Thiol compounds including predominantly glutathione (GSH) are key components of redox homeostasis, which are involved in the protection and regulation of mammalian cells. The assessment of cell redox status by means of in situ analysis of GSH in living cells is often preferable over established assays in cell lysates due to fluctuations of the GSH pool. For this purpose, we propose a microplate assay with monochlorobimane (MCB) as an available fluorescent probe for GSH, although poorly detected in the microplate format. In addition to the new procedure for improved MCB-assisted GSH detection in plate-grown cells and its verification with GSH modulators, this study provides a useful methodology for the evaluation of cell redox status probed through relative GSH content and responsiveness to both supplemented thiols and variation in oxygen pressure. The roles of extracellular interactions of thiols and natural variability of cellular glutathione on the assay performance were emphasized and discussed. The results are of broad interest in cell biology research and should be particularly useful for the characterization of pathological cells with decreased GSH status and increased oxidative status as well as redox-modulating factors.

Phosphorus species are potent modulators of physicochemical and bioactive properties of peptide compounds. O,O-diorganyl dithiophoshoric acids (DTP) form bioactive salts with nitrogen-containing biomolecules; however, their potential as a peptide modifier is poorly known. We synthesized amphiphilic ammonium salts of O,O-dimenthyl DTP with glutathione, a vital tripeptide with antioxidant, protective and regulatory functions. DTP moiety imparted radical scavenging activity to oxidized glutathione (GSSG), modulated the activity of reduced glutathione (GSH) and profoundly improved adsorption and electrooxidation of both glutathione salts on graphene oxide modified electrode. According to NMR spectroscopy and GC–MS, the dithiophosphates persisted against immediate dissociation in an aqueous solution accompanied by hydrolysis of DTP moiety into phosphoric acid, menthol and hydrogen sulfide as well as in situ thiol-disulfide conversions in peptide moieties due to the oxidation of GSH and reduction of GSSG. The thiol content available in dissolved GSH dithiophosphate was more stable during air oxidation compared with free GSH. GSH and the dithiophosphates, unlike DTP, caused a thiol-dependent reduction of MTS tetrazolium salt. The results for the first time suggest O,O-dimenthyl DTP as a redox modifier for glutathione, which releases hydrogen sulfide and induces biorelevant redox conversions of thiol/disulfide groups.

Divalent trace metals (TM), especially copper (Cu), cobalt (Co) and zinc (Zn), are recognized as essential microelements for tissue homeostasis and regeneration. To achieve a balance between therapeutic activity and safety of administered TMs, effective gel formulations of TMs with elucidated regenerative mechanisms are required. We studied in vitro and in vivo effects of biodegradable macroporous cryogels doped with Cu, Co or Zn in a controllable manner. The extracellular ROS generation by metal dopants was assessed and compared with the intracellular effect of soluble TMs. The stimulating ability of TMs in the cryogels for cell proliferation, differentiation and cytokine/growth factor biosynthesis was characterized using HSF and HUVEC primary human cells. Multiple responses of host tissues to the TM-doped cryogels upon subcutaneous implantation were characterized taking into account the rate of biodegradation, production of HIF-1α/matrix metalloproteinases and the appearance of immune cells. Cu and Zn dopants did not disturb the intact skin organization while inducing specific stimulating effects on different skin structures, including vasculature, whereas Co dopant caused a significant reorganization of skin layers, the appearance of multinucleated giant cells, along with intense angiogenesis in the dermis. The results specify and compare the prooxidant and regenerative potential of Cu, Co and Zn-doped biodegradable cryogels and are of particular interest for the development of advanced bioinductive hydrogel materials for controlling angiogenesis and soft tissue growth.

A series of silver-polygalacturonate complexes with improved structure and activity against bacterial infections was developed. Pure sodium polygalacturonate was obtained by saponification of a pectin precursor and identified by NMR as predominantly homogalacturonan (uronide content 95%). Polygalacturonate complexes with ionic and borohydride-reduced silver with a controllable metallic component were synthesized; the role of spontaneous Ag⁺ reduction was revealed. The presence of uniform 5 nm nanoparticles and negligible particulate by-products in the reduced complexes was verified. The complexes showed similar silver-normalized activity against non-resistant bacteria, irrespective of complex stoichiometry/silver state. Pharmaceutical silver proteinate with a similar nanoparticle profile exhibited the same silver-normalized activity, indicating the lack of a ligand effect. The Ag⁺ complex was more effective against some hospital drug-resistant strains. The cytotoxicity of the complexes depended on fibroblast type, silver state, ligand type, exposure time, presumably in association with cellular availability and glutathione depletion. The complexes were administered to rats with excisional wounds persistently infected with S. aureus. Swab/histological analyses of the treated wounds revealed decreased bacterial burden/tissue damage, along with promotion of wound contraction/closure and matrix formation. The nanoparticle complexes that were compared had similar antibacterial/regenerative effects, while the Ag⁺ complex demonstrated higher efficacy in vivo. These results encourage the use of the developed silver-polygalacturonate complexes as antibacterial substances.

Considering the important cytoprotective and signaling roles but relatively narrow therapeutic index of hydrogen sulfide (H₂S), advanced H₂S donors are required to achieve a therapeutic effect. In this study, we proposed glutathione dithiophosphates as new combination donors of H₂S and glutathione. The kinetics of H₂S formation in dithiophosphate solutions suggested a continuous H₂S release by the donors, which was higher for the dithiophosphate of reduced glutathione than oxidized glutathione. The compounds, unlike NaHS, inhibited the proliferation of C2C12 myoblasts at submillimolar concentrations due to an efficient increase in intracellular H₂S. The H₂S donors more profoundly affected reactive oxygen species and reduced glutathione levels in C2C12 myocytes, in which these parameters were elevated compared to myoblasts. Oxidized glutathione dithiophosphate as well as control donors exerted antioxidant action toward myocytes, whereas the effect of reduced glutathione dithiophosphate at (sub-)micromolar concentrations was rather modulating. This dithiophosphate showed an enhanced negative inotropic effect mediated by H₂S upon contraction of the atrial myocardium, furthermore, its activity was prolonged and reluctant for washing. These findings identify glutathione dithiophosphates as redox-modulating H₂S donors with long-acting profile, which are of interest for further pharmacological investigation.

Важным объектом современных экспериментальных исследований являются сероводород (H2S) и его предшественники, выполняющие множественные биологические функции, некоторые из которых представляют интерес для модуляции компонентов системы гемостаза. В работе оценено влияние новых синтетических доноров H2S на основе производных дитиофосфорной кислоты на агрегацию тромбоцитов, их митохондриальный потенциал (Δψm), а также параметры свертывания крови по данным тромбоэластографического анализа. Производные дитиофосфорной кислоты не вызывали изменения Δψm активированных тромбоцитов, а также агрегацию тромбоцитов и деполяризацию их митохондриальной мембраны без активации АДФ. В присутствии АДФ соединения умеренно ингибировали агрегацию, сходным образом с Na2S. Эффект соединений изменялся при понижении плотности тромбоцитов и активации: исследуемые доноры снижали Δψm в 4 – 7 раз (p < 0,05). Соединения в концентрации 1 мМ сокращали время начала формирования сгустка крови на 28 – 29 % (q < 0,05) и проявляли тенденцию к уменьшению продолжительности времени достижения его постоянной прочности, не оказывая значимого влияния на другие параметры коагуляции. Полученные результаты выявляют потенциальную антиагрегантную активность производных дитиофосфорной кислоты, объясняемую их способностью высвобождать H2S, а также указывают на сложный характер их влияния на коагуляционный процесс.