Search Results (174)

Some studies performed in our laboratory on pyrrole and its derivatives pointed towards the enrichment of the evaluations of these promising chemical structures for the potential treatment of neurodegenerative conditions in general and Parkinson’s disease in particular. A classical Paal-Knorr cyclization approach is applied to synthesize the basic hydrazine used for the formation of the designed series of hydrazones (15a–15g). The potential neurotoxic and neuroprotective effects of the newly synthesized derivatives were investigated in vitro using different models of induced oxidative stress at three subcellular levels (rat brain synaptosomes, mitochondria, and microsomes). The results identified as the least neurotoxic molecules, 15a, 15d, and 15f applied at a concentration of 100 µM to the isolated fractions. In addition, the highest statistically significant neuroprotection was observed for 15a and 15d at a concentration of 100 µM using three different injury models on subcellular fractions, including 6-hydroxydopamine in rat brain synaptosomes, tert-butyl hydroperoxide in brain mitochondria, and non-enzyme-induced lipid peroxidation in brain microsomes. The hMAOA/MAOB inhibitory activity of the new compounds was studied at a concentration of 1 µM. The lack of a statistically significant hMAOA inhibitory effect was observed for all tested compounds, except for 15f, which showed 40% inhibitory activity. The most prominent statistically significant hMAOB inhibitory effect was determined for 15a, 15d, and 15f, comparable to that of selegiline. The corresponding selectivity index defined 15f as a non-selective MAO inhibitor and all other new hydrazones as selective hMAOB inhibitors, with 15d indicating the highest selectivity index of >471. The most active and least toxic representative (15d) was evaluated in vivo on Rotenone based model of Parkinson’s disease. The results revealed no microscopically visible alterations in the ganglion and glial cells in the animals treated with rotenone in combination with 15d. Full article

The subcellular partitioning of trace elements (TEs) may depend on their binding preferences, although few field data are available from mining-impacted areas. Northern pike and lake whitefish were collected from different aquatic systems located in the Yellowknife mining area (Northwest Territories, Canada) to examine the subcellular partitioning of TEs in liver cells. Elements belonging to metal classes based on binding affinities were considered: A (Ce, La), borderline (As, Pb), and class B (Ag, Cd). Measurements in the metal-detoxified fractions (granule-like structures and heat-stable proteins and peptides) and in the putative metal-sensitive fractions (heat-denatured proteins, mitochondria and microsomes, and lysosomes) revealed marked differences among metal classes. In both fish species, Cd and Ag accumulated more as detoxified forms (higher than 50%, likely bound to metallothionein-like proteins) than La and Ce (not more than 20%). The two borderline TEs (As and Pb) showed an intermediate behavior between classes A and B. Similar proportions were found in the “sensitive” subcellular fractions for all TEs, where quantitative ion character-activity relationships (QICARs) indicated the covalent index and electronegativity as predictors of the TE contribution in this compartment. This study supports the use of classes of metals to predict the toxicological risk of data-poor metals in mining areas. Full article

So far, synthetic biology approaches for the construction of artificial microorganisms have fostered the transformation of acceptor cells with genomes from donor cells. However, this strategy seems to be limited to closely related bacterial species only, due to the need for a “fit” between donor and acceptor proteomes and structures. “Fitting” of cellular regulation of metabolite fluxes and turnover between donor and acceptor cells, i.e. cybernetic heredity, may be even more difficult to achieve. The bacterial transformation experiment design 1.0, as introduced by Frederick Griffith almost one century ago, may support integration of DNA, macromolecular, topological, cybernetic and cellular heredity: (i) attenuation of donor Pneumococci of (S) serotype fosters release of DNA, and hypothetically of non-DNA structures compatible with subsequent transfer to and transformation of acceptor Pneumococci from (R) to (S) serotype; (ii) use of intact donor cells rather than of subcellular or purified fractions may guarantee maximal diversity of the structural and cybernetic matter and information transferred; (iii) “Blending” or mixing and fusion of donor and acceptor Pneumococci may occur under accompanying transfer of metabolites and regulatory circuits. A Griffith transformation experiment design 2.0 is suggested, which may enable efficient exchange of DNA as well as non-DNA structural and cybernetic matter and information, leading to unicellular hybrid microorganisms with large morphological/metabolic phenotypic differences and major features compared to predeceding cells. The prerequisites of horizontal gene and somatic cell nuclear transfer, the molecular mechanism of transformation, the machineries for the biogenesis of bacterial cytoskeleton, micelle-like complexes and membrane landscapes are briefly reviewed on the basis of underlying conceptions, ranging from Darwin’s “gemmules” to “stirps”, cytoplasmic and “plasmon” inheritance, “rhizene agency”, “communicology”, “transdisciplinary membranology” to up to Kirschner’s “facilitated variation”. Full article

This study aims to evaluate the tolerance of an endophytic fungus isolated from the fibrous roots of Gentiana yunnanensis Franch. to arsenic (As) and elucidate the underlying physiological and molecular mechanisms. The filamentous fungus is identified as Cladosporium cladosporioides based on morphological characteristics and phylogenetic tree analysis, belonging to the family Moniliaceae and Phyla Hyphomycetes. The tolerance of C. cladosporioides to As(V) was assessed by measuring its biomass under varying concentrations of As(V). The fungus exhibited remarkable As(V) tolerance, with an EC₅₀ value of 2051.94 mg/L, and accumulated high concentrations of As in its mycelium. Subcellular distribution analysis revealed that As was predominantly localized in the cell wall fraction, with levels 4.06 times higher than those in the non-cell wall fraction. Notably, the concentrations of total organic As and As(III) in the mycelium were 852.75 μg/g and 24.94 μg/g, respectively, with conversion ratios of 76.64% and 2.24%. The organic As levels significantly surpassed both As(V) and As(III) concentrations in all cellular fractions (cell wall and non-cell wall components), demonstrating particularly efficient As transformation in C. cladosporioides. Under As(V) stress, the membrane antioxidant system, including superoxide dismutase (SOD), metallothionein (MT), glutathione (GSH), and melanin, was activated and significantly enhanced to mitigate oxidative damage. Transcriptomic analysis identified 4771 differentially expressed genes (DEGs; 2527 upregulated), including highly expressed As-responsive genes (CcArsH_1, CcARR_1, CcARR_3, CcGST_1, and CcGST_3). Strong correlations emerged between As speciation (total/organic/As(V)/As(III)), antioxidant levels, and DEG expression patterns. Taken together, these findings demonstrate that C. cladosporioides employs a multi-faceted As detoxification strategy involving subcellular distribution and reductive transformation (As(V) to As(III)/organic As), antioxidant system enhancement, transcriptomic adaptations, and integrated defense strategy. This work highlights C. cladosporioides potential for As bioremediation and elucidates As accumulation mechanisms in G. yunnanensis. Full article

Neurodegenerative diseases involve oxidative stress and enzyme dysregulation, necessitating novel neuroprotective agents. This study evaluates the neuroprotective and antioxidant potential of seven pyrrole-based compounds with predicted radical scavenging activity and inhibitory effects on monoamine oxidase B (MAO-B) and acetylcholinesterase (AChE). The compounds were tested in vitro using SH-SY5Y neuroblastoma cells and subcellular rat brain fractions, including synaptosomes, mitochondria, and microsomes. Neuroprotective and antioxidant effects were assessed in oxidative stress models, including H₂O₂-induced stress in SH-SY5Y cells, 6-hydroxydopamine toxicity in synaptosomes, tert-butyl hydroperoxide-induced stress in mitochondria, and non-enzyme lipid peroxidation in microsomes. In silico screening for lipophilicity, hydrogen bonding, total polar surface area (TPSA), and ionization properties, was performed to evaluate bioavailability. All compounds exhibited a weak neurotoxic effect on the subcellular fractions at a concentration of 100 µM. However, in oxidative stress models, they demonstrated significant neuroprotective and antioxidant effects at 100 µM. In SH-SY5Y cells, compounds 7, 9, 12, 14, and 15 exhibited low toxicity and strong protective effects at concentrations as low as 1 µM. In silico analysis prioritized compounds 1, 7, 9, 12, and 14 for further development based on their favorable bioavailability. The tested pyrrole-based compounds exhibit promising neuroprotective and antioxidant properties, with several candidates showing potential for further development based on both in vitro efficacy and predicted oral bioavailability. Full article

Background/Objectives: ⁸⁹Zr-oxine is an ex vivo cell labeling agent that enables cells to be tracked in vivo by positron emission tomography (PET) over a period of up to two weeks. To better understand where ⁸⁹Zr-oxine binds within cellular components, factors affecting labeling and intracellular distribution of ⁸⁹Zr were examined. Methods: Mouse primary T cells, natural killer cells, dendritic cells, and monocytes, and cell lines EL4 (mouse lymphoma), DC2.4 (mouse dendritic cell), Kit225K6 (human T cell leukemia) and MC38 (mouse colon adenocarcinoma) were labeled with ⁸⁹Zr-oxine or ¹¹¹In-oxine and protein binding within the cellular compartments, the labeling thresholds, and radioactivity retention were subsequently determined. Results: Cell incorporation of ⁸⁹Zr-oxine (27.8–71.8 kBq/10⁶ cells) positively correlated with cellular size and protein mass. Most (>97%) ⁸⁹Zr was protein-bound and primarily localized in the cytoplasm, membrane, and nuclear fractions (>81%) with distribution patterns varying by cell type. By contrast, ¹¹¹In-oxine showed lower protein-binding activity of approximately 59–65%, with 62–65% of ¹¹¹In localized in the cytoplasm. Autoradiography of electrophoresed subcellular fractionated cell samples indicated stable binding by ⁸⁹Zr-oxine to proteins in all subcellular fractions but unstable protein binding by ¹¹¹In. Saturation studies showed that ⁸⁹Zr-oxine labeling was saturable, and further labeling reduced cellular retention. Biodistribution of dendritic cells labeled with either ⁸⁹Zr-oxine or ¹¹¹In-oxine indicated greater retention of ⁸⁹Zr in the labeled cells in vivo than ¹¹¹In. Conclusions: ⁸⁹Zr-oxine stably binds many intracellular proteins and shows much higher and more stable protein binding than ¹¹¹In-oxine. Intracellular protein binding of ⁸⁹Zr accounts for the ability of ⁸⁹Zr-oxine labeling to successfully track cells in vivo long-term on PET. Full article

Pharmaceutical and Personal Care Products (PPCPs), such as carbamazepine, enter the food chain through wastewater irrigation, posing risks to ecosystems and human health. However, research on the translocation and subcellular distribution of carbamazepine in vegetables is limited. Herein, we used ¹⁴C-labeled carbamazepine as a tracer to investigate its removal, accumulation, and subcellular compartmentalization in hydroponic vegetable systems. Results showed carbamazepine accumulated in Chinese flowering cabbage and water spinach with removal efficiencies of 93.0–93.2%. The compound was absorbed by roots and translocated to aboveground tissues, particularly in bottom leaves, reaching 90.3 μmol/kg after 768 h, as confirmed by autoradiography. Subcellular analysis indicated that carbamazepine is predominantly distributed in root organelles and in the soluble fraction of leaves and stems. A strong correlation (R² > 0.800) was observed between root enrichment coefficients and log K_OW for caffeine, carbamazepine, and kresoxim-methyl. Higher lipid content in water spinach roots (2.07%) significantly enhanced upward transport, underscoring lipid content’s role in translocation. Additionally, a higher xylem content in the plant accelerated the transport of carbamazepine. This study provides key insights into the environmental behavior of organic pollutants, supporting efforts in environmental and health protection. Full article

Background: The association of sex hormone-binding globulin (SHBG) with heart failure (HF) remains a topic of ongoing debate, particularly in the light of type 2 diabetes mellitus (T2DM). We aimed to assess the association of SHBG with clinical and echocardiographic parameters of HF in men according to the presence of T2DM. Methods: Data on baseline characteristics, cardiovascular risk factors and medications, laboratory findings including serum SHBG and total testosterone concentrations, and echocardiographic parameters were prospectively collected for 215 male patients consecutively hospitalized for an acute episode of HF. Results: Patients with T2DM were older (p = 0.013), had a greater body mass index (p = 0.009) and NYHA class (p = 0.001), and were more likely to have hypertension (p < 0.001) or hyperlipidemia (p = 0.032). A moderate correlation among SHBG and total testosterone with the left ventricular ejection fraction (LVEF) was observed only in T2DM patients (r = 0.456) but not among non-T2DM patients (r = 0.194). A multivariate analysis revealed the independent association of increased SHBG levels with lower LVEF values among T2DM patients (ß = −0.542, p < 0.0001), whereas in the same group higher total testosterone was an independent predictor of higher LVEF (ß = 0.531, p < 0.0001) and lower LVDD (ß = −0.442, p = 0.0002) levels. Conclusions: In men with HF and T2DM, in contrast to testosterone, SHBG may have an independent adverse impact on the LVEF, which may account for 12.5% of the variance in LVEF levels. The possible subcellular mechanisms of SHBG in men with diabetic myocardial disorder should be additionally explored. Full article

Mitochondrial miRNAs (mitomiRs), which are miRNAs that located within mitochondria, have emerged as crucial regulators in a variety of human diseases, including multiple types of cancers. However, the specific role of mitomiRs in clear cell renal cell carcinoma (ccRCC) remains elusive. In this study, we employed a combination of experimental and bioinformatic approaches to uncover the diverse and abundant subcellular distribution of miRNAs within mitochondria in ccRCC. Notably, RNA sequencing after mitochondrial fractionation identified miR-134-5p as a miRNA predominantly detected in the mitochondria of 786O cells, and its expression is significantly upregulated compared to that in 293T cells. Differential expression and survival analyses from TCGA reveal that the upregulation of miR-134-5p is prevalent and closely associated with poor survival outcomes in ccRCC patients. Functionally, exogenous overexpression of miR-134-5p mimics promotes migration in both 786O and Caki-1 cells. Mechanistically, overexpressing the miR-134-5p mimic dramatically downregulates the mRNA levels of CHST6, SFXN2, and GRIK3, whereas the miR-134-5p inhibitor markedly upregulates their expression. Notably, these target mRNAs also predominantly detected in the mitochondria of 786O cells. The downregulated expression signatures of CHST6, SFXN2, and GRIK3 are also closely correlated with poor survival outcomes in ccRCC patients. Taken together, our work identifies a novel mitomiR, miR-134-5p, in ccRCC, provides potential targets that could serve as effective biomarkers for ccRCC diagnosis and prognosis, and opens new avenues for understanding the mitomiR-directed regulatory network in ccRCC progression. Full article

The development of more effective treatments for neurodegenerative disorders presents a significant challenge in modern medicine. Currently, scientists are focusing on discovering bioactive compounds from plant sources to prevent and treat neurodegenerative diseases. Fifteen flavonoids and saponins from C. foliosum Asch. and C. bonus-henricus L. were tested for their inhibitory activity on hMAO-A and hMAO-B. Five compounds (1 μM) exhibit a weak inhibitory effect on hMAO-A and show good inhibitory activity against the hMAO-B enzyme (30–35%), compared to the positive control selegiline (55%). These active compounds were examined on rat brain synaptosomes and mitochondria obtained by multiple differential centrifugations using a Percoll gradient. Their effects were also monitored on rat brain microsomes obtained by double differential centrifugation. The main parameters characterizing the functional–metabolic status of subcellular fractions are synaptosomal viability, GSH level, and MDA production. All tested compounds (50 μM) demonstrated significant neuroprotective and antioxidant activities across models of induced oxidative stress, including 6-OHDA, t-BuOOH, and Fe^2+/AA-induced lipid peroxidation. The plausible mechanisms of neuroprotection rely on MAO-B inhibition, the scavenging of ROS, stabilizing the cell membrane by reducing MDA production, and neutralizing free radicals by maintaining GSH levels. In addition, we developed and validated a UHPLC-HRMS method for identifying and simultaneously quantificatying flavonoids and saponins in the aerial parts of C. foliosum. Compounds 30-normedicagenic acid- HexA-Hex-TA 22f and medicagenic acid-HexA-Hex-TA 25f were considered new natural compounds. Full article

Caffeine contamination in water sources raises concerns about its transfer to agricultural products and potential risks to human health through the food chain. Despite these concerns, limited research has focused on the accumulation and distribution of exogenous caffeine in tea plants. This study explored the uptake, translocation, targeted accumulation, subcellular distribution, and preliminary metabolism of ¹⁴C-labeled caffeine in a hydroponic tea seedling system. After 192 h of cultivation, more than 83.8% of the caffeine had been removed from the nutrient solution. Within the plants, ¹⁴C-caffeine and its metabolites predominantly accumulated in the roots. Subcellular analysis indicates that in root cells, ¹⁴C was mainly distributed in the soluble fraction, cell walls, and plastids, while in shoot cells, it was concentrated in the soluble fraction and cell walls. Metabolic profiling reveals distinct varietal differences: in Longjing 43 tea seedlings, ¹⁴C was predominantly present as the caffeine parent compound, whereas in Jiaming No. 1 tea seedlings, ¹⁴C was found both as the parent compound and as its metabolite, xanthine. This study revealed differences in the uptake, translocation, and metabolism of exogenous caffeine among different tea plant varieties, providing broader insights into the impact of caffeine pollution on agricultural ecosystems. Full article

Lentinula edodes has a strong cadmium-enrichment ability, posing a potential threat to human health. However, the cadmium tolerance and detoxification mechanisms of Lentinula edodes are not understood. We investigated the physiological responses, subcellular distribution, and chemical forms of cadmium in two Lentinula edodes strains (1504 and L130) with contrasting cadmium tolerance. The results showed that appropriate, low-level cadmium promoted mycelial growth, and higher cadmium exposure induced obvious inhibition of mycelial growth by damaging the cell wall and membrane structure and triggering the overproduction of ROS. Antioxidant enzymes played an important role in cadmium detoxification, as well as functional group modulation. Cadmium was predominantly distributed in the cell wall fraction, and NaCl-extractable cadmium was the main chemical form. Enhanced antioxidant enzyme activities, reduced cadmium accumulation, and increased HAc-extractable cadmium with less toxicity promoted stronger cadmium tolerance and detoxification abilities in L130 compared to 1504. Thus, this study provides new insights into cadmium tolerance and detoxification in Lentinula edodes. Full article

p66Shc is an adaptor protein and one of the cellular fate regulators since it modulates mitogenic signaling pathways, mitochondrial function, and reactive oxygen species (ROS) production. p66Shc is localized mostly in the cytosol and endoplasmic reticulum (ER); however, under oxidative stress, p66Shc is post-translationally modified and relocates to mitochondria. p66Shc was found in the intermembrane space, where it interacts with cytochrome c, contributing to the hydrogen peroxide generation by the mitochondrial respiratory chain. Our previous studies suggested that p66Shc is localized also in mitochondria-associated membranes (MAM). MAM fraction consists of mitochondria and mostly ER membranes. Contact sites between ER and mitochondria host proteins involved in multiple processes including calcium homeostasis, apoptosis, and autophagy regulation. Thus, p66Shc in MAM could participate in processes related to cell fate determination. Due to reports on various and conditional p66Shc intracellular localization, in the present paper, we describe the allocation of p66Shc pools in different subcellular compartments in mouse liver tissue and HepG2 cell culture. We provide additional evidence for p66Shc localization in MAM. In the present study, we use precisely purified subcellular fraction isolated by differential centrifugation-based protocol from control mouse liver tissue and HepG2 cells and from cells treated with hydrogen peroxide to promote mitochondrial p66Shc translocation. We performed controlled digestion of crude mitochondrial fraction, in which the degradation patterns of p66Shc and MAM fraction marker proteins were comparable. Moreover, we assessed the distribution of the individual ShcA isoforms (p46Shc, p52Shc, and p66Shc) in the subcellular fractions and their contribution to the total ShcA in control mice livers and HepG2 cells. In conclusion, we showed that a substantial pool of p66Shc protein resides in MAM in control conditions and after oxidative stress induction. Full article

This experimental study was performed to evaluate the alterations in the expression of a few subunits composing glutamate AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) and NMDA (N-methyl-D-aspartate) receptors in the hippocampal cells of Wistar rats in response to long-term fluoride (F^-) exposure. The animals were given water with background 0.4 (control), 5, 20, and 50 ppm F^- (as NaF) for 12 months. The cognitive capacities of rats were examined by novel object recognition (NOR), Y-maze test, and Morris water maze tests. RT-qPCR and Western blotting techniques were used to evaluate the expression of different AMPA and NMDA subunits at transcriptional and translational levels, respectively. Long-term F^- poisoning disturbed the formation of hippocampus-dependent working spatial and long-term non-spatial memory. The expression of Gria1, Gria2, and Gria3 genes encoding different subunits of AMPA receptors were comparable in hippocampi of control and F^--exposed animals, although the levels of both Grin2a and Grin2b mRNA increased. Long-term F^- intake enhanced the ratio of phospho-GluA1/total-GluA1 proteins in subcellular fraction enriched with cytosolic proteins, while decreased content of GluA2 but elevated level of GluA3 were observed in subcellular fraction enriched with membrane proteins. Such changes were accompanied by increased phosphorylation of GluN2A and GluN2B subunits, higher ratios of GluN2A/GluN1 and GluN2B/GluN1 proteins in the cytosol, and GluN2A/GluN2B ratio in membranes. These changes indicate the predominance of Ca²⁺-permeable AMPARs in membranes and a shift between different NMDARs subunits in hippocampal cells of F^--exposed rats, which is typical for neurodegeneration and can at least partially underly the observed disturbances in cognitive capacities of animals. Full article

Neurosteroids are undeniably regarded as neuroprotective mediators, regulating brain function by rapid non-genomic actions involving interference with microtubules. Conversely, hyperphosphorylated Tau is considered responsible for the onset of a plethora of neurodegenerative diseases, as it dissociates from microtubules, leading to their destabilization, thus impairing synaptic vesicle transport and neurotransmission. Consequently, we aimed to investigate the effects of neurosteroids, specifically allopregnanolone (Allo) and dehydroepiandrosterone (DHEA), on the levels of total and phosphorylated at Serine 404 Tau (p-Tau) in C57BL/6 mice brain slices. In total tissue extracts, we found that neurosteroids elevated both total and p-Tau levels without significantly altering the p-Tau/Tau ratio. In addition, the levels of several enzymes implicated in Tau phosphorylation did not display significant differences between conditions, suggesting that neurosteroids influence Tau distribution rather than its phosphorylation. Hence, we subsequently examined the mitochondria-enriched subcellular fraction where, again, both p-Tau and total Tau levels were increased in the presence of neurosteroids. These effects seem actin-dependent, as disrupting actin polymerization by cytochalasin B preserved Tau levels. Furthermore, co-incubation with high [Ca²⁺] and neurosteroids mitigated the effects of Ca²⁺ overload, pointing to cytoskeletal remodeling as a potential mechanism underlying neurosteroid-induced neuroprotection. Full article