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1.
Pharmaceutics ; 13(4)2021 Apr 20.
Article in English | MEDLINE | ID: mdl-33923881

ABSTRACT

Every day, thousands of patients receive erythrocyte concentrates (ECs). They are indispensable for modern medicine, despite their limited resource. Artificial oxygen carriers (AOCs) represent a promising approach to reduce the need for ECs. One form of AOCs is perfluorodecalin-filled albumin-based nanocapsules. However, these AOCs are not storable and need to be applied directly after production. In this condition, they are not suitable as a medicinal product for practical use yet. Lyophilization (freeze drying) could provide the possibility of durable and applicable nanocapsules. In the present study, a suitable lyophilization process for perfluorodecalin-filled nanocapsules was developed. The nanocapsules were physicochemically characterized regarding capsule size, polydispersity, and oxygen capacity. Even though the perfluorodecalin-filled albumin-based nanocapsules showed a loss in oxygen capacity directly after lyophilization, they still provided a remarkable residual capacity. This capacity did not decline further for over two months of storage. Furthermore, the nanocapsule size remained unaltered for over one year. Therefore, the AOCs were still applicable and functional after long-term storage due to the successful lyophilization.

2.
Redox Biol ; 34: 101570, 2020 07.
Article in English | MEDLINE | ID: mdl-32473461

ABSTRACT

The failure of insulin-producing ß-cells is the underlying cause of hyperglycemia in diabetes mellitus. ß-cell decay has been linked to hypoxia, chronic inflammation, and oxidative stress. Thioredoxin (Trx) proteins are major actors in redox signaling and essential for signal transduction and the cellular stress response. We have analyzed the cytosolic, mitochondrial, and extracellular Trx system proteins in hypoxic and cytokine-induced stress using ß-cell culture, isolated pancreatic islets, and pancreatic islet transplantation modelling low oxygen supply. Protein levels of cytosolic Trx1 and Trx reductase (TrxR) 1 significantly decreased, while mitochondrial Trx2 and TrxR2 increased upon hypoxia and reoxygenation. Interestingly, Trx1 was secreted by ß-cells during hypoxia. Moreover, murine and human pancreatic islet grafts released Trx1 upon glucose stimulation. Survival of transplanted islets was substantially impaired by the TrxR inhibitor auranofin. Since a release was prominent upon hypoxia, putative paracrine effects of Trx1 on ß-cells were examined. In fact, exogenously added recombinant hTrx1 mitigated apoptosis and preserved glucose sensitivity in pancreatic islets subjected to hypoxia and inflammatory stimuli, dependent on its redox activity. Human subjects were studied, demonstrating a transient increase in extracellular Trx1 in serum after glucose challenge. This increase correlated with better pancreatic islet function. Moreover, hTrx1 inhibited the migration of primary murine macrophages. In conclusion, our study offers evidence for paracrine functions of extracellular Trx1 that improve the survival and function of pancreatic ß-cells.


Subject(s)
Thioredoxin-Disulfide Reductase , Thioredoxins , Animals , Auranofin , Humans , Mice , Oxidation-Reduction , Oxidative Stress , Thioredoxin-Disulfide Reductase/metabolism , Thioredoxins/genetics , Thioredoxins/metabolism
3.
Biochim Biophys Acta ; 1850(6): 1274-85, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25735211

ABSTRACT

BACKGROUND: Thioredoxin (Trx) family proteins are crucial mediators of cell functions via regulation of the thiol redox state of various key proteins and the levels of the intracellular second messenger hydrogen peroxide. Their expression, localization and functions are altered in various pathologies. Here, we have analyzed the impact of Trx family proteins in neuronal development and recovery, following hypoxia/ischemia and reperfusion. METHODS: We have analyzed the regulation and potential functions of Trx family proteins during hypoxia/ischemia and reoxygenation of the developing brain in both an animal and a cellular model of perinatal asphyxia. We have analyzed the distribution of 14 Trx family and related proteins in the cerebellum, striatum, and hippocampus, three areas of the rat brain that are especially susceptible to hypoxia. Using SH-SY5Y cells subjected to hypoxia and reoxygenation, we have analyzed the functions of some redoxins suggested by the animal experiment. RESULTS AND CONCLUSIONS: We have described/discovered a complex, cell-type and tissue-specific expression pattern following the hypoxia/ischemia and reoxygenation. Particularly, Grx2 and Trx1 showed distinct changes during tissue recovery following hypoxia/ischemia and reoxygenation. Silencing of these proteins in SH-SY5Y cells subjected to hypoxia-reoxygenation confirmed that these proteins are required to maintain the normal neuronal phenotype. GENERAL SIGNIFICANCE: These findings demonstrate the significance of redox signaling in cellular pathways. Grx2 and Trx1 contribute significantly to neuronal integrity and could be clinically relevant in neuronal damage following perinatal asphyxia and other neuronal disorders.


Subject(s)
Asphyxia Neonatorum/enzymology , Brain/enzymology , Glutaredoxins/metabolism , Hypoxia-Ischemia, Brain/enzymology , Neurons/enzymology , Thioredoxins/metabolism , Animals , Asphyxia Neonatorum/pathology , Brain/pathology , Cell Line, Tumor , Disease Models, Animal , Glutaredoxins/genetics , Humans , Hypoxia-Ischemia, Brain/pathology , Male , Neurons/pathology , Oxidation-Reduction , Oxygen/metabolism , Phenotype , RNA Interference , Rats, Sprague-Dawley , Signal Transduction , Thioredoxins/genetics , Time Factors , Transfection
4.
J Biol Chem ; 285(52): 40699-705, 2010 Dec 24.
Article in English | MEDLINE | ID: mdl-20929858

ABSTRACT

The proteins from the thioredoxin family are crucial actors in redox signaling and the cellular response to oxidative stress. The major intracellular source for oxygen radicals are the components of the respiratory chain in mitochondria. Here, we show that the mitochondrial 2-Cys peroxiredoxin (Prx3) is not only substrate for thioredoxin 2 (Trx2), but can also be reduced by glutaredoxin 2 (Grx2) via the dithiol reaction mechanism. Grx2 reduces Prx3 exhibiting catalytic constants (K(m), 23.8 µmol·liter(-1); V(max), 1.2 µmol·(mg·min)(-1)) similar to Trx2 (K(m), 11.2 µmol·liter(-1); V(max), 1.1 µmol·(mg·min)(-1)). The reduction of the catalytic disulfide of the atypical 2-Cys Prx5 is limited to the Trx system. Silencing the expression of either Trx2 or Grx2 in HeLa cells using specific siRNAs did not change the monomer:dimer ratio of Prx3 detected by a specific 2-Cys Prx redox blot. Only combined silencing of the expression of both proteins led to an accumulation of oxidized protein. We further demonstrate that the distribution of Prx3 in different mouse tissues is either linked to the distribution of Trx2 or Grx2. These results introduce Grx2 as a novel electron donor for Prx3, providing further insights into pivotal cellular redox signaling mechanisms.


Subject(s)
Glutaredoxins/metabolism , Mitochondria/enzymology , Mitochondrial Proteins/metabolism , Peroxiredoxins/metabolism , Thioredoxins/metabolism , Animals , Female , Gene Expression Regulation, Developmental/physiology , Gene Silencing , Glutaredoxins/genetics , HeLa Cells , Humans , Mice , Mitochondria/genetics , Mitochondrial Proteins/genetics , Organ Specificity/physiology , Oxidation-Reduction , Oxidative Stress/physiology , Peroxiredoxin III , Peroxiredoxins/genetics , Protein Multimerization/physiology , Rats , Thioredoxins/genetics
5.
Biochem Biophys Res Commun ; 394(2): 372-6, 2010 Apr 02.
Article in English | MEDLINE | ID: mdl-20226171

ABSTRACT

Mammalian glutaredoxin 3 (Grx3/PICOT) is an essential protein involved in the regulation of signal transduction, for instance during immune cell activation and development of cardiac hypertrophy, presumably in response to redox signals. This function requires the sensing of such stresses by a hitherto unknown mechanism. Here, we characterized Grx3/PICOT as iron-sulfur protein. The protein binds two bridging [2Fe-2S] clusters in a homodimeric complex with the active site cysteinyl residues of its two monothiol glutaredoxin domains and glutathione bound non-covalently to the Grx domains. Co-immunoprecipitation of 55-iron with Grx3/PICOT from Jurkat cells suggested the presence of these cofactors under physiological conditions. The [2Fe-2S]2+ clusters were not redox active, instead they were lost upon treatment of the holo protein with ferricyanide or S-nitroso glutathione. This redox-induced dissociation of the Grx3/PICOT holo complex may be a mechanism of Grx3/PICOT activation in response to reactive oxygen and nitrogen species.


Subject(s)
Carrier Proteins/metabolism , Iron-Sulfur Proteins/metabolism , Iron/metabolism , Carrier Proteins/chemistry , Ferricyanides/metabolism , Humans , Immunoprecipitation , Iron Radioisotopes/metabolism , Iron-Sulfur Proteins/chemistry , Jurkat Cells , Oxidation-Reduction , Protein Conformation , Protein Stability , Protein Structure, Tertiary , S-Nitrosoglutathione/metabolism
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