20 research outputs found
Specyficzność substratowa, aktywność i lokalizacja subkomórkowa ludzkiego białka oporności wielolekowej ABCG2
Białko ABCG2 u człowieka funkcjonuje jako transporter wielu substancji o znaczeniu fizjologicznym – metabolitów, składników pożywienia, leków. Celem niniejszej pracy było opracowanie nowej metody badania specyficzności substratowej ABCG2 wobec szerokiej grupy związków naturalnych, która umożliwiałaby identyfikację substratów lub inhibitorów oraz ilościowy opis kinetyki ich oddziaływania z transporterem; zastosowanie techniki obrazowania czasu życia fluorescencji (FLIM) do badania aktywności transportowej ABCG2 oraz oddziaływań białko-białko i białko-substrat; a także opracowanie opartej o mikroskopię konfokalną metodyki badania przebiegu i mechanizmów internalizacji białek błonowych i określenie związku między przekształceniami strukturalnymi białka ABCG2 a jego lokalizacją subkomórkową.
W wyniku przeprowadzonych prac przystosowano metodę derywatyzacji fluorescencyjnej flawonoidów za pomocą estru 2-aminoetylowego kwasu difenyloborinowego (DPBA) do badania aktywności transportowej białka ABCG2 w komórkach ssaczych. Umożliwiło to identyfikację kilkudziesięciu flawonoidów, związków pochodzenia roślinnego obecnych w żywności, jako nieznanych uprzednio substratów lub inhibitorów białka ABCG2. Flawonoid luteolinę zidentyfikowano i opisano jako dobry nowy substrat modelowy do badania aktywności i inhibicji ABCG2.
Zastosowano FLIM do czasowo-rozdzielczej dekonwolucji obrazu, co wykorzystano przy pomiarach aktywności ABCG2. Wdrożono również metodę pomiarów Försterowskiego rezonansowego transferu energii (FRET) w celu określania powinowactwa substratu do białka transportowego w żywej komórce. Stosując pomiary FRET-FLIM udowodniono eksperymentalnie oddziaływanie białko-białko między cząsteczkami ABCG2.
Dzięki zastosowaniu zaawansowanych technik obrazowania, opartych o mikroskopię konfokalną, udało się odkryć i scharakteryzować nowe interesujące zjawisko: stymulowaną wiązaniem przeciwciała endocytozę białka ABCG2. W wyniku przeprowadzonych badań udało się wyjaśnić, że w wyniku stabilizacji jednej z możliwych konformacji białka następuje szybka internalizacja kompleksu białko-przeciwciało, zachodząca na drodze endocytozy o mechanizmie mieszanym, częściowo zależnym od klatryny, częściowo od cholesterolu
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Synthesis, internalization and visualization of n-(4-carbomethoxy) pyrrolidone terminated PAMAM [G5:G3-TREN] Tecto(dendrimers) in mammalian cells
Tecto(dendrimers) are well-defined, dendrimer cluster type covalent structures. In this article, we present the synthesis of such a PAMAM [G5:G3-(TREN)]-N-(4-carbomethoxy) pyrrolidone terminated tecto(dendrimer). This tecto(dendrimer) exhibits nontraditional intrinsic luminescence (NTIL; excitation 376 nm; emission 455 nm) that has been attributed to three fluorescent components characterized by different fluorescence lifetimes. Furthermore, it has been shown that this PAMAM [G5:G3-(TREN)]-N-(4-carbomethoxy) pyrrolidone terminated tecto(dendrimer) is able to form a polyplex with double stranded DNA, and is nontoxic for HeLa and HMEC-1 cells up to a concentration of 10 mg/mL, even though it accumulates in endosomal compartments as demonstrated by its unique NTIL emission properties. Many of the above features would portend the proposed use of this tecto(dendrimer) as an efficient transfection agent. Quite surprisingly, transfection activity could not be demonstrated in HeLa cells, and the possible reasons are discussed in the article. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)
Differential Quantitative Proteomics of Human Microvascular Endothelial Cells 1 by iTRAQ Reveals Palladin to be a New Biomarker During TGF-β1 Induced Endothelial Mesenchymal Transition
The study uses global quantitative proteomics to investigate the molecular mechanisms behind the induction
of endothelial-mesenchymal transition (EndMT) by transforming growth factor–β (TGF-β). Orbitrap Velos mass
spectrometers and iTRAQ – a labeling-based analysis were used to perform a global and quantitative comparison
of two proteomes of Human Microvascular Endothelial Cells-1 (HMEC-1) treated or not treated by TGF-β1. iTRAQ
analysis identified 43 differentially-expressed proteins in the early stages of EndMT induced by TGF-β1. From 5522
identified proteins, 26 were downregulated and 17 were upregulated, including proteins such as palladin, POTE I,
torsin A and nucleoporin (NDC1). Further analysis of palladin revealed its increased mRNA and protein expression in response to TGF-β and Snail transcription factor. Our findings demonstrate that the newly- identified proteins may be involved in early stages of biological processes leading to EndM
Nanoparticles for Directed Immunomodulation: Mannose-Functionalized Glycodendrimers Induce Interleukin-8 in Myeloid Cell Lines
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
Sugar-Modified Poly(propylene imine) Dendrimers Stimulate the NF-κB Pathway in a Myeloid Cell Line
Purpose: Fourth-generation poly(propylene imine) dendrimers fully surface-modified by maltose (dense shell, PPI-m DS) were shown to be biocompatible in cellular models, which is important for their application in drug delivery. We decided to verify also their inherent bioactivity, including immunomodulatory activity, for potential clinical applications. We tested their effects on the THP-1 monocytic cell line model of innate immunity effectors. Methods: To estimate the cytotoxicity of dendrimers the reasazurin assay was performed. The expression level of NF-κB targets: IGFBP3, TNFAIP3 and TNF was determined by quantitative real-time RT-PCR. Measurement of NF-κB p65 translocation from cytoplasm to nucleus was conducted with a high-content screening platform and binding of NF-κB to a consensus DNA probe was determined by electrophoretic mobility shift assay. The cytokine assay was performed to measure protein concentration of TNFalpha and IL-4. Results: We found that PPI-m DS did not impact THP-1 viability and growth even at high concentrations (up to 100 μM). They also did not induce expression of genes for important signaling pathways: Jak/STAT, Keap1/Nrf2 and ER stress. However, high concentrations of 4th generation PPI-m DS (25–100 μM), but not their 3rd generation counterparts, induced nuclear translocation of p65 NF-κB protein and its DNA-binding activity, leading to NF-κB-dependent increased expression of mRNA for NF-κB targets: IGFBP3, TNFAIP3 and TNF. However, no increase in pro-inflammatory cytokine secretion was detected. Conclusion: We conclude that maltose-modified PPI dendrimers of specific size could exert a modest immunomodulatory effect, which may be advantageous in clinical applications (e.g. adjuvant effect in anti-cancer vaccines)
Integrative systematics and ecology of a new deep-sea family of tanaidacean crustaceans
A new family of paratanaoidean Tanaidacea – Paranarthrurellidae fam. nov. – is erected to accommodate two genera without family classification (Paratanaoidea incertae sedis), namely Armatognathia Kudinova-Pasternak, 1987 and Paranarthrurella Lang, 1971. Seven new species of Paranarthrurella and two of Armatognathia are described from material taken in different deep-sea areas of the Atlantic and Pacific oceans. The type species of Paranarthrurella — P. caudata (Kudinova-Pasternak, 1965) — is redescribed based on the paratype. The genus Cheliasetosatanais Larsen and Araújo-Silva, 2014 originally classified within Colletteidae is synonymised with Paranarthrurella, and Arthrura shiinoi Kudinova-Pasternak, 1973 is transferred to Armatognathia. Amended diagnoses of Armatognathia and Paranarthrurella genera are given. Choosing characters for distinguishing and defining both genera was supported by Principal Component Analysis. Designation of the new family is supported by molecular phylogenetic analysis of COI and 18S datasets. The distribution of all species currently included in the new family was visualised and their bathymetric distribution analysed
Screening the Expression of ABCB6 in Erythrocytes Reveals an Unexpectedly High Frequency of Lan Mutations in Healthy Individuals
Lan is a high-incidence blood group antigen expressed in more than 99.9% of the population. Identification of the human ABC transporter ABCB6 as the molecular basis of Lan has opened the way for studies assessing the relation of ABCB6 function and expression to health and disease. To date, 34 ABCB6 sequence variants have been described in association with reduced ABCB6 expression based on the genotyping of stored blood showing weak or no reactivity with anti-Lan antibodies. In the present study we examined the red blood cell (RBC) surface expression of ABCB6 by quantitative flow cytometry in a cohort of 47 healthy individuals. Sequencing of the entire coding region of the ABCB6 gene in low RBC ABCB6 expressors identified a new allele (IVS9+1G>A, affecting a putative splice site at the boundary of exon 9) and two nonsynonymous SNPs listed in the SNP database (R192Q (rs150221689) and G588 S (rs145526996)). The R192Q mutation showed co-segregation with reduced RBC ABCB6 expression in a family, and we found the G588 S mutation in a compound heterozygous individual with undetectable ABCB6 expression, suggesting that both mutations result in weak or no expression of ABCB6 on RBCs. Analysis of the intracellular expression pattern in HeLa cells by confocal microscopy indicated that these mutations do not compromise overall expression or the endolysosomal localization of ABCB6. Genotyping of two large cohorts, containing 235 and 1039 unrelated volunteers, confirmed the high allele frequency of Lan-mutations. Our results suggest that genetic variants linked to lower or absent cell surface expression of ABCB6/Langereis may be more common than previously thought.This work was supported by the Lendulet Program of the Hungarian Academy of Sciences (GS), OTKA 83533 and by the Polish POIG grant 01.01.02-10-005/08 TESTOPLEK, supported by the EU through the European Regional Development Fund. Hajnalka Andrikovics is a recipient of the Janos Bolyai Research Scholarship from the Hungarian Academy of Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Dr. Camilo Toro and Dr. William Gahl of the NIH
Undiagnosed Diseases Program for an affected patient specimen; that
work was supported by the Intramural Research Program of the National
Human Genome Research Institute and the Office of the Director of the
NIH. We thank Lionel Arnaud (National Institute of Blood Transfusion
(INTS), Paris, France) for helpful discussions
Calcium Dyshomeostasis Alters CCL5 Signaling in Differentiated PC12 Cells
Background. Plasma membrane Ca2+-ATPase (PMCA) is the most sensitive cellular calcium detector. It exists in four main isoforms (PMCA1-4), among which PMCA2 and PMCA3 are considered as fast-acting neuron-specific forms. In the brain, PMCA function declines progressively during aging; thereby impaired calcium homeostasis may contribute to some neurodegenerative diseases. These destructive processes can be propagated by proinflammatory chemokines, including chemokine CCL5, which causes phospholipase C-mediated liberation of Ca2+ from endoplasmic reticulum by IP3-gated channels. Methods. To mimic the changes in aged neurons we used stable transfected differentiated PC12 cells with downregulated PMCA2 or PMCA3 and analyzed the effect of CCL5 on calcium transients with Fluo-4 reagent. Chemokine receptors were evaluated using Western blot, and IP3 receptors expression level was assessed using qRT-PCR and Western blot. Results. In PMCA-reduced cell lines, CCL5 released more Ca2+ by IP3-sensitive receptors, and the time required for Ca2+ clearance was significantly longer. Also, in these lines we detected altered expression level of CCR5 and IP3 receptors. Conclusion. Although modification of PMCAs composition could provide some protection against calcium overload, reduction of PMCA2 appeared to be more detrimental to the cells than deficiency of PMCA3. Under pathological conditions, including inflammatory CCL5 action and long-lasting Ca2+ dyshomeostasis, insufficient cell protection may result in progressive degeneration and death of neurons
PAMAM dendrimer with 4-carbomethoxypyrrolidone - <i>In vitro</i> assessment of neurotoxicity
Synthesis, Internalization and Visualization of N-(4-Carbomethoxy) Pyrrolidone Terminated PAMAM [G5:G3-TREN] Tecto(dendrimers) in Mammalian Cells
Tecto(dendrimers) are well-defined, dendrimer cluster type covalent structures. In this article, we present the synthesis of such a PAMAM [G5:G3-(TREN)]-N-(4-carbomethoxy) pyrrolidone terminated tecto(dendrimer). This tecto(dendrimer) exhibits nontraditional intrinsic luminescence (NTIL; excitation 376 nm; emission 455 nm) that has been attributed to three fluorescent components characterized by different fluorescence lifetimes. Furthermore, it has been shown that this PAMAM [G5:G3-(TREN)]-N-(4-carbomethoxy) pyrrolidone terminated tecto(dendrimer) is able to form a polyplex with double stranded DNA, and is nontoxic for HeLa and HMEC-1 cells up to a concentration of 10 mg/mL, even though it accumulates in endosomal compartments as demonstrated by its unique NTIL emission properties. Many of the above features would portend the proposed use of this tecto(dendrimer) as an efficient transfection agent. Quite surprisingly, transfection activity could not be demonstrated in HeLa cells, and the possible reasons are discussed in the article