Reactive oxygen species impair Na+ transport and renal components of the renin-angiotensin-aldosterone system after paraquat poisoning.

Paraquat (1,1'-dimethyl-4,4'-bipyridyl dichloride) is an herbicide widely used worldwide and officially banned in Brazil in 2020. Kidney lesions frequently occur, leading to acute kidney injury (AKI) due to exacerbated reactive O2 species (ROS) production. However, the consequences of ROS exposure on ionic transport and the regulator local renin-angiotensin-aldosterone system (RAAS) still need to be elucidated at a molecular level. This study evaluated how ROS acutely influences Na+-transporting ATPases and the renal RAAS. Adult male Wistar rats received paraquat (20 mg/kg; ip). After 24 h, we observed body weight loss and elevation of urinary flow and serum creatinine. In the renal cortex, paraquat increased ROS levels, NADPH oxidase and (Na++K+)ATPase activities, angiotensin II-type 1 receptors, tumor necrosis factor-α (TNF-α), and interleukin-6. In the medulla, paraquat increased ROS levels and NADPH oxidase activity but inhibited (Na++K+)ATPase. Paraquat induced opposite effects on the ouabain-resistant Na+-ATPase in the cortex (decrease) and medulla (increase). These alterations, except for increased serum creatinine and renal levels of TNF-α and interleukin-6, were prevented by 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (tempol; 1 mmol/L in drinking water), a stable antioxidant. In summary, after paraquat poisoning, ROS production culminated with impaired medullary function, urinary fluid loss, and disruption of Na+-transporting ATPases and angiotensin II signaling.

HSK3486 Inhibits Colorectal Cancer Growth by Promoting Oxidative Stress and ATPase Inhibitory Factor 1 Activation.

BACKGROUND: HSK3486 (ciprofol), a new candidate drug similar to propofol, exerts sedative and hypnotic effects through gamma-aminobutyric acid type A receptors; however, its potential role in colorectal cancer is currently unknown. AIMS: This study aimed to evaluate the effects of HSK3486 on colorectal cancer cell proliferation. METHODS: Imaging was performed to detect reactive oxygen species and mitochondrial membrane potential. Western blotting was used to determine the expression of target signals. The HSK3486 molecular mechanism was investigated through ATPase inhibitory factor 1 knockdown and xenograft model experiments to assess mitochondrial function in colorectal cancer cells. RESULTS: Cell Counting Kit-8 and Annexin V/propidium iodide double staining assays showed that HSK3486 inhibited colorectal cancer cell proliferation in a concentration-dependent manner. In addition, HSK3486 treatment increased the expression of B-cell lymphoma-2-associated X, cleaved caspase 3, and cleaved poly (ADP-ribose) polymerase, whereas myeloid cell leukemia-1 and B-cell lymphoma 2 expression decreased. HSK3486 promoted mitochondrial dysfunction by inducing ATPase inhibitor factor 1 expression. Furthermore, HSK3486 promoted oxidative stress, as shown by the increase in reactive oxygen species and lactate dehydrogenase levels, along with a decrease in mitochondrial membrane potential and ATP levels. ATPase inhibitor factor 1 small interfering RNA pretreatment dramatically increased the mitochondrial membrane potential and tumor size in a xenograft model following exposure to HSK3486. CONCLUSION: Collectively, our findings revealed that HSK3486 induces oxidative stress, resulting in colorectal cancer cell apoptosis, making it a potential candidate therapeutic strategy for colorectal cancer.

Antibacterial Activity and Mechanism of Polygonatum sibiricum Extract Against Bacillus cereus and Its Application in Pasteurized Milk.

The purpose of this study was to reveal the antibacterial activity and mechanism of Polygonatum sibiricum extract (PSE) against Bacillus cereus and further analyze the application of PSE in pasteurized milk (PM). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values and growth curve analysis were used to evaluate the antibacterial activity of PSE against B. cereus. The changes in contents of intracellular adenosine 5'-triphosphate (ATP) and reactive oxygen species (ROS), activities of ß-galactosidase, adenosine triphosphatase (ATPase) and alkaline phosphatase (AKP), cell membrane potential, protein and nucleic acid leakage, and cell morphology were used to reveal the antibacterial mechanism. The effects of PSE on viable count and sensory evaluation of PM during storage were analyzed. The results showed that the MIC and MBC values of PSE against B. cereus were 2 and 4 mg/mL, respectively. Growth curve analysis showed that PSE with a concentration of 2 MIC could completely inhibit the growth of B. cereus. After treatments with PSE, the levels of intracellular ATP and ROS, and activities of ß-galactosidase, ATPase and AKP of B. cereus were significantly reduced (p < 0.05). Cell membrane was depolarized, amounts of protein and nucleic acid leakage were significantly increased (p < 0.05), and cell morphology was destroyed. Furthermore, PSE significantly reduced the viable count of B. cereus in PM and improved the sensory quality of PM during storage (p < 0.05). Together, our findings suggested that PSE had the desired effect as a natural preservative applied in PM.

ApoA-I Protects Pancreatic ß-Cells From Cholesterol-Induced Mitochondrial Damage and Restores Their Ability to Secrete Insulin.

BACKGROUND: High cholesterol levels in pancreatic ß-cells cause oxidative stress and decrease insulin secretion. ß-cells can internalize apo (apolipoprotein) A-I, which increases insulin secretion. This study asks whether internalization of apoA-I improves ß-cell insulin secretion by reducing oxidative stress. METHODS: Ins-1E cells were cholesterol-loaded by incubation with cholesterol-methyl-ß-cyclodextrin. Insulin secretion in the presence of 2.8 or 25 mmol/L glucose was quantified by radioimmunoassay. Internalization of fluorescently labeled apoA-I by ß-cells was monitored by flow cytometry. The effects of apoA-I internalization on ß-cell gene expression were evaluated by RNA sequencing. ApoA-I-binding partners on the ß-cell surface were identified by mass spectrometry. Mitochondrial oxidative stress was quantified in ß-cells and isolated islets with MitoSOX and confocal microscopy. RESULTS: An F1-ATPase ß-subunit on the ß-cell surface was identified as the main apoA-I-binding partner. ß-cell internalization of apoA-I was time-, concentration-, temperature-, cholesterol-, and F1-ATPase ß-subunit-dependent. ß-cells with internalized apoA-I (apoA-I+ cells) had higher cholesterol and cell surface F1-ATPase ß-subunit levels than ß-cells without internalized apoA-I (apoA-I- cells). The internalized apoA-I colocalized with mitochondria and was associated with reduced oxidative stress and increased insulin secretion. The IF1 (ATPase inhibitory factor 1) attenuated apoA-I internalization and increased oxidative stress in Ins-1E ß-cells and isolated mouse islets. Differentially expressed genes in apoA-I+ and apoA-I- Ins-1E cells were related to protein synthesis, the unfolded protein response, insulin secretion, and mitochondrial function. CONCLUSIONS: These results establish that ß-cells are functionally heterogeneous, and apoA-I restores insulin secretion in ß-cells with elevated cholesterol levels by improving mitochondrial redox balance.

Arjunolic acid reverses fluoxetine-induced alterations in testicular steroidogenic enzymes and membrane bound ionic pump imbalance through suppression of oxido-inflammatory stress and apoptosis.

OBJECTIVE: The impact of the anti-depressant therapy on gonadal function has been recognized and discussed over the years. However, data to supplement our understanding of the impact of arjunolic acid (AA) therapies in protecting against FXT-induced gonadal dysfunction is lacking clear scientific evidence. Hence, this study aimed to investigate the possible effect of AA on fluoxetine-induced altered testicular function in rats. METHODS: After 14 days acclimatization, Thirty-six (36) adult male rats were randomly divided into 6 groups (n=6). Rats in groups 1 received normal saline (10mL/kg); groups 2 & 3 were given AA (1.0mg/kg body weight) and AA (2.0mg/kg body weight), respectively; whereas, rats in group 4 were given FXT (10mg/kg/p.o/day), and groups 5 & 6 were given a combination of FXT (10mg/kg) + AA (1.0mg/kg body weight); and FXT (10mg/kg) + AA (2.0mg/kg body weight), respectively. RESULTS: The results shows that FXT significantly altered testicular steroidogenic enzymes (3ß-HSD and 17ß-HSD) and proton pump ATPase (Na+/K+ ATPase, Ca2+ ATPase and H+ ATPase) activities, as well as testicular architecture when compared with controls. More so, FXT caused oxido-inflammation and apoptosis, as evidence by increases in MDA, MPO, TNF-α, IL-1ß, Caspase 3 and p53. However, AA at a different dose significantly ameliorated the destructive impacts of FXT on steroidogenic enzymes, proton pump ATPase as well as increased Bcl-2, SOD, CAT, GSH and improved testicular architecture in rats. CONCLUSIONS: AA reverses fluoxetine-induced alterations in testicular steroidogenic enzymes and membrane-bound ionic pump through suppression of oxido-inflammatory stress and apoptosis.

Specific targeting to the Mycobacterium tuberculosis P-type ATPase Membrane Transporter, CtpF, of antituberculous compounds obtained by structure-based design.

Background: The resurgence of Mycobacterium tuberculosis (Mtb) strains that resist anti-tuberculosis (anti-TB) drugs used currently stresses the search for more effective low-toxicity drugs against new targets. Due to their role in ion homeostasis and virulence, Mtb plasma membrane P-type ATPases are interesting anti-TB targets, in particular, the Ca2+ transporting P2-type ATPase CtpF which is involved in oxidative stress response and persistence. Methods: In this study, the effect on the transcription level of the ctpF gene and other Mtb P2-type ATPases of two anti-Mtb hits was assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Both anti-Mtb hits ZINC14541509 and ZINC63908257 had been previously identified using pharmacophore-based virtual screening and MM-GBSA binding free energy. In addition, the bacterial activity of both compounds on Mycobacterium bovis was evaluated to see whether or not there is an effect on other mycobacteria of the Mtb complex. Results: qRT-PCR experiments showed that the ctpF transcription level was significantly higher in the presence of both compounds, especially ZINC14541509, strongly suggesting that CtpF may be a specific target of the selected compound. Conclusions: ZINC14541509 should be considered as an alternative for the structural-based design of novel anti-TB drugs.

The protective effect of Luteolin on chicken spleen lymphocytes from ammonia poisoning through mitochondria and balancing energy metabolism disorders.

Ammonia poses a significant challenge in the contemporary intensive breeding industry, resulting in substantial economic losses. Despite this, there is a dearth of research investigating efficacious strategies to prevent ammonia poisoning in poultry. Consequently, the objective of this study was to investigate the molecular mechanisms through which Luteolin (Lut) safeguards mitochondria and restores equilibrium to energy metabolism disorders, thereby shielding chicken spleen lymphocytes from the detrimental effects of ammonia poisoning. Chicken spleen lymphocytes were categorized into 3 distinct groups: the control group, the ammonia group (with the addition of 1 mmol/L of ammonium chloride), and the Lut group (with the treatment of 0.5 µg/mL of Lut for 12 h followed by the addition of 1 mmol/L of ammonium chloride). These groups were then cultured for a duration of 24 h. To investigate the potential protective effect of Lut on lymphocytes exposed to ammonia, various techniques were employed, including CCK-8 analysis, ultrastructural observation, reagent kit methodology, fluorescence microscopy, and quantitative real-time PCR (qRT-PCR). The findings indicate that Lut has the potential to mitigate the morphological damage of mitochondria caused by ammonia poisoning. Additionally, it can counteract the decline in mitochondrial membrane potential, ATP content, and ATPase activities (specifically Na+/K+-ATPase, Ca2+-ATPase, Mg2+-ATPase, and Ca/Mg2+-ATPase) following exposure to ammonia in lymphocytes. Lut also has the ability to regulate the expression of genes involved in mitochondrial fusion (Opa1, Mfn1, and Mfn2) and division (Drp1 and Mff) in spleen lymphocytes after ammonia exposure. This regulation leads to a balanced energy metabolism (HK1, HK2, LDHA, LDHB, PFK, PK, SDHB, and ACO2) and provides protection against ammonia poisoning.

Salinity-dependent mitigation of naphthalene toxicity in migratory Takifugu obscurus juveniles: Implications for survival, oxidative stress, and osmoregulation.

Naphthalene, an environmental pollutant classified as a polycyclic aromatic hydrocarbon (PAH), can induce toxicity in fish and other aquatic organisms. Through our investigation, we determined how Takifugu obscurus juveniles were affected by naphthalene (0, 2 mg L-1) exposure in terms of oxidative stress biomarkers and Na+/K+-ATPase activity in various tissues (gill, liver, kidney and muscle) under dissimilar salinities (0, 10 psu). Results suggest that naphthalene exposure significantly affects the survival of T. obscurus juveniles and leads to significant changes in the levels of malondialdehyde, superoxide dismutase, catalase, glutathione, and Na+/K+-ATPase activity, which are indicative of oxidative stress and emphasized the risks associated with osmoregulatory function. The higher salinity affected on the noxious effects of naphthalene can be observed, resulting in decreased biomarker levels and increased Na+/K+-ATPase activity. Salinity levels affected the uptake of naphthalene and its impact on different tissues, with high salinity conditions having mitigating effects on oxidative stress and naphthalene uptake in the liver and kidney tissues. Increased Na+/K+-ATPase activity was observed in all tissues treated with 10 psu and 2 mg L-1 naphthalene. Our findings deepen the understanding of T. obscurus juveniles' physiological responses to naphthalene exposure, and highlight the potential mitigating effects of salinity. These insights can inform the development of appropriate conservation and management practices to protect aquatic organisms from susceptibility.

Sub­chronic administration of lead alters markers of oxidative stress, acetylcholinesterase and Na+K+­ATPase activities in rat brain.

This study investigated the effects of sub­chronic administration of lead (Pb) acetate on thiobarbituric acid reactive substances (TBA­RS), total sulfhydryl content, protein carbonyl content, antioxidant enzymes (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GSH­Px]), acetylcholinesterase (AChE), and Na+K+­ATPase in the cerebral structures of rats. Male Wistar rats aged 60 days were treated with saline (control group) or Pb (treatment group), at various doses, by gavage, once a day for 35 days. The animals were sacrificed twelve hours after the last administration, and the cerebellum, hippocampus and cerebral cortex were removed. The results showed that Pb did not alter the evaluated oxidative stress parameters. Furthermore, Pb (64 and/or 128 mg/kg) altered SOD in the cerebellum, cerebral cortex and hippocampus. Pb (128 mg/kg) altered CAT in the cerebellum and cerebral cortex and GSH­Px in the cerebral cortex. Also, Pb (64 mg/kg and 128 mg/kg) altered GSH­Px in the cerebellum. Moreover, Pb (128 mg/kg) increased AChE in the hippocampus and decreased Na+K+­ATPase in the cerebellum and hippocampus. In conclusion, sub­chronic exposure to Pb (occupational and environmental intoxication) altered antioxidant enzymes, AChE, and Na+K+­ATPase, contributing to cerebral dysfunction.

The Oncogenic Protein Kinase/ATPase RIOK1 Is Up-Regulated via the c-myc/E2F Transcription Factor Axis in Prostate Cancer.

The atypical protein kinase/ATPase RIO kinase (RIOK)-1 is involved in pre-40S ribosomal subunit production, cell-cycle progression, and protein arginine N-methyltransferase 5 methylosome substrate recruitment. RIOK1 overexpression is a characteristic of several malignancies and is correlated with cancer stage, therapy resistance, poor patient survival, and other prognostic factors. However, its role in prostate cancer (PCa) is unknown. In this study, the expression, regulation, and therapeutic potential of RIOK1 in PCa were examined. RIOK1 mRNA and protein expression were elevated in PCa tissue samples and correlated with proliferative and protein homeostasis-related pathways. RIOK1 was identified as a downstream target gene of the c-myc/E2F transcription factors. Proliferation of PCa cells was significantly reduced with RIOK1 knockdown and overexpression of the dominant-negative RIOK1-D324A mutant. Biochemical inhibition of RIOK1 with toyocamycin led to strong antiproliferative effects in androgen receptor-negative and -positive PCa cell lines with EC50 values of 3.5 to 8.8 nmol/L. Rapid decreases in RIOK1 protein expression and total rRNA content, and a shift in the 28S/18S rRNA ratio, were found with toyocamycin treatment. Apoptosis was induced with toyocamycin treatment at a level similar to that with the chemotherapeutic drug docetaxel used in clinical practice. In summary, the current study indicates that RIOK1 is a part of the MYC oncogene network, and as such, could be considered for future treatment of patients with PCa.

In vitro galactose impairs energy metabolism in the brain of young rats: protective role of antioxidants.

We, herein, investigated the in vitro effects of galactose on the activity of pyruvate kinase, succinate dehydrogenase (SDH), complex II and IV (cytochrome c oxidase) of the respiratory chain and Na+K+-ATPase in the cerebral cortex, cerebellum and hippocampus of 30-day-old rats. We also determined the influence of the antioxidants, trolox, ascorbic acid and glutathione, on the effects elicited by galactose. Galactose was added to the assay at concentrations of 0.1, 3.0, 5.0 and 10.0 mM. Control experiments were performed without galactose. Galactose, at 3.0, 5.0 and 10.0 mM, decreased pyruvate kinase activity in the cerebral cortex and at 10.0 mM in the hippocampus. Galactose, at 10.0 mM, reduced SDH and complex II activities in the cerebellum and hippocampus, and reduced cytochrome c oxidase activity in the hippocampus. Additionally, decreased Na+K+-ATPase activity in the cerebral cortex and hippocampus; conversely, galactose, at 3.0 and 5.0 mM, increased this enzyme's activity in the cerebellum. Data show that galactose disrupts energy metabolism and trolox, ascorbic acid and glutathione addition prevented the majority of alterations in the parameters analyzed, suggesting the use of antioxidants as an adjuvant therapy in Classic galactosemia.

Effect of alpha-lipoic acid and caffeine-loaded chitosan nanoparticles on obesity and its complications in liver and kidney in rats.

The present work investigated the effect of α-lipoic acid (ALA) and caffeine-loaded chitosan nanoparticles (CAF-CS NPs) on obesity and its hepatic and renal complications in rats. Rats were divided into control, rat model of obesity induced by high fat diet (HFD), and obese rats treated with ALA and/or CAF-CS NPs. At the end of the experiment, the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) and the levels of urea, creatinine, interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) were determined in the sera of animals. In addition, malondialdehyde (MDA), nitric oxide (NO), and reduced glutathione (GSH) were measured in hepatic and renal tissues. Renal Na+, K+-ATPase was assessed. The histopathological changes were examined in the hepatic and renal tissues. Obese rats showed a significant increase in AST, ALT, ALP, urea, and creatinine. This was associated with a significant increase in IL-1ß, TNF-α, MDA, and NO. A significant decrease in hepatic and renal GSH and renal Na+, K+-ATPase activity was recorded in obese rats. Obese rats also showed histopathological alterations in hepatic and renal tissues. Treatment with ALA and/or CAF-CS NPs reduced the weight of obese rats and ameliorated almost all the hepatic and renal biochemical and histopathological changes induced in obese rats. In conclusion, the present findings indicate that ALA and/or CAF-CS NPs offered an effective therapy against obesity induced by HFD and its hepatic and renal complications. The therapeutic effect of ALA and CAF-CS NPs could be mediated through their antioxidant and anti-inflammatory properties.

Chronic fluoxetine treatment in socially-isolated rats modulates the prefrontal cortex synaptoproteome.

Exposure to chronic social isolation (CSIS) and synapse dysfunction have been implicated in the etiology of major depressive disorder (MDD). Fluoxetine (Flx) has been widely used to treat MDD, but its mechanisms of action remain elusive. We employed comparative synaptoproteomics to investigate the changes in the levels of proteins and molecular signaling pathways in prefrontal cortical samples of adult male Wistar rats exposed to CSIS, a rat model of depression, and CSIS rats treated with chronic Flx and controls, using liquid chromatography coupled to tandem mass spectrometry. Flx-treated control rats showed a decreased level of proteins involved in vesicle-mediated transport, and a predominantly increased level of exocytosis-associated proteins. CSIS significantly reduced the level of proteins involved in the ATP metabolic process, clathrin-dependent endocytosis, and proteolysis. Flx treatment in CSIS rats stimulated synaptic vesicle trafficking by increasing the regulation of exo/endocytosis-associated proteins, proteins involved in synaptic plasticity including neurogenesis, Cox5a, mitochondria-associated proteins involved in oxidative phosphorylation, and ion transport proteins (Slc8a2, Atp1b2). Flx treatment resulted in an increased synaptic vesicle dynamic, plasticity and mitochondrial functionality, and a suppression of CSIS-induced impairment of these processes. BIOLOGICAL SIGNIFICANCE: Identifying biomarkers of MDD and treatment response is the goal of many studies. Contemporary studies have shown that many molecular alterations associated with the pathophysiology of MDD reside within the synapse. As part of this research, a growing importance is the use of proteomics, as monitoring the changes in protein levels enables the identification of (possible) biochemical pathways and processes of importance for the development of depressive-like behavior and the efficacy of antidepressant treatments. We profiled proteomic changes representative of the development of CSIS-induced depressive-like behavior and the antidepressant effects of Flx. Our study has identified synaptosomal proteins and altered molecular pathways that may be potential markers of prefrontal cortical synaptic dysfunction associated with depressive-like behavior, and further clarified the mechanisms of depressive-like behavior and mode of action of Flx. Our findings indicate potential PFC synaptic targets for antidepressant treatment.

Disturbance of gut microbiota aggravates cadmium-induced neurotoxicity in zebrafish larvae through V-ATPase.

Cadmium (Cd) is a harmful environmental pollutant that causes damage to the nervous system, and exposure to Cd also disrupts the gut microbiota. However, it is still unclear whether Cd-induced neurotoxicity is related to alteration of the microbiota. In this study, we first established a germ-free (GF) zebrafish model to avoid the effects of gut microbiota disturbances caused by Cd exposure, and found that Cd-induced neurotoxic effects were weak in GF zebrafish. RNA sequencing showed that expression levels of V-ATPase family genes (atp6v1g1, atp6v1b2, and atp6v0cb) were significantly decreased in Cd-treated conventionally reared (CV) zebrafish, while this inhibition could be avoided in GF zebrafish. Overexpression of atp6v0cb in the V-ATPase family could partially rescue Cd-induced neurotoxicity. Our study shows that the disturbance of gut microbiota aggravates Cd-induced neurotoxicity, and that this may be associated with the expression of several genes in the V-ATPase family.

Regulation of proteolysis in bovine cumulus cells with possible inclusion of proton pump activators.

The aim of this study was to reveal the effects of V-ATPase proton pump activation on lysosomal acidity and protein degradation in cultured cumulus cells. Cumulus cells from bovine ovaries were cultured in the presence of 10 and 50 µM doses of V-ATPase proton pump activators PIP2, PMA and DOG for 12 and 24 h. At the end of the culture period, the level of protein degradation was evaluated through DQ-Red-BSA analysis and the lysosomes were detected through a fluorescent probe. In addition, total and phosphorylated MAPK1/3 and AKT protein levels of cumulus cells were determined through Western blotting. PIP2 and PMA were shown to increase protein degradation and lysosomal acidity in cultured bovine cumulus cells, whereas DOG did not have any significant effects on these cells. Total and phosphorylated MAPK and AKT protein levels were higher in PIP2 and PMA groups compared with the control and DOG. It was concluded that particular proton pump activators can enhance protein degradation and lysosomal acidification in cultured bovine cumulus cells without having detrimental effects on cell signalling members required for cell viability and proper functioning. Due to the cellular interactions, increasing the lysosomal activity in cumulus cells in the culture environment could also affect the removal of protein aggregates in the oocytes. This strategy could be effective for improving in vitro maturation of the oocytes by providing proteostasis.

Antiseizure effect of 2-deoxyglucose is not dependent on the presynaptic vacuole ATP pump or the somatic ATP-sensitive K+ channel.

Inhibition of glycolysis with 2-deoxyglucose (2-DG) produces antiseizure effects in brain slices and animal models, yet the mechanisms remain elusive. Here, we examined two glycolysis-derived ATP-associated mechanisms: vacuole ATP pump (V-ATPase) and ATP-sensitive K+ channel (KATP). Epileptiform bursts were generated in the CA3 area of hippocampal slices by 0 Mg2+ and 4-aminopyridine. 2-DG consistently abolished epileptiform bursts in the presence of pyruvate (to sustain tricarboxylic acid cycle for oxidative ATP production) at 30-33°C but not at room temperature (22°C). Under physiological conditions, 2-DG did not reduce the amplitude of evoked excitatory postsynaptic currents (EPSCs) or the paired-pulse ratio in CA3 neurons. During repetitive high-frequency (20 Hz, 20-50 pulses) stimulation, 2-DG did not accelerate the decline of EPSCs (i.e., depletion of transmitter release), even when preincubated with 8 mM K+ to enhance activity-dependent uptake of 2-DG. In addition, in 2-DG tetanic stimulation (200 Hz, 1 s) dramatically increased rather than diminished the occurrence of spontaneous EPSCs immediately after stimulation (i.e., no transmitter depletion). Moreover, a V-ATPase blocker (concanamycin) failed to block epileptiform bursts that were subsequently abolished by 2-DG. Furthermore, 2-DG did not induce detectable KATP current in hippocampal neurons. Finally, epileptiform bursts were not affected by either a KATP opener (diazoxide) or a KATP blocker (glibenclamide) but were blocked by 2-DG in the same slices. Altogether, these data suggest that 2-DG's antiseizure action is temperature dependent and achieved exclusively by inhibition of glycolysis and is not likely to be mediated by the two membrane-bound ATP-associated machinery mechanisms, V-ATPase and KATP.NEW & NOTEWORTHY Inhibition of glycolysis with 2-deoxyglucose (2-DG) represents a novel metabolic antiseizure approach, yet the mechanisms remain elusive. Here, we show that 2-DG's antiseizure action is both glycolysis and temperature dependent but not mediated by the vacuole ATP pump (V-ATPase) or ATP-sensitive K+ channel (KATP). Our data provide new insights to understand 2-DG's cellular mechanisms of action and, more broadly, neuronal metabolism and excitability.

https://elsevier.proofcentral.com/en-us/landing-page.html?token=baf280639f2773e07701834b1c13daInhibition of spermatogenesis by hypoxia is mediated by V-ATPase via the JNK/c-Jun pathway in mice.

Spermatocyte apoptosis is the primary cause of a poor outcome after hypoxia-triggered spermatogenesis reduction (HSR). Vacuolar H+-ATPase (V-ATPase) is involved in the regulation of hypoxia-induced spermatocyte apoptosis; however, the underlying mechanism remains to be elucidated. The aim of this study was to investigate the effect of V-ATPase deficiency on spermatocyte apoptosis and the relationship between c-Jun and apoptosis in primary spermatocytes induced by hypoxia. We found that mice under hypoxia exposure for 30 days demonstrated a marked spermatogenesis reduction and downregulation of V-ATPase expression, which were assessed by a TUNEL assay and western blotting, respectively. V-ATPase deficiency resulted in more severe spermatogenesis reduction and spermatocyte apoptosis after hypoxia exposure. We also observed that silencing V-ATPase expression enhanced JNK/c-Jun activation and death receptor-mediated apoptosis in primary spermatocytes. However, inhibition of c-Jun attenuated V-ATPase deficiency-induced spermatocyte apoptosis in primary spermatocytes. In conclusion, the data in this study suggest that V-ATPase deficiency aggravated hypoxia-induced spermatogenesis reduction by promoting spermatocyte apoptosis in mice via the JNK/c-Jun pathway.

[Short-term exposure to gossypol causes reversible reproductive toxicity and nephrotoxicity in mice].

OBJECTIVE: To study the toxic effects of short-term exposure to gossypol on the testis and kidney in mice and whether these effects are reversible. METHODS: Twenty 7 to 8-week-old male mice were randomized into blank control group, solvent control group, gossypol treatment group and drug withdrawal group. In the former 3 groups, the mice were subjected to daily intragastric administration of 0.3 mL of purified water, 1% sodium carboxymethylcellulose solution, and 30 mg/mL gossypol solution for 14 days, respectively; In the drug withdrawal group, the mice were treated with gossypol solution in the same manner for 14 days followed by treatment with purified water for another 14 days. After the last administration, the mice were euthanized and tissue samples were collected. The testicular tissue was weighed and observed microscopically with HE and PAS staining; the kidney tissue was stained with HE and examined for mitochondrial ATPase activity. RESULTS: Compared with those in the control group, the mice with gossypol exposure showed reduced testicular seminiferous epithelial cells with rounded seminiferous tubules, enlarged space between the seminiferous tubules, interstitium atrophy of the testis, and incomplete differentiation of the spermatogonia. The gossypol-treated mice also presented with complete, non-elongated spermatids, a large number of cells in the state of round spermatids, and negativity for acrosome PAS reaction; diffuse renal mesangial cell hyperplasia, increased mesangial matrix, and adhesion of the mesangium to the wall of the renal capsule were observed, with significantly shrinkage or even absence of the lumens of the renal capsules and reduced kidney mitochondrial ATPase activity. Compared with the gossypol-treated mice, the mice in the drug withdrawal group showed obvious recovery of morphologies of the testis and the kidney, acrosome PAS reaction and mitochondrial ATPase activity. CONCLUSIONS: Shortterm treatment with gossypol can cause reproductive toxicity and nephrotoxicity in mice, but these toxic effects can be reversed after drug withdrawal.

Nanocracker capable of simultaneously reversing both P-glycoprotein and tumor microenvironment.

Here, we describe a multidrug-resistant nanocracker (MDRC) that can treat multi-drug resistant (MDR) cancer by recognizing the acidic microenvironment and inhibiting two mechanisms of MDR such as P-glycoprotein (P-gp) and vacuolar-type ATPase (V-ATPase). MDRC is a liposome formulation co-loading pantoprazole (PZ) and paclitaxel (PTX). PZ acts as a chemosensitizer that enhances the MDR cancer treatment effect of PTX by disrupting the pH gradient and inhibiting P-gp. MDRC-encapsulated PZ and PTX have different release rates, with PZ released within 12 h and PTX sustained release for 48 h in the plasma. MDRC could increase cell uptake by inhibiting the P-gp overexpressed MCF-7/mdr cells and UV-2237M cells, which are human breast MDR cancer cells and murine fibrosarcoma cells, respectively. MDRC can also increase the cytotoxic efficacy of PTX by increasing intracellular pH. MDRC has a 10.5-fold reduced IC50 value in the P-gp overexpressed human breast adenocarcinoma and a 6.3- to 9.5-fold reduced IC50 value in the P-gp non-expressed human breast adenocarcinoma compared to the mixture of PZ and PTX, respectively. Intravenous injection of MDRC did not cause weight loss, liver dysfunction, or major organ toxicity. MDRC exhibited 80% complete remission of murine fibrosarcoma. The excellent therapeutic effect of MDRC on MDR tumors was accompanied by an increase in dendritic cell maturation and cytotoxic T cells. In other words, MDRC has the potential to terminate MDR therapy through the complete remission of MDR tumors.

Undesirable consequences of the metallic nanoparticles action on the properties and functioning of Escherichia coli, Bacillus cereus and Staphylococcus epidermidis membranes.

Controversial and inconsistent findings on the toxicity of metallic nanoparticles (NPs) against many bacteria are common in recorded studies; therefore, further advanced experimental work is needed to elucidate the mechanisms underlying nanotoxicity. This study deciphered the direct effects of Ag-NPs, Cu-NPs, ZnO-NPs and TiO2-NPs on membrane permeability, cytoplasmic leakage, ATP level, ATPase activity and fatty acid profiling of Escherichia coli, Bacillus cereus and Staphylococcus epidermidis as model microorganisms. A multifaceted analysis of all collected results indicated the different influences of individual NPs on the measured parameters depending on their type and concentration. Predominantly, membrane permeability was correlated with increased cytoplasmic leakage, reduced total ATP levels and ATPase activity. The established fatty acid profiles were unique and concerned various changes in the percentages of hydroxyl, cyclopropane, branched and unsaturated fatty acids. Decisively, E. coli was more susceptible to changes in measured parameters than B. cereus and S. epidermidis. Also, it was established that ZnO-NPs and Cu-NPs had a major differentiating impact on studied parameters. Additionally, bacterial cell imaging using scanning electron microscopy elucidated different NPs distributions on the cell surface. The presented results are believed to provide novel, valuable and accumulated knowledge in the understanding of NPs action on bacterial membranes.