Search Results (11)

As part of our ongoing research on new anti-diabetic compounds from ethnopharmacologically consumed plants, two previously undescribed lupane-type triterpenoids (1 and 2) with dicarboxylic groups, an undescribed nor-taraxastane-type triterpenoid (3), and 14 known compounds (4–17) were isolated from the leaves of Cleistocalyx operculatus. Extensive spectroscopic analysis (IR, HRESIMS, 1D, and 2D NMR) was used for structure elucidation, while the known compounds were compared to reference data reported in the scientific literature. All the isolates (1–17) were evaluated for their inhibitory effects on the protein tyrosine phosphatase 1B (PTP1B) enzyme. Compounds 6, 9, and 17 showed strong PTP1B inhibitory activities. The mechanism of PTP1B inhibition was studied through enzyme kinetic experiments. A non-competitive mechanism of inhibition was determined using Lineweaver–Burk plots for compounds 6, 9, and 17. Additionally, Dixon plots were employed to determine the inhibition constant. Further insights were gained through a structure–activity relationship study and molecular docking analysis of isolated compounds with the PTP1B crystal structure. Moreover, all isolates (1–17) were tested for their stimulatory effects on the uptake of 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose (2-NBDG) in differentiated 3T3-L1 adipocyte cells. Compounds 6, 13, and 17 exhibited strong glucose absorption stimulation activity in a dose-dependent manner. Full article

Reactive oxygen and nitrogen species (RONS), including 3-nitro-l-tyrosine, play a dual role in human health, inducing oxidative damage and regulating cellular functions. Early and accurate detection of such molecules, such as L-tyrosine in urine, can serve as critical biomarkers for various cancers. In this study, we aimed to enhance the electrochemical detection of these molecules through the synthesis of La₂Sn₂O₇/f-HNT nanocomposites via a simple hydrothermal method. Detailed structural and morphological characterizations confirmed successful synthesis, consistent with our expected outcomes. The synthesized nanocomposites were utilized as nanocatalysts in electrochemical sensors, showing a notable limit of the detection of 0.012 µM for the real-time detection of 3-nitro-l-tyrosine. These findings underscore the potential of nanomaterial-based sensors in advancing early disease detection with high sensitivity, furthering our understanding of cellular oxidative processes. Full article

To study the molecular basis of the toxicological effect of a dietary nitrosated amino acid, namely, 3-nitrotyrosine (3-NT), differentiated human enterocytes were exposed to dietary concentrations of this species (200 μM) and analyzed for flow cytometry, protein oxidation markers and MS-based proteomics. The possible protective role of a dietary phytochemical, ellagic acid (EA) (200 μM), was also tested. The results revealed that cell viability was significantly affected by exposure to 3-NT, with a concomitant significant increase in necrosis (p < 0.05). 3-NT affected several biological processes, such as histocompatibility complex class II (MHC class II), and pathways related to type 3 metabotropic glutamate receptors binding. Addition of EA to 3-NT-treated cells stimulated the toxicological effects of the latter by reducing the abundance of proteins involved in mitochondrial conformation. These results emphasize the impact of dietary nitrosated amino acids in intestinal cell physiology and warn about the potential negative effects of ellagic acid when combined with noxious metabolites. Full article

Nitrobindins (Nbs) are all-β-barrel heme proteins and are present in prokaryotes and eukaryotes. Although their function(s) is still obscure, Nbs trap NO and inactivate peroxynitrite. Here, the kinetics of peroxynitrite scavenging by ferric Danio rerio Nb (Dr-Nb(III)) in the absence and presence of CO₂ is reported. The Dr-Nb(III)-catalyzed scavenging of peroxynitrite is facilitated by a low pH, indicating that the heme protein interacts preferentially with peroxynitrous acid, leading to the formation of nitrate (~91%) and nitrite (~9%). The physiological levels of CO₂ dramatically facilitate the spontaneous decay of peroxynitrite, overwhelming the scavenging activity of Dr-Nb(III). The effect of Dr-Nb(III) on the peroxynitrite-induced nitration of L-tyrosine was also investigated. Dr-Nb(III) inhibits the peroxynitrite-mediated nitration of free L-tyrosine, while, in the presence of CO₂, Dr-Nb(III) does not impair nitro-L-tyrosine formation. The comparative analysis of the present results with data reported in the literature indicates that, to act as efficient peroxynitrite scavengers in vivo, i.e., in the presence of physiological levels of CO₂, the ferric heme protein concentration must be higher than 10⁻⁴ M. Thus, only the circulating ferric hemoglobin levels appear to be high enough to efficiently compete with CO₂/HCO₃⁻ in peroxynitrite inactivation. The present results are of the utmost importance for tissues, like the eye retina in fish, where blood circulation is critical for adaptation to diving conditions. Full article

Genetically encoded caged amino acids can be used to control the dynamics of protein activities and cellular localization in response to external cues. In the present study, we revealed the structural basis for the recognition of O-(2-nitrobenzyl)-L-tyrosine (oNBTyr) by its specific variant of Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (oNBTyrRS), and then demonstrated its potential availability for time-resolved X-ray crystallography. The substrate-bound crystal structure of oNBTyrRS at a 2.79 Å resolution indicated that the replacement of tyrosine and leucine at positions 32 and 65 by glycine (Tyr32Gly and Leu65Gly, respectively) and Asp158Ser created sufficient space for entry of the bulky substitute into the amino acid binding pocket, while Glu in place of Leu162 formed a hydrogen bond with the nitro moiety of oNBTyr. We also produced an oNBTyr-containing lysozyme through a cell-free protein synthesis system derived from the Escherichia coli B95. ΔA strain with the UAG codon reassigned to the nonnatural amino acid. Another crystallographic study of the caged protein showed that the site-specifically incorporated oNBTyr was degraded to tyrosine by light irradiation of the crystals. Thus, cell-free protein synthesis of caged proteins with oNBTyr could facilitate time-resolved structural analysis of proteins, including medically important membrane proteins. Full article

Heavy metal pollution causes many soils to become a toxic environment not only for plants, but also microorganisms; however, little is known how heavy metal contaminated environment affects metabolism of phytopathogens and their capability of infecting host plants. In this study the oomycete Phytophthora infestans (Mont.) de Bary, the most harmful pathogen of potato, growing under moderate cadmium stress (Cd, 5 mg/L) showed nitro-oxidative imbalance associated with an enhanced antioxidant response. Cadmium notably elevated the level of nitric oxide, superoxide and peroxynitrite that stimulated nitrative modifications within the RNA and DNA pools in the phytopathogen structures. In contrast, the protein pool undergoing nitration was diminished confirming that protein tyrosine nitration is a flexible element of the oomycete adaptive strategy to heavy metal stress. Finally, to verify whether Cd is able to modify P. infestans pathogenicity, a disease index and molecular assessment of disease progress were analysed indicating that Cd stress enhanced aggressiveness of vr P. infestans towards various potato cultivars. Taken together, Cd not only affected hyphal growth rate and caused biochemical changes in P. infestans structures, but accelerated the pathogenicity as well. The nitro-oxidative homeostasis imbalance underlies the phytopathogen adaptive strategy and survival in the heavy metal contaminated environment. Full article

Quantification with satisfactory specificity and sensitivity of free 3-Nitro-l-tyrosine (3-NT), 3-Chloro-l-tyrosine (3-CT), and 3-Bromo-l-tyrosine (3-BT) in biological samples as potential inflammation, oxidative stress, and cancer biomarkers is analytically challenging. We aimed at developing a liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based method for their simultaneous analysis without an extract purification step by solid-phase extraction. Validation of the developed method yielded the following limits of detection (LOD) and quantification (LOQ) for 3-NT, 3-BT, and 3-CT: 0.030, 0.026, 0.030 ng/mL (LODs) and 0.100, 0.096, 0.098 ng/mL (LOQs). Coefficients of variation for all metabolites and tested concentrations were <10% and accuracy was within 95–105%. Method applicability was tested on colorectal cancer patients during the perioperative period. All metabolites were significantly higher in cancer patients than healthy controls. The 3-NT was significantly lower in advanced cancer and 3-BT showed a similar tendency. Dynamics of 3-BT in the early postoperative period were affected by type of surgery and presence of surgical site infections. In conclusion, a sensitive and specific LC-MS/MS method for simultaneous quantification of free 3-NT, 3-BT, and 3-CT in human plasma has been developed. Full article

Metal-polluted areas, especially where municipal sewage is used as fertilizer, often have high concentrations of more than one metal. The development of the root system is regulated by a complex signaling network, which includes reactive oxygen and nitrogen species. The delicate balance of the endogenous signal system can be affected by various environmental stimuli including heavy metals (HMs) in excess. Our goal was to analyze the microelement homeostasis, root architecture, and to determine the underlying changes in the nitro-oxidative status in the root system of rapeseed (Brassica napus L.) and sunflower (Helianthus annuus L.) subjected to combined HM treatments. The effect of model-sewage in two different layouts was simulated in rhizotron system by only supplementing the highest HM concentrations (Cd, Cr, Cu, Hg, Ni, Pb, and Zn) legally allowed. The two species reacted differently to combined HM treatment; compared to the relatively sensitive sunflower, rapeseed showed better metal translocation capability and root growth even at the more severe treatment, where the pattern of protein tyrosine nitration was reorganized. The obtained results, especially the increased nitric oxide content and changed pattern of tyrosine nitration in rapeseed, can indicate acclimation and species-specific nitro-oxidative responses to combined HM stress. Full article

Seven pimarane diterpenes (1–7) were isolated from Orthosiphon stamineus Benth. by assay-guided isolation. All of the isolates possessed a 2-deoxy-2-((7-nitro-2,1,3-benzoxadiazol-4-yl)amino)-d-glucose uptake effect in 3T3-L1 adipocytes at concentrations of 5 and 10 μM. Most of them showed potent inhibition against protein tyrosine phosphatase 1B with IC₅₀ values ranging from 0.33 to 9.84 μM. In the kinetic study, all inhibition types were exposed for the examined potencies, including mixed-competitive (1), non-competitives (3 and 5), competitive (6), and uncompetitive (7). The results suggested that O. stamineus and its pimarane diterpenes might exert the hypoglycemic effect via the insulin signaling pathway targeting inhibition of protein tyrosine phosphatase 1B (PTP1B) activity. Full article

Tyrosinemia type 1 (TT1) treatment with 2-(2-nitro-4-trifluormethyl-benzyl)-1,3-cyclohexanedione (NTBC) and a phenylalanine-tyrosine restricted diet is associated with low phenylalanine concentrations. Phenylalanine supplementation is prescribed without comprehensive consideration about its effect on metabolic control. We investigated the effect of phenylalanine supplementation on bloodspot phenylalanine, tyrosine, NTBC and succinylacetone. Eleven TT1 patients received 0, 20 and 40 mg/kg/day phenylalanine supplementation with the phenylalanine-tyrosine free L-amino acid supplements. Bloodspots were collected before breakfast, midday and evening meal. Differences between study periods, sample times and days within a study period were studied using (generalized) linear mixed model analyses. Twenty and 40 mg/kg/day phenylalanine supplementation prevented daytime phenylalanine decreases (p = 0.05) and most low phenylalanine concentrations, while tyrosine concentrations increased (p < 0.001). Furthermore, NTBC and succinylacetone concentrations did not differ between study periods. To conclude, 20 mg/kg/day phenylalanine supplementation can prevent most low phenylalanine concentrations without increasing tyrosine to concentrations above the target range or influencing NTBC and succinylacetone concentrations, while 40 mg/kg/day increased tyrosine concentrations to values above the targeted range. Additionally, this study showed that the effect of phenylalanine supplementation, and a possible phenylalanine deficiency, should be assessed using pre-midday meal blood samples that could be combined with an overnight fasted sample when in doubt. Full article

A simple and rapid As³⁺ detection method using 3-nitro-L-tyrosine (N-Tyr) is reported. We discovered the specific property of N-Tyr, which specifically chelates As³⁺. The reaction between As³⁺ and N-Tyr induces a prompt color change to vivid yellow, concomitantly increasing the absorbance at 430 nm. The selectivity for As³⁺ is confirmed by competitive binding experiments with various metal ions (Hg²⁺, Pb²⁺, Cd²⁺, Cr³⁺, Mg²⁺, Ni²⁺, Cu²⁺, Fe²⁺, Ca²⁺, Zn²⁺, and Mn²⁺). Also, the N-Tyr binding site, binding affinity, and As³⁺/N-Tyr reaction stoichiometry are investigated. The specific reaction is utilized to design a sensor that enables the quantitative detection of As³⁺ in the 0.1–100 μM range with good linearity (R² = 0.995). Furthermore, the method’s applicability for the analysis of real samples, e.g., tap and river water, is successfully confirmed, with good recoveries (94.32–109.15%) using As³⁺-spiked real water samples. We believe that our discovering and its application for As³⁺ analysis can be effectively utilized in environmental analyses such as those conducted in water management facilities, with simplicity, rapidity, and ease. Full article