ATPase Protein Research Articles

Ca2+ATPase enzyme activities and lipid and protein oxidations of grass carp (Ctenopharyngodon idellus) stored at 4 °C for 30 min under electromagnetic fields

This work studied the effects of electromagnetic fields (EMF) with frequencies between 100 and 400 Hz and a fixed strength of 12 mT on cold storage of grass carp at 4 °C for 30 min, and Ca2+ATPase enzyme activities, and lipid and protein oxidations in samples were measured to assess changes in intracellular Ca2+ concentration and oxidative stability. Results showed higher Ca2+ATPase activities in samples treated with EMF frequencies. Significant (p < 0.05) decreases occurred in protein oxidation for samples treated between 100 and 300 Hz, but an increase was observed for treatment with 400 Hz. However, the lipid oxidation increased for samples treated up to 200 Hz and decreased with further increase in frequency to 300 and 400 Hz. Nuclear magnetic resonance analysis showed that exposure to different frequencies of EMF could reduce the association of water molecules with protein for both bound and immobilized water. Overall, treatments of EMF between 100 and 400 Hz could improve grass carp quality during cold storage.

Effects of Allanblackia floribunda stem-bark Extracts on Oxidative stress and Mitochondrial Dysfunction in Rats Exposed to Crude Oil

The development of degenerative diseases has been linked to oxidative stress and mitochondrial dysfunction. Additionally, it has been demonstrated that the changes caused by crude oil exposure contribute to the onset of degenerative diseases. There is little or no information on the anti-degeneration properties of Allanblackia floribunda. This study was carried out to investigate brain mitochondrial tissues exposed to crude oil and the effects of A. floribunda as a preventive and therapeutic intervention against oxidative stress and mitochondrial dysfunction. Twenty-five Wistar rats were divided into five groups: the control group received distilled water, the second group received ethanol extracts of A. floribunda, the third group received crude oil (5 mL/kg), the fourth and fifth groups received crude oil and A. floribunda extract concurrently (200 and 400 mg/kg, respectively). The results showed that crude oil treatment caused a significant increase in brain mitochondrial MDA levels and induced significant alterations in brain mitochondrial antioxidant enzyme activities compared with control. Treatment with the extract alone revealed a significant increase in protein carbonyl, conjugated dienes, and ATPases compared with crude oil-treated rats. This indicates that A. floribunda inhibited crude oil-induced mitochondrial lipid peroxidation and improved ATPase and antioxidant status in the rat brain mitochondria, which further suggests that A. floribunda possesses potential and may efficiently inhibit crude oil-induced oxidative damage and improve mitochondrial functions.

Mechanism exploration of Solanum nigrum L. hyperaccumulating Cd compared to Zn from the perspective of metabolic pathways based on differentially expressed proteins using iTRAQ

It is challenging to determine the mechanism involved in only Cd hyperaccumulation by Solanum nigrum L. owing to the uniqueness of the process. Isobaric tags for relative and absolute quantitation (iTRAQ) were used to explore the mechanism by which S. nigrum hyperaccumulates Cd by comparing the differentially expressed proteins (DEPs) for Cd and Zn accumulation (non-Zn hyperaccumulator). Based on the comparison between the DEPs associated with Cd and Zn accumulation, the relative metabolic pathways reflected by 17 co-intersecting specific proteins associated with Cd and Zn accumulation included phagosome, aminoacyl-tRNA biosynthesis, and carbon metabolism. Apart from the 17 co-intersecting specific proteins, the conjoint metabolic pathways reported by 21 co-intersecting specific proteins associated with Cd accumulation and 30 co-intersecting specific proteins associated with Zn accumulation, the most differentially expressed metabolic pathways might cause Cd TF (Translocation factor)> 1 and Zn TF< 1, including protein export, ribosome, amino sugar, and nucleotide sugar metabolism. The determined DEPs were verified using qRT-PCR with the four key proteins M1CW30, A0A3Q7H652, A0A0V0IFB9, and A0A0V0IAC4. The plasma membrane H+-ATPase protein was identified using western blotting. Some physiological indices for protein-related differences indirectly confirmed the above results. These results are crucial to further explore the mechanisms involved in Cd hyperaccumulation.

Formation of multiple flagella caused by a mutation of the flagellar rotor protein FliM in Vibrio alginolyticus.

Marine bacterium Vibrio alginolyticus forms a single flagellum at a cell pole. In Vibrio, two proteins (GTPase FlhF and ATPase FlhG) regulate the number of flagella. We previously isolated the NMB155 mutant that forms multiple flagella despite the absence of mutations in flhF and flhG. Whole-genome sequencing of NMB155 identified an E9K mutation in FliM that is a component of C-ring in the flagellar rotor. Mutations in FliM result in defects in flagellar formation (fla) and flagellar rotation (che or mot); however, there are a few reports indicating that FliM mutations increase the number of flagella. Here, we determined that the E9K mutation confers the multi-flagellar phenotype and also the che phenotype. The co-expression of wild-type FliM and FliM-E9K indicated that they were competitive in regard to determining the flagellar number. The ATPase activity of FlhG has been correlated with the number of flagella. We observed that the ATPase activity of FlhG was increased by the addition of FliM but not by the addition of FliM-E9K in vitro. This indicates that FliM interacts with FlhG to increase its ATPase activity, and the E9K mutation may inhibit this interaction. FliM may control the ATPase activity of FlhG to properly regulate the number of the polar flagellum at the cell pole.

Comparative transcriptome analysis reveals the resistance regulation mechanism and fungicidal activity of the fungicide phenamacril in Fusarium oxysporum

Fusarium oxysporum (Fo) is an important species complex of soil-borne pathogenic fungi that cause vascular wilt diseases of agricultural crops and some opportunistic diseases of humans. The fungicide phenamacril has been extensively reported to have antifungal activity against Fusarium graminearum and Fusarium fujikuroi. In this study, we found that the amino acid substitutions (V151A and S418T) in Type I myosin FoMyo5 cause natural low resistance to phenamacril in the plant pathogenic Fo isolates. Therefore, we compared the transcriptomes of two phenamacril-resistant Fo isolates FoII5, Fo1st and one phenamacril-sensitive isolate Fo3_a after 1 μg/mL phenamacril treatment. Among the 2728 differentially expressed genes (DEGs), 14 DEGs involved in oxidation–reduction processes and MFS transporters, were significantly up-regulated in phenamacril-resistant isolates. On the other hand, 14 DEGs involved in ATP-dependent RNA helicase and ribosomal biogenesis related proteins, showed significantly down-regulated expression in both phenamacril-resistant and -sensitive isolates. These results indicated that phenamacril not only seriously affected the cytoskeletal protein binding and ATPase activity of sensitive isolate, but also suppressed ribosome biogenesis in all the isolates. Hence, this study helps us better understand resistance regulation mechanism and fungicidal activity of phenamacril and provide reference for the development of new fungicides to control Fo.

Abstract 5680: Hypoxia-responsive and HIF1α-regulated AAA+ ATPase ATAD2 shows high oncogenic potential in stomach cancer

Abstract The burden of stomach cancer incidence and death is a global challenge. Treating stomach cancer remains immensely difficult due to the lack of available markers to detect the disease at the early stages. Therefore, there is an immense demand for finding out an early detection biomarker for stomach cancer. Recently, ATAD2 is found to be overexpressed in stomach cancer with high prognostic significance and recognised as a promising biomarker for stomach cancer. ATAD2 is a unique cancer/testis antigen (CTA) that belongs to both AAA+ ATPase and bromodomain family proteins. However, nothing much is known about the mechanism of ATAD2-mediated stomach carcinogenesis. Using bioinformatics analyses as well as studies with stomach cancer cells and stomach biopsy tissue samples, we address the regulation and function of ATAD2 expression in stomach cancer. We show that ATAD2 is overexpressed in the most common intestinal type of stomach adenocarcinoma, and enhanced expression of ATAD2 is observed in all stages and grades of stomach cancer. Our immunofluorescence microscopy study with stomach biopsy tissues confirms the high expression of ATAD2 both in stomach adenocarcinoma and metastatic stomach tissue samples. Survival analysis indicates that upregulated ATAD2 expression drastically affects the survival of stomach cancer patients. Since H. pylori infection, and hypoxia are the major contributing factors for stomach carcinogenesis; we further study their role in ATAD2-mediated stomach malignancy. An enhanced expression of ATAD2 is observed in H. pylori-infected stomach cancer cells. We identify ATAD2 as a hypoxia-responsive and HIF1α-regulated gene and elucidate that upregulated expression of ATAD2 enhances proliferation and migration of hypoxic stomach cancer cells. We further recognize the protein-protein interaction (PPI) network of ATAD2 with the top-ranked partners like ESR1 (or ERα), NCOA3 (or ACTR/SRC3/AIB1), SUMO2, HDA11 (HDAC11), SPTN2 (SPTBN2), and MYC. Among them, ESR1, MYC, NCOA3, and HDA11 are oncoproteins. Most strikingly, we report the participation of two druggable targets of ATAD2, i.e., AAA+ ATPase and bromodomain in the ATAD2-PPI network; ESR1, SUMO2, SPTN2, and MYC show a preference for bromodomain, whereas NCOA3 and HDA11 prefer ATPase domain of ATAD2. The importance of such findings in terms of targeting bromodomain-PPI and/or ATPase-PPI interfaces to curtail stomach cancer is elucidated. Citation Format: Anasuya Roychowdhury, Aditi Nayak, Sugandh Kumar, Anshuman Dixit, Asima Bhattacharyya. Hypoxia-responsive and HIF1α-regulated AAA+ ATPase ATAD2 shows high oncogenic potential in stomach cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5680.

Expanding the phenotype of ATP6AP1 deficiency.

Vacuolar ATPases (V-ATPases) are large multisubunit proton pumps conserved among all eukaryotic cells that are involved in diverse functions including acidification of membrane-bound intracellular compartments. The ATP6AP1 gene encodes an accessory subunit of the vacuolar (V)-ATPase protein pump. Pathogenic variants in ATP6AP1 have been described in association with a congenital disorder of glycosylation (CDG), which are highly variable, but often characterized by immunodeficiency, hepatopathy, and neurologic manifestations. Although the most striking and common clinical feature is hepatopathy, the phenotypic and genotypic spectrum of ATP6AP1-CDG continues to expand. Here, we report identical twins who presented with acute liver failure and jaundice. Prenatal features included cystic hygroma, atrial septal defect, and ventriculomegaly. Postnatal features included pectus carinatum, connective tissue abnormalities, and hypospadias. Whole-exome sequencing (WES) revealed a novel de novo in-frame deletion in the ATP6AP1 gene (c.230_232delACT;p.Tyr77del). Although both twins have the commonly reported clinical feature of hepatopathy seen in other individuals with ATP6AP1-CDG-related disorder, they do not have neurological sequelae. This report expands the phenotypic spectrum of ATP6AP1-CDG-related disorder with both probands exhibiting unique prenatal and postnatal features, including fetal ventriculomegaly, umbilical hernia, pectus carinatum, micropenis, and hypospadias. Furthermore, this case affirms that neurological features described in the initial case series on ATP6AP1-CDG do not appear to be central, whereas the prenatal and connective tissue manifestations may be more common than previously thought. This emphasizes the importance of long-term clinical follow-up and variant interpretation using current updated recommendations.

Enhancement performance of CO2 on the organic toxicity removal of sludge by Scenedesmus obliquus with proteomics analysis

Microalgae have strong adaptability to toxic environments. The accumulation of toxic organic matter and secondary metabolites during the biologically treatment of wastewater can cause sludge toxicity. Scenedesmus obliquus was cultivated in the sludge extract from coal-gasification synthetic wastewater contained 100 mg/L hydroquinone, with 15% CO2, to investigate the removal of sludge toxicity together with CO2 fixation. Results showed that S. obliquus could utilize the organic matter and CO2 in the mixed cultivation mode. On the 30th day, the dry cell weight was 1.46 ± 0.07 g/L, and chlorophyll-a was 24.76 ± 0.21 mg/L. S. obliquus reduced the sludge toxicity by heterotrophically utilizing the organic matter of sludge extract. TOC decreased from 393.52 ± 0.65 mg/L to 113.78 ± 0.43 mg/L, and organic matter with molecular weights of < 500 Da and 500–5KDa was significantly consumed, reducing the sludge toxicity from 83.46 ± 0.27% to 29.37 ± 0.19% in terms of inhibition rate of luminescent bacteria. S. obliquus mainly utilized HCO3- for autotrophic growth and the CO2 removal (%) was up to the maximum 35.08 ± 0.19% on the 10th day. Proteomics analysis results showed that 15% CO2 improved autotrophic ability of S. obliquus and the activity of the functional protein, thereby improving its antioxidant capacity against toxicity stress. By increasing its protein kinase and ATPase activity, S. obliquus enhanced the oxidative phosphorylation process to heterotrophically utilize organic matter to reduce the sludge toxicity.

Pharmacogenomics of in vitro response of the NCI-60 cancer cell line panel to Indian natural products

BackgroundIndian natural products have been anecdotally used for cancer treatment but with limited efficacy. To better understand their mechanism, we examined the publicly available data for the activity of Indian natural products in the NCI-60 cell line panel.MethodsWe examined associations of molecular genomic features in the well-characterized NCI-60 cancer cell line panel with in vitro response to treatment with 75 compounds derived from Indian plant-based natural products. We analyzed expression measures for annotated transcripts, lncRNAs, and miRNAs, and protein-changing single nucleotide variants in cancer-related genes. We also examined the similarities between cancer cell line response to Indian natural products and response to reference anti-tumor compounds recorded in a U.S. National Cancer Institute (NCI) Developmental Therapeutics Program database.ResultsHierarchical clustering based on cell line response measures identified clustering of Phyllanthus and cucurbitacin products with known anti-tumor agents with anti-mitotic mechanisms of action. Curcumin and curcuminoids mostly clustered together. We found associations of response to Indian natural products with expression of multiple genes, notably including SLC7A11 involved in solute transport and ATAD3A and ATAD3B encoding mitochondrial ATPase proteins, as well as significant associations with functional single nucleotide variants, including BRAF V600E.ConclusionThese findings suggest potential mechanisms of action and novel associations of in vitro response with gene expression and some cancer-related mutations that increase our understanding of these Indian natural products.

LMPA Regulates Lesion Mimic Leaf and Panicle Development Through ROS-Induced PCD in Rice.

Leaf and panicle are important nutrient and yield organs in rice, respectively. Although several genes controlling lesion mimic leaf and panicle abortion have been identified, a few studies have reported the involvement of a single gene in the production of both the traits. In this study, we characterized a panicle abortion mutant, lesion mimic leaf and panicle apical abortion (lmpa), which exhibits lesions on the leaf and causes degeneration of apical spikelets. Molecular cloning revealed that LMPA encodes a proton pump ATPase protein that is localized in the plasma membrane and is highly expressed in leaves and panicles. The analysis of promoter activity showed that the insertion of a fragment in the promoter of lmpa caused a decrease in the transcription level. Cellular and histochemistry analysis indicated that the ROS accumulated and cell death occurred in lmpa. Moreover, physiological experiments revealed that lmpa was more sensitive to high temperatures and salt stress conditions. These results provide a better understanding of the role of LMPA in panicle development and lesion mimic formation by regulating ROS homeostasis.

Enhancement effects of dissolved organic matter leached from sewage sludge on microbial reduction and immobilization of Cr(VI) by Geobacter sulfurreducens

Sewage sludge has a high concentration of dissolved organic matter (DOM) which contains compounds that can serve as electron donors or shuttles for metal reduction by dissimilatory metal reducing bacteria (DMRB). In this study, Cr(VI) removal by G. sulfurreducens, a common DMRB present in anaerobic soils, was examined in the presence or absence of sludge DOM. Two different types of sludge DOM were tested; composted sludge DOM (C-DOM) and anaerobically digested sludge DOM (A-DOM). Both sludge DOMs enhanced Cr(VI) reduction by G. sulfurreducens, but C-DOM was more effective likely because it had higher concentrations of humic substances that served as electron shuttles. Transcriptomic studies indicated that G. sulfurreducens utilizes several different mechanisms to tolerate chromium including extracellular Cr(VI) reduction and immobilization by outer membrane c-type cytochromes and electrically conductive pili, intracellular Cr(VI) reduction by triheme cytochromes and NAD(P)H FMN reductase proteins, and chromium efflux by several P-type ATPase and RND transporter proteins. Microscopy experiments also showed that Cr(III) crystals formed on the surface of the cells, indicating that extracellular Cr(VI) reduction and adsorption was involved in the chromium removal process. These results help provide insight into the potential use of sewage sludge as an additive to enhance the bioremediation of chromium contaminated soils.

Proteome profiling reveals changes in energy metabolism, transport and antioxidation during drought stress in Nostoc flagelliforme

BackgroundDrought is an important abiotic stress that constrains the growth of many species. Despite extensive study in model organisms, the underlying mechanisms of drought tolerance in Nostoc flagelliforme remain elusive.ResultsWe characterized the drought adaptation of N. flagelliforme by a combination of proteomics and qRT-PCR. A total of 351 differentially expressed proteins involved in drought stress adaptation were identified. It was found that the expression of several nutrient influx transporters was increased, including molybdate ABC transporter substrate binding protein (modA), sulfate ABC transporter substrate-binding protein (sbp) and nitrate ABC transporter (ntrB), while that of efflux transporters for toxic substances was also increased, including arsenic transporting ATPase (ArsA), potassium transporter (TrkA) and iron ABC transporter substrate-binding protein (VacB). Additionally, photosynthetic components were reduced while sugars built up during drought stress. Non-enzymatic antioxidants, orange carotenoid protein (OCP) homologs, cytochrome P450 (CYP450), proline (Pro) and ascorbic acid (AsA) were all altered during drought stress and may play important roles in scavenging reactive oxygen species (ROS).ConclusionIn this study, N. flagelliforme may regulates its adaptation to drought stress through the changes of protein expression in photosynthesis, energy metabolism, transport, protein synthesis and degradation and antioxidation.Highlights• A total of 351 DEPs involved in adaptation to drought stress were identified.• Changes in the expression of six OCP homologs were found in response to drought stress.• Differential expression of transporters played an important role in drought stress adaptation.• Most PSII proteins were downregulated, while PSI proteins were unchanged in response to drought stress.• Sugar metabolism was upregulated in response to drought stress.

Potential mechanisms of Na+/K+-ATPase attenuation by heat and pesticides co-exposure in goldfish: role of cellular apoptosis, oxidative/nitrative stress, and antioxidants in gills.

In this study, we examined the dose-dependent effects of an environmentally relevant pesticide cocktail (metalachlor, linuron, isoproturon, tebucanazole, aclonifen, atrazine, pendimethalin, and azinphos-methyl) and temperature change (22 vs. 32°C for 4-week exposure) on Na+/K+-ATPase, 3-nitrotyrosine protein (NTP), dinitrophenyl protein (DNP), catalase (CAT), and superoxide dismutase (SOD) expressions in gills of goldfish (Carassius auratus). Histopathological analysis showed widespread damage to gill in elevated temperature (32°C) and pesticide co-exposure groups, including fusion of secondary lamellae, club-shaped primary lamellae, rupture of epithelial layer, loss of normal architecture, and hemorrhaging. Immunohistochemical and qRT-PCR analyses showed significant decreases in Na+/K+-ATPase protein and mRNA expressions in gills exposed to higher temperature and pesticides; however, combined exposure to heat and pesticides significantly increases NTP, DNP, CAT, and SOD expressions. In situ TUNEL assay revealed elevated levels of apoptotic cells in response to combined exposure. Collectively, our results suggest the combined effects of heat and pesticide stress cause cellular damage, upregulate oxidative/nitrative stress biomarkers, and increase apoptotic cells, downregulate Na+/K+-ATPase expression in gills. This provides new evidence for oxidant/antioxidant-dependent mechanisms for downregulation of Na+/K+-ATPase expression in gills during combined exposure.

Single and joint toxicity assessment of acetamiprid and thiamethoxam neonicotinoids pesticides on biochemical indices and antioxidant enzyme activities of a freshwater fish Catla catla

Neonicotinoids pesticides are extensively used in many countries due to their high insect selectivity. Acetamiprid and thiamethoxam are the neonicotinoids most commonly detected in the aquatic environment. This work examined the single and joint toxicity of acetamiprid and thiamethoxam in a freshwater fish Catla catla. Fish were exposed to acetamiprid (0.5 mg/L and 1 mg/L), thiamethoxam (0.01 mg/L and 0.5 mg/L) and their binary mixtures (0.5 mg/L of acetamiprid and 0.01 mg/L of thiamethoxam) for 96 h. The stress biomarkers such as glucose, protein, electrolytes, Na+/K+ -ATPase and oxidative stress were evaluated. Among the biochemical parameters, plasma protein, electrolytes (sodium, potassium and chloride) and gill ATPase activity were decreased in response to individual and binary mixtures treatments. In contrast, blood glucose level showed significant increase in all the treatments. Exposure to various concentrations of acetamiprid and thiamethoxam resulted in significant decrease in superoxide dismutase (SOD) activity in the gill tissue. However, SOD activity was significantly elevated during binary mixtures treatment. Glutathione peroxidase (GPx), catalase (CAT), glutathione-S-transferase (GST) and reduced glutathione (GSH) levels in gills were decreased significantly after individual and binary mixtures treatments. Fish exposed at individual and binary mixtures significantly elevated the level of LPO in gill tissue. Our findings suggest that multi-biomarker approach can be effectively used to assess the effects of joint toxicity of pesticides and to monitor the neonicotinoids pesticides in the aquatic environment.

Hydrogen Attenuates Thyroid Hormone-Induced Cardiac Hypertrophy in Rats by regulating angiotensin II type 1 receptor and NADPH oxidase 2 mediated oxidative stress

Cardiac hypertrophy occurs as a result of high levels of thyroid hormone, which may contribute to heart failure and is closely related to oxidative stress. Hydrogen is a good antioxidant. In this study, we found that intragastric levothyroxine administration for two weeks caused obvious cardiac hypertrophy without reduced systolic function. Additionally, hydrogen inhalation ameliorated the levothyroxine-induced metabolic increase and cardiac hypertrophy in rats. Serum brain natriuretic peptide expression was also attenuated by hydrogen treatment. However, hydrogen had no significant effect on levothyroxine -induced serum troponin I and serum thyroid hormone changes.Hydrogen treatment also reduced the levothyroxine-induced increase in cardiac malondialdehyde, 8-hydroxy-2-deoxyguanosine and serum hydrogen peroxide levels and upregulated superoxide dismutase and glutathione peroxidase activity. Additionally, western blotting results showed that hydrogen inhalation inhibited the expression of cardiac nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2), angiotensin II type 1 receptor, sarcoplasmic reticulum Ca2+-ATPase (SERCA2), phospho-phospholamban and α-myosin heavy chain proteins. In conclusion, the present study revealed a protective effect of hydrogen on levothyroxine -induced cardiac hypertrophy by regulating angiotensin II type 1 receptors and NOX2-mediated oxidative stress in rats.

Evaluation of the TRIP13 level in breast cancer and insights into potential molecular pathways

TRIP13 is a member of the large superfamily of the AAA + ATPase proteins and is associated with a variety of activities. Emerging evidence has shown that TRIP13 may serve as an oncogene. However, the function of TRIP13 in breast cancer (BC) has not yet been elucidated. Here, a variety of bioinformatic tools and laboratory experiments were combined to analyse the expression patterns, prognostic value and functional network of TRIP13 in BC. Multiple databases and immunohistochemistry (IHC) indicated a higher TRIP13 expression in BC tissue compared with normal tissue. TRIP13 was highly expressed in lung metastatic lesions compared with primary tumours in a 4T1 cell implantation BALB/c mouse model of BC. Kaplan–Meier plots also revealed that high TRIP13 expression correlated with poor survival in patients with BC. Furthermore, gene set enrichment analysis revealed that TRIP13 was primarily enriched in the signalling pathway of PI3K‐AKT‐mTOR. Suppressing TRIP13 could inhibit the expression of related genes, as well as the proliferation and migration of BC cell. Finally, 10 hub genes with a high score of connectivity were filtered from the protein–protein interaction (PPI) network, including MAD2L1, CDC20, CDC5L, CDK1, CCNA2, BUB1B, RAD51, SPO11, KIF11 and AURKB. Thus, TRIP13 may be a promising prognostic biomarker and an effective therapeutic target for BC.

PPAR-α knockout leads to elevated blood pressure response to angiotensin II infusion associated with an increase in renal α-1 Na+/K+ ATPase protein expression and activity

Peroxisome proliferator activated receptor alpha (PPAR-α) deletion has been shown to increase blood pressure (BP). We hypothesized that the BP increase in PPAR-α KO mice was mediated by increased expression and activity of basolateral Na+/K+ ATPase (NKA) pump. To address this hypothesis, we treated wild-type (WT) and PPAR-α knockout (KO) mice with a slow-pressor dose of angiotensin II (400 ng/kg·min) for 12 days by osmotic minipump. Radiotelemetry showed no significant differences in baseline mean arterial pressure (MAP) between WT and PPAR-α KO mice; however, by day 12 of infusion, MAP was significantly higher in PPAR-α KO mice (156 ± 16) compared to WT mice (138 ± 11 mmHg). NKA activity and protein expression (α1 subunit) were significantly higher in PPAR-α KO mice compared to WT mice. There was no significant difference in NKA mRNA levels. Angiotensin II further increased the expression and activity of the NKA in both genotypes along with the water channel, aquaporin 1 (Aqp1). In contrast, angiotensin II decreased the expression (64–97% reduction in band density) of sodium‑hydrogen exchanger-3 (NHE3), NHE regulatory factor-1 (NHERF1, Slc9a3r1), sodium‑potassium-2-chloride cotransporter (NKCC2), and epithelial sodium channel (ENaC) β- and γ- subunits in the renal cortex of both WT and PPAR-α KO mice, with no difference between genotypes. The sodium-chloride cotransporter (NCC) was also decreased by angiotensin II, but significantly more in PPAR-α KO (59% WT versus 77% KO reduction from their respective vehicle-treated mice). Our results suggest that PPAR-α attenuates angiotensin II-mediated increased blood pressure potentially via reducing expression and activity of the NKA.

Effect of hyperhomocysteinemia on rat cardiac sarcoplasmic reticulum.

Increased concentration of plasma homocysteine (Hcy) is an independent risk factor of cardiovascular disease, yet the mechanism by which hyperhomocysteinemia (HHcy) causes cardiac dysfunction is largely unknown. The aim of present study was to investigate the contribution of sarcoplasmic reticulum to impaired cardiac contractile function in HHCy. HHcy-induced by subcutaneous injection of Hcy (0.45μmol/g of body weight) twice a day for a period of 2weeks resulted in significant decrease in developed left ventricular pressure and maximum rate of ventricular relaxation. Our results show that abundances of SR Ca2+-handling proteins, Ca2+-ATPase (SERCA2), calsequestrin and histidine-rich calcium-binding protein are significantly reduced while the content of phospholamban is unchanged. Moreover, we found that increased PLN:SERCA2 ratio results in the inhibition of SERCA2 activity at low free Ca2+ concentrations. We further discovered that HHcy is not associated with increased oxidative stress in SR. Taken together, these findings suggest that disturbances in SR Ca2+ handling, caused by altered protein contents but not oxidative damage, may contribute to impaired cardiac contractility in HHcy.

Elucidating the acid-base mechanisms underlying otolith overgrowth in fish exposed to ocean acidification

Over a decade ago, ocean acidification (OA) exposure was reported to induce otolith overgrowth in teleost fish. This phenomenon was subsequently confirmed in multiple species; however, the underlying physiological causes remain unknown. Here, we report that splitnose rockfish (Sebastes diploproa) exposed to ~1600 μatm pCO2(pH ~7.5) were able to fully regulated the pH of both blood and endolymph (the fluid that surrounds the otolith within the inner ear). However, while blood was regulated around pH 7.80, the endolymph was regulated around pH ~8.30. These different pH setpoints result in increased pCO2diffusion into the endolymph, which in turn leads to proportional increases in endolymph [HCO3−] and [CO32−]. Endolymph pH regulation despite the increased pCO2suggests enhanced H+removal. However, a lack of differences in inner ear bulk and cell-specific Na+/K+-ATPase and vacuolar type H+-ATPase protein abundance localization pointed out to activation of preexisting ATPases, non-bicarbonate pH buffering, or both, as the mechanism for endolymph pH-regulation. These results provide the first direct evidence showcasing the acid-base chemistry of the endolymph of OA-exposed fish favors otolith overgrowth, and suggests that this phenomenon will be more pronounced in species that count with more robust blood and endolymph pH regulatory mechanisms.

Xanthine Oxidoreductase Inhibitors Suppress the Onset of Exercise-Induced AKI in High HPRT Activity Urat1-Uox Double Knockout Mice.

Hereditary renal hypouricemia type 1 (RHUC1) is caused by URAT1/SLC22A12 dysfunction, resulting in urolithiasis and exercise-induced AKI (EIAKI). However, because there is no useful experimental RHUC1 animal model, the precise pathophysiologic mechanisms underlying EIAKI have yet to be elucidated. We established a high HPRT activity Urat1-Uox double knockout (DKO) mouse as a novel RHUC1 animal model for investigating the cause of EIAKI and the potential therapeutic effect of xanthine oxidoreductase inhibitors (XOIs). The novel Urat1-Uox DKO mice were used in a forced swimming test as loading exercise to explore the onset mechanism of EIAKI and evaluate related purine metabolism and renal injury parameters. Urat1-Uox DKO mice had uricosuric effects and elevated levels of plasma creatinine and BUN as renal injury markers, and decreased creatinine clearance observed in a forced swimming test. In addition, Urat1-Uox DKO mice had increased NLRP3 inflammasome activity and downregulated levels of Na+-K+-ATPase protein in the kidney, as Western blot analysis showed. Finally, we demonstrated that topiroxostat and allopurinol, XOIs, improved renal injury and functional parameters of EIAKI. Urat1-Uox DKO mice are a useful experimental animal model for human RHUC1. The pathogenic mechanism of EIAKI was found to be due to increased levels of IL-1β via NLRP3 inflammasome signaling and Na+-K+-ATPase dysfunction associated with excessive urinary urate excretion. In addition, XOIs appear to be a promising therapeutic agent for the treatment of EIAKI.