Two single cysteine substitution mutants at helix surface sites in T4 lysozyme (D72C and V131C) have been modified with a series of nitroxide methanethiosulfonate reagents to investigate the structural dynamical origins their electron paramagnetic resonance spectra. The novel include 4-substituted derivatives either pyrroline or pyrrolidine nitroxides. spectral line shapes were analyzed as function side chain structure temperature using simulation method order parameter diffusion rates about...

The traditional site-directed spin labeling (SDSL) method, which utilizes cysteine residues and sulfhydryl-reactive nitroxide reagents, can be challenging for proteins that contain functionally important native or disulfide bonds. To make SDSL amenable to any protein, we introduce an orthogonal strategy, i.e., one does not rely on of the functional groups found in common 20 amino acids. In this genetically encoded unnatural acid p-acetyl-L-phenylalanine (p-AcPhe) is reacted with a...

In plants experiencing environmental stress, the formation of reactive oxygen is often presumed. this study, singlet was detected in broad bean (Vicia faba) leaves that were photoinhibited vivo. Detection based on reaction with DanePy (dansyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole) yielding a nitroxide radical (DanePyO) which EPR active and also features lower fluorescence compared to DanePy. The two (fluorescent spin) sensor fuctions are commensurate, makes detecting possible...

In order to understand the physiological functions of reactive oxygen species (ROS) generated in leaves, their direct measurement vivo is special importance. Here we report experiments with two dansyl-based ROS sensors, singlet specific DanePy and HO-1889NH, which both superoxide radicals. detection 1O2 O2–• by fluorescence quenching was O2–•. The sensors were administered spinach leaves through a pinhole, then exposed either excess photosynthetically active radiation or UV (280–360 nm)...

A disulfide-linked nitroxide side chain (R1) is the most widely used spin label for determining protein topology, mapping structural changes, and characterizing nanosecond backbone motions by site-directed labeling. Although internal motion of R1 number preferred rotamers are limited, translating interspin distance measurements spatial orientation information into constraints challenging. Here, we introduce a highly constrained designated RX as an alternative to these applications. formed...

The protective effects of stable nitroxides, as well their hydroxylamine and amine precursors, have been tested in Chinese hamster V79 cells subjected to H2O2 exposure at fixed concentration or ionizing radiation. Cytotoxicity was evaluated by monitoring the viability assessed clonogenic assay. compounds varied terms ring size, oxidation state, substituents. Electrochemical studies were carried out measure redox midpoint potentials. show that case protection against exposure, determined In...

Abstract A nitroxide side chain (R1) has been substituted at single sites along a helix–turn–helix motif in T4 lysozyme (residues 114–135). Together with previously published data, the new reported complete continuous scan through motif. Mutants R1 115 and 118 were selected for crystallographic analysis to identify structural origins of corresponding two‐component EPR spectra. At 115, is shown occupy two rotamers room temperature crystal structure, one which not reported. The components...

A series of 3,5-bis(arylidene)-4-piperidone (DAP) compounds are considered as synthetic analogues curcumin for anticancer properties. We performed structure-activity relationship studies by synthesizing a number DAPs N-alkylated or acylated with nitroxides their amine precursors potent antioxidant moieties. Both subtituents on arylidene rings and piperidone nitrogen (five- six-membered, 2- 3-substituted 3,4-disubstituted isoindoline nitroxides) were varied. The efficacy the new DAP was...

Abstract The purpose of this study was to evaluate the anticancer potency and mechanism a novel difluorodiarylidenyl piperidone (H-4073) its N-hydroxypyrroline modification (HO-3867) in human ovarian cancer. Studies were done using established cancer cell lines (A2870, A2780cDDP, OV-4, SKOV3, PA-1, OVCAR3) as well murine xenograft tumor (A2780) model. Both compounds comparably significantly cytotoxic A2780 cells. However, HO-3867 showed preferential toxicity toward cells while sparing...

Cisplatin resistance is a major obstacle in the treatment of ovarian cancer. Drug combinations with synergistic or complementary functions are promising strategy to overcome this issue. We studied anticancer efficacy novel compound, HO-3867, used combination cisplatin against chemotherapy-resistant A2780R cells, cisplatin-resistant human cancer cell line, were exposed 1, 5, 10 uM HO-3867 alone (10 ug/ml) for 24 hours. Cell viability (MTT), proliferation (BrdU), cell-cycle analysis (FACS),...

Previous studies have shown that the mobility of nitroxide side chains in a protein, inferred from electron paramagnetic resonance (EPR) spectra, can be used to classify particular sites as helix surface sites, tertiary contact buried or loop sites. In addition, sequence dependence identify regular secondary structure. However, most widely chain, an apparent interaction ring with protein at some gives rise EPR spectra degenerate those present study, we use selected T4 lysozyme evaluate novel...

The capacity of ultraviolet (UV) radiation to generate reactive oxygen species (ROS) in spinach leaves was studied with fluorescent sensors. Leaf segments were exposed short-term (15–30 min), narrow-band UV irradiation various wavelengths between 290–390 nm corresponding equal numbers photons and, depending on the wavelength, 18–36 μmol m–2 s–1 quantum flux. This caused 5–30% loss photosynthesis measured as variable chlorophyll fluorescence. In accordance previous results, less affected by...

Aims Oxidative stress followed by abnormal signalling can play a critical role in the development of long-term, high blood pressure-induced cardiac remodelling heart failure (HF).Since oxidative stress-induced poly(ADP-ribose)polymerase (PARP) activation and cell death have been observed several experimental models, we investigated possibility that inhibition nuclear PARP improves performance delays transition from hypertensive cardiopathy to HF spontaneously rat (SHR) model HF.Methods...

The flavoprotein Cytochrome P450 reductase (CPR) is the unique electron pathway from NADPH to (CYPs). conformational dynamics of human CPR in solution, which involves transitions a "locked/closed" an "unlocked/open" state, crucial for transfer. To date, however, factors guiding these changes remain unknown. By Site-Directed Spin Labelling coupled Electron Paramagnetic Resonance spectroscopy, we have incorporated non-canonical amino acid onto flavin mononucleotide (FMN) and adenine...

This study introduces a method for the quantitative analysis of cross-section scanning electron micrographs graphene oxide membranes. involves segmentation using neural network built on UNet architecture that was trained synthetic data. Subsequently, OrientLayer algorithm is applied to calculate orientational order parameters lamellae forming membrane. The ordering compared with oxidized planes determined by spin probe technique. High prospects joint application microscopy and paramagnetic...

Abstract Understanding protein structure requires studying its dynamics, which is critical to elucidating functional role. Biophysical techniques have revolutionized this field over time, providing remarkable insights into structure‐function relationships. Among these, Site‐Directed Spin Labelling (SDSL) combined with Electron Paramagnetic Resonance (EPR) a powerful method delivering structural data at the residue level, irrespective of size or environment. Traditional nitroxide labels...

Fatty acid synthase (FAS) and focal adhesion kinase (FAK), which are overexpressed in a variety of human epithelial tumors, play key role the migration invasion cancer cells. Hence, strategies targeted at inhibiting FAS/FAK proteins may have therapeutic potential for treatment. The goal present study was to determine effect HO-3867, synthetic compound, on migratory ability ovarian cells understand mechanistic pathways including involvement FAS, FAK, associated signaling proteins. done using...

Phosphatase and tensin homolog (PTEN), a tumor suppressor gene, has been shown to play vital role in vascular smooth muscle cell (SMC) proliferation hence is potential therapeutic target inhibit remodeling. The goal of this study was evaluate the efficacy mechanism HO-3867 [((3<i>E</i>,5<i>E</i>)-3,5-bis[(4-fluorophenyl)methylidene]-1-[(1-hydroxy-2,2,5,5-tetramethyl-2,5-dihydro-1<i>H</i>-pyrrol-3-yl)methyl]piperidin-4-one)], new synthetic curcuminoid, inhibition SMC restenosis. Experiments...

Doxorubicin (DOX) is a drug commonly used for the treatment of cancer. The development resistance to DOX common, and high cumulative doses cause potentially lethal cardiac side effects. HO-3867 (3,5-bis(4-fluorobenzylidene)-1-[(2,2,5,5-tetramethyl-2,5-dihydro-1-hydroxy-pyrrol-3-yl)methyl]piperidin-4-one), synthetic curcumin analog, has been shown exhibit both anticancer cardioprotective However, its cardioprotection in setting conventional cancer therapy not established. This work...

Curcumin, a naturally-occurring compound found in the rhizome of Curcuma longa plant, is known for its antitumor activities. However, clinical efficacy limited due to poor bioabsorption. A new class synthetic analogs curcumin, namely diarylidenylpiperidone (DAP), has been developed with substantially higher anticancer activity than curcumin. cellular uptake and bioabsorption have not evaluated. In this study we determined absorption representative DAP compound, HO-3867, using optical...