Decrease In ATPase Activity Research Articles

Exploration of the mechanisms of biofilm formation by Enterococcus faecalis in glucose starvation environments.

Enterococcus faecalis is the most common single species present in teeth after failed root canal therapy. This is mainly due to its ability to maintain viability for a long time in filled root canals where nutrients are normally sparse. The aim of this study was to explore the mechanism of E. faecalis survival and biofilm formation in glucose-starved environments. Enterococcus faecalis ATCC 33186 was inoculated in energy starvation media for biofilm formation. Confocal laser scanning microscopy and fluorescent DNA-binding agents were employed to assess biofilm-forming ability. The physiochemical properties of the biofilm cell wall were investigated by measuring the hydrophobicity, extracellular polysaccharide and ATPase activity. The expression of stress and virulence genes was quantified by real-time quantitative polymerase chain reaction. The ability for biofilm formation decreased with a decreasing concentration of glucose. The cell surface hydrophobicity increased dramatically with decreasing glucose concentration. Water-soluble exopolysaccharide (WSE) synthesis decreased in glucose starvation media, whereas water-insoluble exopolysaccharide (WIE) synthesis increased. Amarked decrease in ATPase activity was observed only in a no glucose medium. In addition, transcription of ace, fsrB and gelE genes increased under glucose starvation stress while atpE, salB and esp genes were down-regulated. Enterococcus faecalis survival and biofilm formation under glucose starvation stress may be attributed to an increase in cell-surface hydrophobicity and additionally to the up-regulation of some genes involved in stress response and biofilm formation. These characteristics may explain why E. faecalis can maintain viability for a long time in an energy-starved environment and why it is frequently isolated from persistently infected root canals.

Effects of benzo(a)pyrene exposure on the ATPase activity and content of Ca²⁺ in the hippocampus of neonatal SD rats

To investigate the effect of benzo(α)pyrene on the ATPase activity and content of Ca²⁺ in the hippocampus of neonatal SD rats. Sixty male and 60 female 4-days-old neonatal SD rats were randomly divided into 5 groups (n=24): a blank control group, a vehicle control group (peanut oil), 3 benzo(α)pyrene groups (0.02, 0.2 and 2 mg/kg, respectively). SD rats were given benzo(α)pyrene (dissolved in peanut oil) by gavage daily from postnatal day 4 (PND4) to PND20. The nerve reflex, the condition of neuro-muscle development and motion function were examined in the period of treatment. The colorimetric technique was used to detect the activity of Ca²⁺-ATPase and Ca²⁺-Mg²⁺-ATPase in hippocampus after the treatment. The concentration of Ca²⁺ of synapse in the hippocampus of rats was detected by fluorescent labeling. The results from the behavior tests showed that duration of surface reflex latency in rats with medium dose of benzo(α)pyrene was longer compared with that in the control group in PND12. The duration of surface reflex latency in rats with high dose of benzo(α) pyrene is longer in PND 14 and PND 16 compared with that in the control group (P<0.05). Compared with the rats in the control group, the activities of Ca²⁺-Mg²⁺-ATPase and Ca²⁺-ATPase in hippocampus in rats with high dose benzo(α) pyrene were significantly decreased, and the degree in the decrease of Ca²⁺-ATPase activity was dose-dependent (P<0.05). The contents of Ca²⁺ in the hippocampus in rats with medium or high dose of benzo(α) pyrene were significantly increased compared with that in the control group (P<0.05), which showed a dose-dependent manner (P<0.05). Benzo(α)pyrene exposure led to the decrease in ATPase activity as well as Ca²⁺ overload of the synapse in the hippocampal tissue, which in turn results in the nerve damage of newborn SD rats.

Chelerythrine inhibits the sarco/endoplasmic reticulum Ca2+-ATPase and results in cell Ca2+ imbalance

The isoquinoline alkaloid chelerythrine is described as an inhibitor of SERCA. The ATPase inhibition presented two non-competitive components, Ki1=1, 2μM and Ki2=26μM. Conversely, chelerythrine presented a dual effect on the p-nitrophenylphosphatase (pNPPase) of SERCA. Ca2+-dependent pNPPase was activated up to ∼5μM chelerythrine with inhibition thereafter. Ca2+-independent pNPPase was solely inhibited. The phosphorylation of SERCA with ATP reached half-inhibition with 10μM chelerythrine and did not parallel the decrease of ATPase activity. In contrast, chelerythrine up to 50μM increased the phosphorylation by Pi. Cross-linking of SERCA with glutaraldehyde was counteracted by high concentrations of chelerythrine. The controlled tryptic digestion of SERCA shows that the low-affinity binding of chelerythrine evoked an E2-like pattern. Our data indicate a non-competitive inhibition of ATP hydrolysis that favors buildup of the E2-conformers of the enzyme. Chelerythrine as low as 0.5–1.5μM resulted in an increase of intracellular Ca2+ on cultured PBMC cells. The inhibition of SERCA and the loss of cell Ca2+ homeostasis could in part be responsible for some described cytotoxic effects of the alkaloid. Thus, the choice of chelerythrine as a PKC-inhibitor should consider its potential cytotoxicity due to the alkaloid’s effects on SERCA.

Photo-Control of Mitotic Kinesin Eg5 using Thiol Group Reactive Fulgimide Derivative

It is believed that the loop L5 of kinesin is important region for motor function. Interestingly mitotic kinesin Eg5 has a several times longer L5 in comparison with other kinesins. It has been demonstrated that the L5 of Eg5 performed as a stabilizer for the Eg5-specific inhibitors (STLC, monastrol) complexes. Aim of our study is to control the function of Eg5 photo-reversibly using photochromic molecules incorporated into L5. Previously, we have prepared Eg5 mutants (E116C,E118C,T125C,W127C,D130C) which have a single cysteine residue in L5 in order to incorporate photochromic molecules. We also synthesized thiol reactive photochromic molecules 4-phenylazomaleinanil(PAM) and Iodoacetyl-spiropyran (IASP). PAM and IASP were incorporated into the mutants stoichiometrically. Some of the Eg5 mutants modified with PAM and IASP showed reversible alteration of ATPase activity upon ultraviolet (UV) and visible (VIS) light irradiations. In this study, we synthesized a novel thiol reactive photochromic molecules monoiodoacetyl-flugide(IAFG). Fulgimide performs photoreversible isomerzation between non-polar opened-ring form and polar closed-ring form upon visible light and ultraviolet light. IAFG was incorporated into Eg5 mutant W127C stoichiometrically. Althought the modified Eg 5 mutant W127C-IAFG showed slightly decreased ATPase activity, the ATPase activity showed photoreversible alteration upon UV and visible light irradiations. Alteration in the ATPase activity of W127C-IAFG in the presence of STLC upon UV and VIS light irradiations was also examined.

DDX41 Is a Tumor Suppressor Gene Associated with Inherited and Acquired Mutations

Familial MDS is rare and usually occurs at a young age. Distinguishing familial from sporadic disease at the typical late age of disease onset is difficult. While investigating the genetic background in familial AML cases, we identified 5 families characterized by germ line (GL) mutations in the DEAD-box RNA helicase DDX41 gene. In one family, a father, son, daughter and paternal grandmother were affected. Sequencing revealed a GL DDX41 mutation (c.419insGATG, p.D140fs; prevalence in controls <.0001%) co-segregating with the disease. This mutation was recurrent in three other AML families, while in another family containing identical twins with congruent MDS, a c.T1187C mutation (not detected in controls) was identified. Further screening (N=1052) identified 15 patients with GL DDX41 alterations. We previously reported recurrent somatic (c.G1574A, p.R525H) DDX41 mutations in MDS. Further analysis revealed that somatic and GL mutations coincided, with 50% of patients carrying GL mutations having biallelic lesions of DDX41, suggesting a strong predisposition to second allele lesions. Isolated recurrent somatic p.D140fs was found in another 12 patients. In total, 25 cases had DDX41 mutations: 15 GL and 17 somatic of which 7 were biallelic. In addition to DDX41 mutations, we observed 15 somatic mutations (5% of all cases) in other members of the DEAD/H-box RNA helicase family (DDX11,17,23,53,50,60 andDHX29,32,33,34,37,58). GL DDX41 mutations could be considered founder lesions, while somatic DDX41 mutations were ancestral in some cases and secondary in others. GL and somatic DDX41 mutations were more commonly associated with normal karyotype (81% vs. 47%; P=.02), while biallelic cases lacked other typical AML mutations. Among 112 patients treated with lenalidomide, 8/8 of the DDX41MT cases responded (P=.006) per IWG criteria. The most frequent mutations coincident with DDX41 lesions involved TP53 and RUNX1.DDX41 is expressed in myeloid cells, consistent with a function in hematopoiesis. GL mutations were predominantly out of frame insertions early in the gene, likely functionally equivalent to deletions. Deletion of DDX41 is found in 26% of all del5q cases. Such deletions of the DDX41 locus lead to haploinsufficient expression, which is also observed in 5% of diploid cases. DDX41 appears to be an essential gene, as the inactivating GL c.419insGATG mutation was not found among del5q35.3 patients and no DDX41-/- pups have been observed to date in the offspring of DDX41 knockout mice. For somatic DDX41R525H, we showed decreased ATPase activity in vitro, suggestive of a possible dominant negative effect.Lentiviral shRNA DDX41 knockdown (40-50%) in K562 cells enhanced proliferation compared to mock transduced cells. Forced DDX41 over-expression in U937 cells, which express low levels of DDX41, inhibited growth. Over-expression of either WT or DDX41R525H in HEK293 cells led to the p.R525H mutant increasing soft agar colony formation compared to DDX41WT or DDX41KD cells. Previous data suggested that DDX41 is a component of the catalytic spliceosome and becomes stably associated at a late step (Complex C) immediately prior to catalysis of the first splicing reaction. We have also verified this result through proteomic analysis of complexes associated with epitope tagged DDX41. The co-precipitated proteins included many mature spliceosomal components, but few that are associated with early forming complexes, suggesting that DDX41 is distinct from other MDS-associated splicing factors that function early in the pathway. In DDX41KD HEK293 cells (80% reduction), using qRT-PCR analysis of the spliced to unspliced RNA ratio, we demonstrated that splicing of a subset of introns is indeed significantly reduced. In patient samples with DDX41MT, using deep RNA NGS, we found that multiple alternative exons were altered in their inclusion frequency.In summary, we identified GL mutations in DDX41 that are associated with the development of hereditary MDS/AML. The strong family history and late onset suggest high penetrance and long latency. GL DDX41 defects strongly predispose to somatic DDX41 mutations. Our results indicate that DDX41 lesions lead to altered splicing of many genes likely responsible for down-stream leukemogenic effects of DDX41MT. DDX41 mutations, along with hemizygous DDX41 deletions, constitute a new type of leukemogenic defect and highlight a new class of tumor suppressor genes. DisclosuresSekeres:Celgene Corp.: Membership on an entity’s Board of Directors or advisory committees; Amgen: Membership on an entity’s Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity’s Board of Directors or advisory committees.

Rescuing Noise‐Induced Permanent Threshold Shift by D‐Methionine in Guinea Pigs

Objectives: This study examined the effectiveness of D-methionine (D-met) in rescuing a noise-induced permanent threshold shift (PTS) and cochlear biochemistry following noise exposure. Methods: One hour after being exposed to continuous white noise at 105 ± 2 dB SPL for 6 hours, guinea pigs were treated 5 times at 12-hour intervals with 200, 400, or 600 mg/kg D-met or sterile 0.9% saline by intraperitoneal injection. Auditory brainstem responses were examined before and 14 days after noise exposure. Six guinea pigs with normal hearing that were not exposed to noise served as control animals. The level of D-met in rescuing noise-induced hearing loss and the changes in cochlear biochemistry, including membranous Na+, K+-ATPase, and Ca2+-ATPase, lipid peroxidation, and nitric oxide, were evaluated. Results: Hearing acuity had no significant differences before and 14 days after noise exposure for the animals treated with 600 mg/kg D-met, compared with the control animals, whereas significant differences occurred among the animals in the other treatment groups. The level of rescue from noise-induced PTS following treatment with D-met was dose-dependent. No significant differences in the mean enzyme activities were observed between the controls and the D-met 600 mg/kg treatment group. The attenuation of the noise-induced decreases in the enzyme activities was dose-dependent. Likewise, D-met-dose-dependent decreases in oxidative stress were observed in the D-met treated groups. Conclusions: Treatment with D-met significantly rescued noise-induced PTS in a dose-dependent manner. Significant attenuation of increased oxidative stress and decreased ATPase activities were concurrent with the D-met-mediated improvements in noise-induced auditory dysfunction.

Quality Changes and Establishment of Predictive Models for Bighead Carp (Aristichthys nobilis) Fillets During Frozen Storage

The objectives of this study were to investigate quality changes of bighead carp fillets during frozen storage by measuring free fatty acids (FFA), salt extractable protein (SEP) content, Ca2+-ATPase activity, and total sulfhydryl (SH) content and to develop predictive models based on them. FFA increased with the extension of storage time. Meanwhile, SEP content, SH content, and Ca2+-ATPase activity all decreased during storage. A marked inhibition (p < 0.05) of FFA formation and SEP content, Ca2+-ATPase activity and SH content decrease was observed during storage of bighead carp fillets at −30 °C, compared to those stored at −10 and −20 °C. Additionally, the relative errors of models based on Ca2+-ATPase and SH were all below 10 %. Thus, the lower frozen temperature can inhibit quality damage effectively, and a reliable estimation of quality changes could be performed by models based on Ca2+-ATPase and SH.

Mitigation of lead-induced neurotoxicity by the naringin: erythrocytes as neurons substitute markers.

This study aimed to investigate the effect of lead (Pb) on neuronal nitric oxide synthase (nNOS) activity using erythrocytes as neurons surrogate markers. Moreover, the protective effect of naringin (NAR) against lead acetate (PbAc)-induced neurotoxicity was investigated. Human erythrocytes were incubated with L-arginine (L-Arg), Nω-nitro-L-Arginine methyl ester ( L-NAME), NAR, PbAc, PbAc+L-Arg, PbAc+NAR, or PbAc+L-Arg+NAR. The present results revealed that incubation of erythrocytes with PbAc inhibited NOS activity and decreased nitrite levels as an index for nitric oxide (NO) production to values similar that of L-NAME as known NOS inhibitor. Likewise, PbAc induced a significant decrease in activities of ATPases and acetylcholinesterase compared to control cells. Furthermore, PbAc exposure significantly increased protein carbonyl content (PCC) and malondialdehyde (MDA) levels while significantly decrease the levels of reduced glutathione (GSH). On the contrary, incubation of erythrocytes with PbAc in the presence of L-Arg+NAR synergistically ameliorated the investigated parameters compared to erythrocytes incubated with PbAc alone. These data suggest that NAR can restore NO bioavailability in a situation of Pb-induced cellular damage. This attributed to antioxidant activity and restoration NOS activity.

Biochemical characterization of sporadic/familial hemiplegic migraine mutations

Sporadic hemiplegic migraine type 2 (SHM2) and familial hemiplegic migraine type 2 (FHM2) are rare forms of hemiplegic migraine caused by mutations in the Na+,K+-ATPase α2 gene. Today, more than 70 different mutations have been linked to SHM2/FHM2, randomly dispersed over the gene. For many of these mutations, functional studies have not been performed. Here, we report the functional characterization of nine SHM2/FHM2 linked mutants that were produced in Spodoptera frugiperda (Sf)9 insect cells. We determined ouabain binding characteristics, apparent Na+ and K+ affinities, and maximum ATPase activity. Whereas membranes containing T345A, R834Q or R879W possessed ATPase activity significantly higher than control membranes, P796S, M829R, R834X, del 935–940 ins Ile, R937P and D999H membranes showed significant loss of ATPase activity compared to wild type enzyme. Further analysis revealed that T345A and R879W showed no changes for any of the parameters tested, whereas mutant R834Q possessed significantly decreased Na+ and increased K+ apparent affinities as well as decreased ATPase activity and ouabain binding. We hypothesize that the majority of the mutations studied here influence interdomain interactions by affecting formation of hydrogen bond networks or interference with the C-terminal ion pathway necessary for catalytic activity of Na+,K+-ATPase, resulting in decreased functionality of astrocytes at the synaptic cleft expressing these mutants.

Analysis of helicase domain mutations in the hepatitis E virus derived from patients with fulminant hepatic failure: Effects on enzymatic activities and virus replication

Fulminant hepatic failure (FHF) is the severe form of hepatitis E virus infection. Virus sequence analyses from severe cases have shown presence of unique and highly conserved mutations in the helicase domain of genotype 1, 3 and 4 viruses. We evaluated role of two amino acid replacements (L1110F) and (V1120I); found to be frequent in genotype 1 FHF-E viruses from India. Three mutant helicase proteins (two with single point mutations and one with dual mutations) were expressed in Escherichia coli and evaluated for their ATPase and RNA unwinding activities. Both L1110F and V1120I helicase mutants showed marginal decrease in ATPase activity, while L1110F/V1120I dual mutant showed normal ATPase activity. All three mutants proteins showed RNA unwinding activities comparable to wild type protein. Corresponding mutations were made in the helicase domain of HEV RLuc replicon and replication efficiencies were tested in the S10-3 (Huh 7) cells. The mutant replicon V1120I showed lower replication as compared to L1110F and L1110F/V1120I mutants. However, all three replicon mutants showed lower replication efficiencies as compared to the wild type replicon. Walker A and Walker B motif mutant HEV replicons were unable to replicate indicating essential role of the virus encoded helicase domain during HEV replication. FHF-E associated helicase mutations resulted in only marginal decrease in the virus replication suggesting alternate function/s of the helicase protein. Mutations in the helicase domain of FHF-E viruses may be responsible for changing virus or host-virus protein–protein interactions, causing alterations in the host responses, eventually leading to more severe disease manifestations.

Effects of chlorogenic acid, caffeine, and coffee on behavioral and biochemical parameters of diabetic rats

Diabetes mellitus (DM) is associated with brain alterations that may contribute to cognitive dysfunctions. Chlorogenic acid (CGA) and caffeine (CA), abundant in coffee (CF), are natural compounds that have showed important actions in the brain. The present study aimed to evaluate the effect of CGA, CA, and CF on acetylcholinesterase (AChE), Na(+), K(+)-ATPase, aminolevulinate dehydratase (δ-ALA-D) activities and TBARS levels from cerebral cortex, as well as memory and anxiety in streptozotocin-induced diabetic rats. Animals were divided into eight groups (n=5-10): control; control/CGA 5mg/kg; control/CA 15mg/kg; control/CF 0.5g/kg; diabetic; diabetic/CGA 5mg/kg; diabetic/CA 15mg/kg; and diabetic/CF 0.5g/kg. Our results demonstrated an increase in AChE activity and TBARS levels in cerebral cortex, while δ-ALA-D and Na(+), K(+)-ATPase activities were decreased in the diabetic rats when compared to control water group. Furthermore, a memory deficit and an increase in anxiety in diabetic rats were observed. The treatment with CGA and CA prevented the increase in AChE activity in diabetic rats when compared to the diabetic water group. CGA, CA, and CF intake partially prevented cerebral δ-ALA-D and Na(+), K(+)-ATPase activity decrease due to diabetes. Moreover, CGA prevented diabetes-induced TBARS production, improved memory, and decreased anxiety. In conclusion, among the compounds studied CGA proved to be a compound which acts better in the prevention of brain disorders promoted by DM.

Modulation of H+,K+-ATPase activity by the molecular chaperone ERp57 highly expressed in gastric parietal cells

ERp57 is a ubiquitous ER chaperone that has disulfide isomerase activity. Here, we found that both ERp57 and gastric H+,K+-ATPase are expressed in a sample derived from the apical canalicular membranes of parietal cells. Overexpression of ERp57 in HEK293 cells stably expressing H+,K+-ATPase significantly increased the ATPase activity without changing the expression level of H+,K+-ATPase. Interestingly, overexpression of a catalytically inactive mutant of ERp57 (C57S/C60S/C406S/C409S) in the cells also increased H+,K+-ATPase activity. In contrast, knockdown of endogenous ERp57 in H+,K+-ATPase-expressing cells significantly decreased ATPase activity without changing the expression level of H+,K+-ATPase. Overexpression and knockdown of ERp57 had no significant effect on the expression and function of Na+,K+-ATPase. These results suggest that ERp57 positively regulates H+,K+-ATPase activity apart from its chaperoning function.

A Novel SERCA Inhibitor Demonstrates Synergy with Classic SERCA Inhibitors and Targets Multidrug-Resistant AML

Drug resistance exists as a major obstacle in the treatment of cancer, and drug molecules that retain effectiveness against resistant cancers are a high clinical priority. Ethyl 2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (CXL017) was recently identified as a promising lead for the treatment of multidrug-resistant leukemia, which elicits its cytotoxic effect, in part, through inhibition of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA). Herein initial experiments with SERCA1a and CXL017 demonstrated no significant effect on calcium affinity, competed with ATP, and induced a dose-dependent decrease in ATPase activity. Among all CXLs tested, (-)-CXL017 exhibited the greatest SERCA inhibition with an IC50 = 13.5 ± 0.5 μM. Inhibitor combination studies were used to assess potential interactions between (-)-CXL017 and well-known SERCA inhibitors: thapsigargin, cyclopiazonic acid, and 2,5-di-tert-butylhydroquinone. Surprisingly, (-)-CXL017 exhibited marked synergy with each of the known SERCA inhibitors, whereas all combinations of the known inhibitors yielded additive effects, indicating that (-)-CXL017 may bind at a unique allosteric site. Treatment of parental (HL60) and multidrug-resistant (HL60/MX2) acute myeloid leukemia cells with the known SERCA inhibitors revealed that all of these inhibitors demonstrate selective cytotoxicity (7.7-400-fold) for the resistant cell line. Within the CXL series, a positive correlation exists between SERCA inhibition and cytotoxicity in HL60/MX2 but not HL60. (-)-CXL017 was also shown to enhance the cytotoxicity of thapsigargin in HL60/MX2 cells. Given the elevated SERCA levels and ER calcium content in HL60/MX2, SERCA likely plays a significant role in the collateral sensitivity of this multidrug-resistance cell line to CXL molecules as well as known SERCA inhibitors.

Dose-dependent effects of d-methionine for rescuing noise-induced permanent threshold shift in guinea-pigs

The purpose of this study was to examine the dose–response effectiveness of d-methionine (d-met) in rescuing a noise-induced permanent threshold shift (PTS) and cochlear biochemistry following noise exposure. One hour after being exposed to continuous broadband white noise at 105dB sound pressure level (SPL) for 6h, guinea-pigs were treated five times at 12-h intervals with 200, 400, or 600mg/kg d-met or sterile 0.9% saline (each group, N=6) by intraperitoneal injection. Six guinea-pigs with normal hearing that were not exposed to noise served as control animals. Although administration of d-met 200mg/kg did not significantly reduce the mean PTS, treatment with d-met 600mg/kg achieved a complete rescue response. The level of rescue from noise-induced PTS following treatment with 200, 400, or 600mg/kg d-met was dose dependent. The attenuation of the noise-induced decreases in the activities of the Na+, K+-ATPase and Ca2+-ATPase following treatment with 200, 400, or 600mg/kg d-met was also dose dependent. Likewise, d-met-dose-dependent decreases in mean lipid peroxidation and nitric oxide levels were observed in the d-met treated groups. Significant attenuation of increased oxidative stress and decreased ATPase activities was concurrent with the d-met-mediated improvements in noise-induced auditory dysfunction.

Dibenzofuran-induced mitochondrial dysfunction: Interaction with ANT carrier

Exposure to environmental pollutants such as dibenzofurans and furans is linked to the pathophysiology of several diseases. Dibenzofuran (DBF) is listed as a pollutant of concern due to its persistence in the environment, bioaccumulation and toxicity to humans, being associated with the development of lung diseases and cancers, due to its extremely toxic properties such as carcinogenic and teratogenic.Mitochondria play a key role in cellular homeostasis and keeping a proper energy supply for eukaryotic cells is essential in the fulfillment of the tissues energy-demand. Therefore, interference with mitochondrial function leads to cell death and organ failure. In this work, the effects of DBF on isolated rat liver mitochondria were analyzed.DBF exposure caused a markedly increase in the lag phase that follows depolarization induced by ADP, indicating an effect in the phosphorylative system. This was associated with a dose-dependent decrease in ATPase activity. Moreover, DBF also increased the threshold to the induction of the mitochondrial permeability transition (MPT) by calcium. Pretreatment of mitochondria with DBF also increased the concentration of carboxyatractyloside (CAT) necessary to abolish ADP phosphorylation and to induce the MPT, suggesting that DBF may interfere with mitochondria through an effect on the adenine nucleotide translocase (ANT). By co-immunoprecipitating ANT and Cyclophilin D (CypD) following MPT induction, we observed that in the presence of DBF, the ratio CypD/ANT was decreased. This demonstrates that DBF interferes with the ANT and so prevents CypD binding to the ANT, causing decreased phosphorylative capacity and inhibiting the MPT, which is also reflected by an increase in calcium retention capacity.Clarifying the role of pollutants in some mechanisms of toxicity, such as unbalance of bioenergetics status and mitochondrial function, may help to explain the progressive and chronic evolution of diseases derived from exposure to environmental pollutants.

Functional studies and proteomics in platelets and fibroblasts reveal a lysosomal defect with increased cathepsin-dependent apoptosis in ATP1A3 defective alternating hemiplegia of childhood

Alternating hemiplegia of childhood (AHC) is a rare syndrome with repeated hemiplegic episodes, paroxysmal events and global neurological impairment. Recently, heterozygous de novo ATP1A3 missense mutations have been identified in AHC patients, but the underlying pathogenesis mechanism remains unknown. Mutation analysis of ATP1A3 in 9 unrelated AHC cases revealed mostly D801N or E815K variants. As platelets represent a good cellular model to study defects in neuropathologies, morphological and functional experiments were performed in these subjects. Platelets from the AHC patients presented with structural and functional abnormalities of granules positive for the lysosomal marker CD63. Similar structural granule abnormalities were detected in patients' fibroblasts. Proteomic analysis of platelets and fibroblasts showed a total of 93 differentially expressed proteins in AHC mainly involved in metabolism. Interestingly, 7 of these proteins were detected in both cell types, including the lysosomal protein cathepsin. AHC fibroblasts revealed significantly increased levels of activated cathepsin B, which induces a stronger activation of apoptosis. Our study is the first to link ATP1A3 defects in AHC to a platelet and fibroblast lysosomal defect with evidence of increased apoptosis. Further studies are needed to define how this lysosomal defect is related to decreased ATPase activity. Biological Significance Only recently, the genetic cause of AHC was identified as heterozygous ATP1A3 mutations, but the underlying pathophysiological mechanism still remains unknown. By performing functional, morphological and proteomic studies in AHC patients we found a structural and functional granule defect in AHC platelets and fibroblasts that was specifically found in granules positive for the lysosomal marker CD63. In particular, proteomics identified several differentially expressed proteins in fibroblasts and platelets from AHC cases that are predicted to have an important role in cell function and maintenance, a pathway typically attributed to lysosomes. The lysosomal protein cathepsin was found to be differentially expressed in both platelets and fibroblasts of AHC patients, inducing a stronger activation of mainly the intrinsic apoptosis. Despite the precise mechanism for the increased lysosomal cathepsin B-dependent apoptosis detected in AHC in relation to impaired ATP1A3 deserves further studies, we could here show some evidence for a defective regulation of apoptosis in AHC, a disease that still has no biochemical or neuroradiological parameters for diagnosis.

Pentylenetetrazol-induced seizures are associated with Na+,K+-ATPase activity decrease and alpha subunit phosphorylation state in the mice cerebral cortex

The present study aimed to investigate whether Na(+),K(+)-ATPase activity and phosphorylation state of the catalytic α subunit are altered by pentylenetetrazol (PTZ)-induced seizures. PTZ (30, 45 or 60 g/kg, i.p.) was administered to adult male Swiss mice, and Na(+),K(+)-ATPase activity and phosphorylation state were measured in the cerebral cortex 15 min after PTZ administration. Na(+),K(+)-ATPase activity significantly decreased after PTZ-induced seizures (60 mg/kg). Immunoreactivity of phosphorylated Ser943 at α subunit was increased after PTZ-induced seizures. A significant positive correlation between Na(+),K(+)-ATPase activity and latency to myoclonic jerks and generalized seizures was found. Conversely, a strong negative correlation between Ser943 phosphorylation and latency to generalized seizures was detected. Given the role of Na(+),K(+)-ATPase as a major regulator of brain excitability, Ser943 at Na(+),K(+)-ATPase α subunit may represent a potentially valuable new target for drug development for seizure disorders.

Energy cost of force production is reduced after active stretch in skinned muscle fibres

Residual force enhancement has been observed consistently in skeletal muscles. Despite an abundance of experimental observations, there has been no information about the metabolic cost of the force observed after stretch. Our aim was to investigate the energy cost of force production after active stretch in skinned fibres isolated from rabbit psoas muscle, by quantifying the ATPase activity using an enzyme-coupled assay. Fibres were actively stretched from an average sarcomere length of 2.4μm to average sarcomere lengths of 2.8 and 3.2μm. Purely isometric reference contractions were performed at average sarcomere lengths of 2.8 and 3.2μm. Simultaneously with the force measurements, the ATP cost per unit of force produced was measured during the last 40s of isometric contraction. Results showed that ATPase activity per unit of force was reduced by 17.2±4.1% in the isometric contractions after active stretch, compared to the purely isometric contraction at the corresponding lengths for both stretch magnitudes. Fibres stretched to an average sarcomere length of 3.2μm showed a higher reduction in ATPase activity per unit of force compared to fibres stretched to an average sarcomere length of 2.8μm (20.7±4.4 versus 12.4±3.2% respectively). Passive force enhancement was observed in all fibres and was correlated with the decrease in ATPase activity. No difference in stiffness was observed between reference and active stretch contractions. These results suggest that skeletal muscles become more efficient after stretch, either by increasing the amount of force produced per cross bridge or by engaging a passive element.

The Tropomyosin Binding Region of Cardiac Troponin T Modulates Crossbridge Recruitment Dynamics in Rat Cardiac Muscle Fibers

The cardiac muscle comprises dynamically interacting components that use allosteric/cooperative mechanisms to yield unique heart-specific properties. An essential protein in this allosteric/cooperative mechanism is cardiac muscle troponin T (cTnT), the central region (CR) and the T2 region of which differ significantly from those of fast skeletal muscle troponin T (fsTnT). To understand the biological significance of such sequence heterogeneity, we replaced the T1 or T2 domain of rat cTnT (RcT1 or RcT2) with its counterpart from rat fsTnT (RfsT1or RfsT2) to generate RfsT1-RcT2 and RcT1-RfsT2 recombinant proteins. In addition to contractile function measurements, dynamic features of RfsT1-RcT2- and RcT1-RfsT2-reconstituted rat cardiac muscle fibers were captured by fitting the recruitment–distortion model to the force response of small-amplitude (0.5%) muscle length changes. RfsT1-RcT2 fibers showed a 40% decrease in tension and a 44% decrease in ATPase activity, but RcT1-RfsT2 fibers were unaffected. The magnitude of length-mediated increase in crossbridge (XB) recruitment (E0) decreased by ~33% and the speed of XB recruitment (b) increased by ~100% in RfsT1-RcT2 fibers. Our data suggest the following: (1) the CR of cTnT modulates XB recruitment dynamics; (2) the N-terminal end region of cTnT has a synergistic effect on the ability of the CR to modulate XB recruitment dynamics; (3) the T2 region is important for tuning the Ca2+ regulation of cardiac thin filaments. The combined effects of CR–tropomyosin interactions and the modulating effect of the N-terminal end of cTnT on CR–tropomyosin interactions may lead to the emergence of a unique property that tunes contractile dynamics to heart rates.

Benzodiazepines and Benzothiazepines as Modulators of the Sarcoplasmic Reticulum Calcium ATP-ase and Ryanodine Receptors in Striated Muscle

We have reported that CGP-37157, a benzothiazepine (BZT) derivative of clonazepam utilized as a blocker of the mitochondrial Na+/Ca2+ exchanger, also activates ryanodine receptors (RyRs) and inhibits the sarcoplasmic reticulum (SR) Ca2+-stimulated ATPase (SERCA). We extended the studies to other BZT (e.g., clonazepan, diltiazem) as well as to benzodiazepines (BZD; e.g. diazepan, lorazepan). We aimed to determine if these drug classes have as a common trait the ability to modulate RyRs and/or SERCA. The effects of BZD and BZT on RyRs activity were tested in SR microsomes with a Ca2+ leak assay as well as after reconstituting RyRs into lipid bilayers. The agents tested had variable potency to increase RyR-mediated Ca2+ leak from skeletal SR microsomes. As an example, while diazepam significantly increased RyR activity, most of the others (clonazepam, lorazepam) had minor effects at high doses. In contrast, diltiazem produced moderate inhibition. Planar bilayer studies confirmed the leak observations with both cardiac and skeletal RyR. In the presence of ruthenium red, most agents decreased the rate of loading which indicates inhibitory effects on SERCA activity. The effects of BZT and BZD on loading correlated with a decrease in ATPase activity of SERCA-enriched skeletal SR fractions. In summary, most BZT and BZD utilized in therapeutics or as pharmacological tools have an inhibitory action on SERCA. In contrast, they show a variety of effects on RyRs ranging from inhibition to activation. Hence, the pharmacological action of BZT and BZD on cellular Ca2+ homeostasis reported in the literature of cardiac and skeletal muscle as well as other non-muscle systems may require taking into consideration the contributions of all drug-sensitive intracellular Ca2+ transporters. (Supported by NIH-GM078665 and AHA-MWA 12180038).