGCase peak production occurred in Days 5 and 6 of lactation, with levels being 35 times greater than on Day 9. An ELISA quantification assay and an enzymatic activity assay for the recombinant hGCase are currently in development. In conclusion, the use of the rat for hGCase transient expression in the milk was proven a valid model for testing the potential use of a mammary gland expression system for the production of a functional human glucocerebrosidase protein.

Glyco-engineered HEK 293-F cell lines for the production of therapeutic glycoproteins with human N-glycosylation and improved pharmacokinetics

Glycobiology ◽  
2021 ◽  
Author(s):  
Rico Uhler ◽  
Ruth Popa-Wagner ◽  
Mario Kröning ◽  
Anja Brehm ◽  
Paul Rennert ◽  
...  
Keyword(s):  
Cell Line ◽  
Therapeutic Proteins ◽  
Coagulation Factor ◽  
Factor Vii ◽  
Hek 293 ◽  
Knock Out ◽  
Hek 293 Cells ◽  
Coagulation Factor Vii ◽  
293 Cells

Abstract N-glycosylated proteins produced in human embryonic kidney 293 (HEK 293) cells often carry terminal N-acetylgalactosamine (GalNAc) and only low levels of sialylation. On therapeutic proteins, such N-glycans often trigger rapid clearance from the patient bloodstream via efficient binding to asialoglycoprotein receptor (ASGP-R) and mannose receptor (MR). This currently limits the use of HEK 293 cells for therapeutic protein production. To eliminate terminal GalNAc, we knocked-out GalNAc transferases B4GALNT3 and B4GALNT4 by CRISPR/Cas9 in FreeStyle 293-F cells. The resulting cell line produced a coagulation factor VII-albumin fusion protein without GalNAc but with increased sialylation. This glyco-engineered protein bound less efficiently to both the ASGP-R and MR in vitro and it showed improved recovery, terminal half-life and area under the curve in pharmacokinetic rat experiments. By overexpressing sialyltransferases ST6GAL1 and ST3GAL6 in B4GALNT3 and B4GALNT4 knock-out cells, we further increased factor VII-albumin sialylation; for ST6GAL1 even to the level of human plasma-derived factor VII. Simultaneous knock-out of B4GALNT3 and B4GALNT4, and overexpression of ST6GAL1 further lowered factor VII-albumin binding to ASGP-R and MR. This novel glyco-engineered cell line is well-suited for the production of factor VII-albumin and presumably other therapeutic proteins with fully human N-glycosylation and superior pharmacokinetic properties.

scholarly journals The Effect of Bacterial Peptide p28 on Viability and Apoptosis Statusof P53-null HeLa Cells

2019 ◽  
Vol 9 (4) ◽  
pp. 668-673
Author(s):  
Haniyeh Abuei ◽  
Abbas Behzad-Behbahani ◽  
Fatemeh Faghihi ◽  
Ali Farhadi ◽  
Gholam Reza Rafiei Dehbidi ◽  
...  
Keyword(s):  
Hela Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Health Concern ◽  
Target Cells ◽  
Bacterial Host ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Anti Cancer

Purpose: Despite all the efforts for discovery of efficient anti-cancer therapeutics, cancer is stilla major health concern worldwide. p28 is a bacterial small peptide which has been widelyinvestigated due to its preferential cell internalization and anti-cancer activities. Intracellularly,p28 offers its anti-cancer traits by impeding the degradation of tumor-suppressor protein "p53".In this study, we investigated the potency of p28 in inducing apoptosis or decreasing cellviability in p53-null "HeLa" cell line.Methods: The coding sequence for p28 peptide was obtained from Pseudomonas aeruginosaby PCR amplification of the p28 gene. The coding gene was cloned in pET-28a vector andtransformed into E. coli bacterial host. Subsequently, the expressed peptide was purified usingNi-NTA chromatography system and introduced into the target cells. The anti-proliferative andapoptotic activity of p28 on HeLa and HEK-293 cells were investigated using MTT and PEAnnexinV Flowcytometry assays.Results: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Westernblotting confirmed the expression of p28 peptide in the bacterial host. Bradford assay revealeda concentration of 0.05 mg/mL for the purified p28. MTT assay of cells treated with p28 atconcentrations of 0, 0.5, 1, 2 and 2.5 μM indicated 24h viability values of 97%, 89%, 88%,87% and 84% for HeLa cells, respectively. Data obtained from flowcytometry analyses revealed24h apoptosis rate of 7.17%, 8.05%, 8.63% and 8.84% for HeLa cells treated with 0, 0.5, 1,and 2 μM p28, respectively.Conclusion : MTT and flowcytometry apoptosis assays suggest no statistically significant effectof p28 on the viability and apoptosis status of p53-null HeLa cells when results compared todata obtained from HEK-293 cells (P > 0.05). These results imply that anti-cancer efficacy of p28is directly dependent on the presence of p53, suggesting p28 as an inappropriate therapeuticagent for treatment of cancers with negative p53 status.<br />

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