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End date: 2024 × Start date: 2020 × DDC: 540 × Subject: Cell culture ×

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Now showing 1 - 4 of 4
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    Quantification of Dolichyl Phosphates Using Phosphate Methylation and Reverse-Phase Liquid Chromatography-High Resolution Mass Spectrometry
    (Columbus, Ohio : American Chemical Society, 2023) Kale, Dipali; Kikul, Frauke; Phapale, Prasad; Beedgen, Lars; Thiel, Christian; Brügger, Britta
    Dolichyl monophosphates (DolPs) are essential lipids in glycosylation pathways that are highly conserved across almost all domains of life. The availability of DolP is critical for all glycosylation processes, as these lipids serve as membrane-anchored building blocks used by various types of glycosyltransferases to generate complex post-translational modifications of proteins and lipids. The analysis of DolP species by reverse-phase liquid chromatography-mass spectrometry (RPLC-MS) remains a challenge due to their very low abundance and wide range of lipophilicities. Until now, a method for the simultaneous qualitative and quantitative assessment of DolP species from biological membranes has been lacking. Here, we describe a novel approach based on simple sample preparation, rapid and efficient trimethylsilyl diazomethane-dependent phosphate methylation, and RPLC-MS analysis for quantification of DolP species with different isoprene chain lengths. We used this workflow to selectively quantify DolP species from lipid extracts derived of Saccharomyces cerevisiae, HeLa, and human skin fibroblasts from steroid 5-α-reductase 3- congenital disorders of glycosylation (SRD5A3-CDG) patients and healthy controls. Integration of this workflow with global lipidomics analyses will be a powerful tool to expand our understanding of the role of DolPs in pathophysiological alterations of metabolic pathways downstream of HMG-CoA reductase, associated with CDGs, hypercholesterolemia, neurodegeneration, and cancer.
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    Time-resolved luminescence detection of peroxynitrite using a reactivity-based lanthanide probe
    (Cambridge : RSC, 2020) Breen, Colum; Pal, Robert; Elsegood, Mark R.J.; Teat, Simon J.; Iza, Felipe; Wende, Kristian; Buckley, Benjamin R.; Butler, Stephen
    Peroxynitrite (ONOO-) is a powerful and short-lived oxidant formed in vivo, which can react with most biomolecules directly. To fully understand the roles of ONOO- in cell biology, improved methods for the selective detection and real-time analysis of ONOO- are needed. We present a water-soluble, luminescent europium(iii) probe for the rapid and sensitive detection of peroxynitrite in human serum, living cells and biological matrices. We have utilised the long luminescence lifetime of the probe to measure ONOO- in a time-resolved manner, effectively avoiding the influence of autofluorescence in biological samples. To demonstrate the utility of the Eu(iii) probe, we monitored the production of ONOO- in different cell lines, following treatment with a cold atmospheric plasma device commonly used in the clinic for skin wound treatment. This journal is © The Royal Society of Chemistry.
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    Redox-triggerable firefly luciferin-bioinspired hydrogels as injectable and cell-encapsulating matrices
    (Cambridge : RSC Publ., 2022) Jin, Minye; Gläser, Alisa; Paez, Julieta I.
    Stimuli-responsive hydrogels are smart materials that respond to variations caused by external stimuli and that are currently exploited for biomedical applications such as biosensing, drug delivery and tissue engineering. The development of stimuli-responsive hydrogels with defined user control is relevant to realize materials with advanced properties. Recently, our group reported firefly luciferin-inspired hydrogel matrices for 3D cell culture. This platform exhibited advantages like rapid gelation rate and tunability of mechanical and biological properties. However, this first molecular design did not allow fine control of the gelation onset, which restricts application as a cell-encapsulating matrice with injectable and processable properties. In this article, we endow the firefly luciferin-inspired hydrogels with redox-triggering capability, to overcome the limitations of the previous system and to widen its application range. We achieve this goal by introducing protected macromers as hydrogel polymeric precursors that can be activated in the presence of a mild reductant, to trigger gel formation in situ with a high degree of control. We demonstrate that the regulation of molecular parameters (e.g., structure of the protecting group, reductant type) and environmental parameters (e.g., pH, temperature) of the deprotection reaction can be exploited to modulate materials properties. This redox-triggerable system enables precise control over gelation onset and kinetics, thus facilitating its utilization as an injectable hydrogel without negatively impacting its cytocompatibility. Our findings expand the current toolkit of chemically-based stimuli-responsive hydrogels.
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    Nanoparticles for Directed Immunomodulation: Mannose-Functionalized Glycodendrimers Induce Interleukin-8 in Myeloid Cell Lines
    (Columbus, Ohio : American Chemical Society, 2021) Jatczak-Pawlik, Izabela; Gorzkiewicz, Michał; Studzian, Maciej; Zinke, Robin; Appelhans, Dietmar; Klajnert-Maculewicz, Barbara; Pułaski, Łukasz
    New therapeutic strategies for personalized medicine need to involve innovative pharmaceutical tools, for example, modular nanoparticles designed for direct immunomodulatory properties. We synthesized mannose-functionalized poly(propyleneimine) glycodendrimers with a novel architecture, where freely accessible mannose moieties are presented on poly(ethylene glycol)-based linkers embedded within an open-shell maltose coating. This design enhanced glycodendrimer bioactivity and led to complex functional effects in myeloid cells, with specific induction of interleukin-8 expression by mannose glycodendrimers detected in HL-60 and THP-1 cells. We concentrated on explaining the molecular mechanism of this phenomenon, which turned out to be different in both investigated cell lines: in HL-60 cells, transcriptional activation via AP-1 binding to the promoter predominated, while in THP-1 cells (which initially expressed less IL-8), induction was mediated mainly by mRNA stabilization. The success of directed immunomodulation, with synthetic design guided by assumptions about mannose-modified dendrimers as exogenous regulators of pro-inflammatory chemokine levels, opens new possibilities for designing bioactive nanoparticles. © 2021 The Authors. Published by American Chemical Society.