F1FO ATP Synthase Is Expressed at the Surface of Embryonic Rat Heart‐Derived H9c2 Cells and Is Affected by Cardiac‐Like Differentiation

Taking advantage from the peculiar features of the embryonic rat heart‐derived myoblast cell line H9c2, the present study is the first to provide evidence for the expression of F₁F_O ATP synthase and of ATPase Inhibitory Factor 1 (IF₁) on the surface of cells of cardiac origin, together documenting that they were affected through cardiac‐like differentiation. Subunits of both the catalytic F₁ sector of the complex (ATP synthase‐β) and of the peripheral stalk, responsible for the correct F₁‐F_O assembly/coupling, (OSCP, b, F6) were detected by immunofluorescence, together with IF₁. The expression of ATP synthase‐β, ATP synthase‐b and F6 were similar for parental and differentiated H9c2, while the levels of OSCP increased noticeably in differentiated cells, where the results of in situ Proximity Ligation Assay were consistent with OSCP interaction within ecto‐F₁F_O complexes. An opposite trend was shown by IF₁ whose ectopic expression appeared greater in the parental H9c2. Here, evidence for the IF₁ interaction with ecto‐F₁F_O complexes was provided. Functional analyses corroborate both sets of data. i) An F₁F_O ATP synthase contribution to the exATP production by differentiated cells suggests an augmented expression of holo‐F₁F_O ATP synthase on plasma membrane, in line with the increase of OSCP expression and interaction considered as a requirement for favoring the F₁‐F_O coupling. ii) The absence of exATP generation by the enzyme, and the finding that exATP hydrolysis was largely oligomycin‐insensitive, are in line in parental cells with the deficit of OSCP and suggest the occurrence of sub‐assemblies together evoking more regulation by IF₁. J. Cell. Biochem. 9999: 1–13, 2015. © 2015 Wiley Periodicals, Inc. J. Cell. Biochem. 117: 470–482, 2016. © 2015 Wiley Periodicals, Inc.     

Evaluation of prokaryotic and eukaryotic cells as food source for Balamuthia mandrillaris

Balamuthia mandrillaris is a recently identified free-living protozoan pathogen that can cause fatal granulomatous encephalitis in humans. Recent studies have shown that B. mandrillaris consumes eukaryotic cells such as mammalian cell cultures as food source. Here, we studied B. mandrillaris interactions with various eukaryotic cells including, monkey kidney fibroblast-like cells (COS-7), human brain microvascular endothelial cells (HBMEC) and Acanthamoeba (an opportunistic protozoan pathogen) as well as prokaryotes, Escherichia coli. B. mandrillaris exhibited optimal growth on HBMEC compared with Cos-7 cells. In contrast, B. mandrillaris did not grow on bacteria but remained in the trophozoite stage. When incubated with Acanthamoeba trophozoites, B. mandrillaris produced partial Acanthamoeba damage and the remaining Acanthamoeba trophozoites underwent encystment. However, B. mandrillaris were unable to consume Acanthamoeba cysts. Next, we observed that B. mandrillaris-mediated Acanthamoeba encystment is a contact-dependent process that requires viable B. mandrillaris. In support, conditioned medium of B. mandrillaris did not stimulate Acanthamoeba encystment nor did lysates of B. mandrillaris. Overall, these studies suggest that B. mandrillaris target Acanthamoeba in the trophozoite stage; however, Acanthamoeba possess the ability to defend themselves by forming cysts, which are resistant to B. mandrillaris. Further studies will examine the mechanisms associated with food selectivity in B. mandrillaris.     

Acidosis Is a key regulator of osteoblast ecto‐nucleotidase pyrophosphatase/phosphodiesterase 1 (NPP1) expression and activity

Previous work has shown that acidosis prevents bone nodule formation by osteoblasts in vitro by inhibiting mineralisation of the collagenous matrix. The ratio of phosphate (P_i) to pyrophosphate (PP_i) in the bone microenvironment is a fundamental regulator of bone mineralisation. Both P_i and PP_i, a potent inhibitor of mineralisation, are generated from extracellular nucleotides by the actions of ecto‐nucleotidases. This study investigated the expression and activity of ecto‐nucleotidases by osteoblasts under normal and acid conditions. We found that osteoblasts express mRNA for a number of ecto‐nucleotidases including NTPdase 1–6 (ecto‐nucleoside triphosphate diphosphohydrolase) and NPP1‐3 (ecto‐nucleotide pyrophosphatase/phosphodiesterase). The rank order of mRNA expression in differentiating rat osteoblasts (day 7) was Enpp1 > NTPdase 4 > NTPdase 6 > NTPdase 5 > alkaline phosphatase > ecto‐5‐nucleotidase > Enpp3 > NTPdase 1 > NTPdase 3 > Enpp2 > NTPdase 2. Acidosis (pH 6.9) upregulated NPP1 mRNA (2.8‐fold) and protein expression at all stages of osteoblast differentiation compared to physiological pH (pH 7.4); expression of other ecto‐nucleotidases was unaffected. Furthermore, total NPP activity was increased up to 53% in osteoblasts cultured in acid conditions (P < 0.001). Release of ATP, one of the key substrates for NPP1, from osteoblasts, was unaffected by acidosis. Further studies showed that mineralised bone formation by osteoblasts cultured from NPP1 knockout mice was increased compared with wildtypes (2.5‐fold, P < 0.001) and was partially resistant to the inhibitory effect of acidosis. These results indicate that increased NPP1 expression and activity might contribute to the decreased mineralisation observed when osteoblasts are exposed to acid conditions. J. Cell. Physiol. 230: 3049–3056, 2015. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Molecular architecture of the N‐type ATPase rotor ring from Burkholderia pseudomallei

The genome of the highly infectious bacterium Burkholderia pseudomallei harbors an atp operon that encodes an N‐type rotary ATPase, in addition to an operon for a regular F‐type rotary ATPase. The molecular architecture of N‐type ATPases is unknown and their biochemical properties and cellular functions are largely unexplored. We studied the B. pseudomallei N₁N_o‐type ATPase and investigated the structure and ion specificity of its membrane‐embedded c‐ring rotor by single‐particle electron cryo‐microscopy. Of several amphiphilic compounds tested for solubilizing the complex, the choice of the low‐density, low‐CMC detergent LDAO was optimal in terms of map quality and resolution. The cryoEM map of the c‐ring at 6.1 Å resolution reveals a heptadecameric oligomer with a molecular mass of ~141 kDa. Biochemical measurements indicate that the c₁₇ ring is H⁺ specific, demonstrating that the ATPase is proton‐coupled. The c₁₇ ring stoichiometry results in a very high ion‐to‐ATP ratio of 5.7. We propose that this N‐ATPase is a highly efficient proton pump that helps these melioidosis‐causing bacteria to survive in the hostile, acidic environment of phagosomes.     

An in vitro screening method to evaluate chemicals as potential chemotherapeutants to control Aeromonas hydrophila infection in channel catfish

Aims: To develop an in vitro screening method to be used for identifying potential effective chemotherapeutants to control Aeromonas hydrophila infections. Methods and Results: Using catfish gill cells G1B and four chemicals (hydrogen peroxide, sodium chloride, potassium permanganate and d ‐mannose), the feasibility of using an in vitro screening method to identify potential effective chemotherapeutants was evaluated in this study. In vitro screening results revealed that, at concentration of 100 mg l^?1, H₂O₂ was the only chemical tested that was able to completely abolish the attachment and invasion of Aer. hydrophila to catfish gill cells. In vivo virulence studies using live channel catfish through bath immersion confirmed that H₂O₂ was the only chemical tested that was able to significantly (P < 0·001) reduce the mortality (from 90 or 100% to 0 or 20%) caused by Aer. hydrophila infections. Conclusions: The in vitro screening method using catfish gill cells G1B could be used to initially identify potential effective chemotherapeutants to control Aer. hydrophila. Significance and Impact of the Study: An in vitro screening method using catfish gill cells to identify potential effective chemotherapeutants described here will cut cost in research compared with the method of using live fish to screen lead compounds for fish disease control.     

Neuronal cell surface ATP synthase mediates synthesis of extracellular ATP and regulation of intracellular pH

The ATP synthase is known to play important roles in ATP generation and proton translocation within mitochondria. Here, we now provide evidence showing the presence of functional ecto‐ATP synthase on the neuronal surface. Immunoblotting revealed that the α, β subunits of ATP synthase F₁ portion are present in isolated fractions of plasma membrane and biotin‐labelled surface protein from primary cultured neurons; the surface distribution of α, β subunits was also confirmed by immunofluorescence staining. Moreover, α and β subunits were also found in fractions of plasma membrane and lipid rafts isolated from rat brain, and flow cytometry analysis showed α subunits on the surface of acutely isolated brain cells. Activity assays showed that the extracellular ATP generation of cultured neurons could be compromised by α, β subunit antibodies and ATP synthase inhibitors. pH_i (intracellular pH) analysis demonstrated that at low extracellular pH, α or β subunit antibodies decreased pH_i of primary cultured neurons. Therefore, ATP synthase on the surface of neurons may be involved in the machineries of extracellular ATP generation and pH_i homoeostasis.     

Prothoracicotropic hormone induces tyrosine phosphorylation in prothoracic glands of the silkworm,Bombyx mori

In the present study, we investigated the tyrosine phosphorylation of Bombyx mori prothoracic glands using phosphotyrosine‐specific antibodies and Western blot analysis. Results showed that prothoracicotropic hormone (PTTH) stimulates a rapid increase in tyrosine phosphorylation of at least 2 proteins in prothoracic glands, one of which was identified as extracellular signal‐regulated kinase (ERK). The phosphorylation of another 120‐kDa protein showed dose‐ and time‐dependent stimulation by PTTH in vitro. In vitro activation of tyrosine phosphorylation was also verified by in vivo experiments: injection of PTTH into day‐6 last‐instar larvae greatly increased tyrosine phosphorylation. Treatment of prothoracic glands with the protein tyrosine phosphatase inhibitor, sodium orthovanadate, also resulted in tyrosine phosphorylation of several proteins and increased ecdysteroidogenesis. The PTTH‐stimulated phosphorylation of the 120‐kDa protein was markedly attenuated by genistein, a broad‐spectrum tyrosine kinase inhibitor, but not by HNMPA‐(AM)₃, a specific inhibitor of insulin receptor tyrosine kinase. PP2, a more‐selective inhibitor of the Src‐family tyrosine kinases, partially inhibited PTTH‐stimulated tyrosine phosphorylation, but not ecdysteroidogenesis. This result implies the possibility that in addition to ERK, the phosphorylation of the 120‐kDa protein, which is not Src‐family tyrosine kinase, is likely also involved in PTTH‐stimulated ecdysteroidogenesis in B. mori. © 2010 Wiley Periodicals, Inc.     

Mechanistic basis for differential inhibition of the F1Fo‐ATPase by aurovertin

The mitochondrial F₁F_o‐ATPase performs the terminal step of oxidative phosphorylation. Small molecules that modulate this enzyme have been invaluable in helping decipher F₁F_o‐ATPase structure, function, and mechanism. Aurovertin is an antibiotic that binds to the β subunits in the F₁ domain and inhibits F₁F_o‐ATPase‐catalyzed ATP synthesis in preference to ATP hydrolysis. Despite extensive study and the existence of crystallographic data, the molecular basis of the differential inhibition and kinetic mechanism of inhibition of ATP synthesis by aurovertin has not been resolved. To address these questions, we conducted a series of experiments in both bovine heart mitochondria and E. coli membrane F₁F_o‐ATPase. Aurovertin is a mixed, noncompetitive inhibitor of both ATP hydrolysis and synthesis with lower K_i values for synthesis. At low substrate concentrations, inhibition is cooperative suggesting a stoichiometry of two aurovertin per F₁F_o‐ATPase. Furthermore, aurovertin does not completely inhibit the ATP hydrolytic activity at saturating concentrations. Single‐molecule experiments provide evidence that the residual rate of ATP hydrolysis seen in the presence of saturating concentrations of aurovertin results from a decrease in the binding change mechanism by hindering catalytic site interactions. The results from these studies should further the understanding of how the F₁F_o‐ATPase catalyzes ATP synthesis and hydrolysis. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 830–840, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

PfeT,a P1B4‐type ATPase,effluxes ferrous iron and protects Bacillus subtilis against iron intoxication

Iron is an essential element for nearly all cells and limited iron availability often restricts growth. However, excess iron can also be deleterious, particularly when cells expressing high affinity iron uptake systems transition to iron rich environments. Bacillus subtilis expresses numerous iron importers, but iron efflux has not been reported. Here, we describe the B. subtilis PfeT protein (formerly YkvW/ZosA) as a P_1B4‐type ATPase in the PerR regulon that serves as an Fe(II) efflux pump and protects cells against iron intoxication. Iron and manganese homeostasis in B. subtilis are closely intertwined: a pfeT mutant is iron sensitive, and this sensitivity can be suppressed by low levels of Mn(II). Conversely, a pfeT mutant is more resistant to Mn(II) overload. In vitro, the PfeT ATPase is activated by both Fe(II) and Co(II), although only Fe(II) efflux is physiologically relevant in wild‐type cells, and null mutants accumulate elevated levels of intracellular iron. Genetic studies indicate that PfeT together with the ferric uptake repressor (Fur) cooperate to prevent iron intoxication, with iron sequestration by the MrgA mini‐ferritin playing a secondary role. Protection against iron toxicity may also be a key role for related P_1B4‐type ATPases previously implicated in bacterial pathogenesis.     

Characterization of ectonucleoside triphosphate diphosphohydrolase (E‐NTPDase; EC 3.6.1.5) activity in mouse peritoneal cavity cells

This study aimed to characterize the activity of ectonucleoside triphosphate diphosphohydrolase (E‐NTPDase; EC 3.6.1.5) in peritoneal cavity cells from BALB/c mice. E‐NTPDase was activated in the presence of both calcium (1.5mM) and magnesium (1.5mM) ions. However, the activity was higher in the presence of Ca²⁺. A pH of 8.5 and temperature of 37°C were the optimum conditions for catalysis. The apparent Km values were 0.51mM and 0.66mM for the hydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate (ADP), respectively. The Vmax values were 136.4 and 120.8 nmol Pi/min/mg of protein for ATPase and ADPase activity, respectively. Nucleotide hydrolysis was inhibited in the presence of sodium azide (20mM, ATP: P < .05; ADP: P < .001), sodium fluoride (20mM; ATP and ADP: P < .001), and suramin (0.3mM; ATP: P < .01; ADP: P < .05), which is a known profile for NTPDase inhibition. Although all of the diphosphate and triphosphate nucleotides that were tested were hydrolyzed, enzyme activity was increased when adenine nucleotides were used as substrates. Finally, we stress that knowledge of the E‐NTPDase catalytic biochemical properties in mouse peritoneal cavity cells is indispensable for properly determining its activity, as well as to fully understand the immune response profile in both healthy and sick cells.     

Purification and biochemical characterization of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione reductase from rat lung and inhibition effects of some antibiotics

G6PD, 6PGD and GR have been purified separately in the single step from rat lung using 2′, 5′-ADP Sepharose 4B affinity chromatography. The purified enzymes showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of the enzymes were estimated to be 134?kDa for G6PD, 107?kDa for 6PGD and 121?kDa for GR by Sephadex G-150 gel filtration chromatography, and the subunit molecular weights was respectively found to be 66, 52 and 63?kDa by SDS-PAGE. Optimum pH, stable pH, optimum ionic strength, optimum temperature, K_M and V_max values for substrates were determined. Product inhibition studies were also performed. The enzymes were inhibited by levofloxacin, furosemide, ceftazidime, cefuroxime and gentamicin as in vitro with IC₅₀ values in the range of 0.07–30.13?mM. In vivo studies demonstrated that lung GR was inhibited by furosemide and lung 6PGD was inhibited by levofloxacin.     

The plasma membrane ATPase of Kloeckera apiculata: purification,characterization and effect of ethanol on activity

Partially (6-fold) purified plasma membrane ATPase from an ethanol-sensitive yeast, Kloeckera apiculata, had an optimum pH of 6.0, an optimum temperature of 35°C, a K _m of 3.6 mm ATP and a V _max of 11 mol P_i/min.mg protein. SDS-PAGE of the semi-purified plasma membrane showed a major band of 106 kDa. No in vivo activation of the ATPase by glucose was observed. Although 4% (v/v) ethanol decreased the growth rate by 50% it did not affect the ATPase. Concentrations of ethanol 2% (v/v) did, however, inhibit the enzyme in vitro. The characteristics of the enzyme did not change during growth in the presence of ethanol.     

Supramolecular organization of the yeast F1Fo-ATP synthase

Background information. The yeast mitochondrial F₁F_o‐ATP synthase is a large complex of 600 kDa that uses the proton electrochemical gradient generated by the respiratory chain to catalyse ATP synthesis from ADP and P_i. For a large range of organisms, it has been shown that mitochondrial ATP synthase adopts oligomeric structures. Moreover, several studies have suggested that a link exists between ATP synthase and mitochondrial morphology. Results and discussion. In order to understand the link between ATP synthase oligomerization and mitochondrial morphology, more information is needed on the supramolecular organization of this enzyme within the inner mitochondrial membrane. We have conducted an electron microscopy study on wild‐type yeast mitochondria at different levels of organization from spheroplast to isolated ATP synthase complex. Using electron tomography, freeze‐fracture, negative staining and image processing, we show that cristae form a network of lamellae, on which ATP synthase dimers assemble in linear and regular arrays of oligomers. Conclusions. Our results shed new light on the supramolecular organization of the F₁F_o‐ATP synthase and its potential role in mitochondrial morphology.     

Production of L-leucine aminopeptidase by selected Streptomyces isolates

Aims: To screen various Streptomyces cultures producing l ‐leucine aminopeptidase (LAP). Methods and Results: Twenty‐one Streptomyces strains were screened for LAP production. The best three producers were found to be Streptomyces mobaraensis NRRL B‐3729, Streptomyces gedanensis IFO 13427, and Streptomyces platensis NRRL 2364. pH optima of the three enzymes were in the range of 8·0–8·5 and the temperature optima varied between 50 and 65°C. LAP of S. mobaraensis was stable at 60°C and pH 8·5 for 60 min. Metal ion salts, CoCl₂.6H₂O and ZnSO₄.7H₂O in 0·7 mmol l^?1 concentration enhanced the relative enzyme activity in all three enzymes. Molecular mass of LAP of S. mobaraensis was found to be approx. 37 kDa. Conclusions: Streptomyces mobaraensis NRRL B‐3729, S. gedanensis IFO 13427, and S. platensis NRRL 2364 were found to be good producers of extracellular LAP. The approx. 37 kDa enzyme of S. mobaraensis is considerably thermostable. Significance and Impact of the Study: A good number of Streptomyces were screened and the ability of the aminopeptidases to release a particular N‐terminal amino acid along with its good thermal stability makes them interesting for controlling the degree of hydrolysis and flavour development for a wide range of substrate.     

Interaction between thapsigargin and ATP4− in the regulation of the intracellular calcium in rat submandibular glands

Rat submandibular glands were digested with crude collagenase, and the intracellular calcium concentration of the cellular suspension was measured using fura-2. In the absence of extracellular magnesium and calcium ([Ca²⁺]_o), ATP had no effect; the response to ATP peaked at 1–2.5 mM [Ca²⁺]_o and was inhibited at 5 mM. One millimolar (mM) extracellular ATP did not increase the leak of LDH or fura-2; 10 m?M Coomassie brilliant blue G specifically inhibited the effect of ATP on [Ca²⁺]_in. Depleting intracellular calcium pools with thapsigargin did not affect the response to ATP. Using a Ca²⁺-free/Ca²⁺ reintroduction protocol, it was shown that ATP and thapsigargin increase the uptake of extracellular calcium. The effect of the two agonists was synergistic. Removal of extracellular sodium inhibited the effect of carbachol on [Ca²⁺]_in and the calcium uptake but potentiated the response to ATP. These results suggest that, after binding to purinergic receptors, extracellular ATP^4- increases [Ca²⁺]_in. ATP^4- does not mobilize thapsigargin-sensitive intracellular calcium pools (among which is the IP₃-sensitive calcium pool) but stimulates the uptake of extracellular calcium by a mechanism inhibited by extracellular sodium, probably by opening a nonselective cation channel. © 1994 Wiley-Liss, Inc.     

Induction of Paenibacillus lentimorbus biofilm by sodium alginate and CaCl2 alleviates drought stress in chickpea

Drought is a major environmental factor that limits chickpea production. An improvement in the adaption of crop to the fluctuating environmental conditions is therefore a major aim in chickpea breeding. However, the complexity of the trait has allowed only marginal progress. Our findings provide a solution to the current situation in the form of improved plant‐growth‐promoting effects caused by the biofilm formation of Paenibacillus lentimorbus B‐30488 (B‐30488) under water‐limiting conditions. In vitro assays demonstrating the biofilm‐forming ability of B‐30488 and the factors enhancing it were studied. Greenhouse experiments were conducted for validating the in vitro results and assessing the effect of seed coating supplements in alleviating drought stress effects in chickpea seedlings. The chickpea seed bacterisation with B‐30488 along with sodium alginate (1%) and CaCl₂ (1 mM) caused an increase in germination percent and increased colony‐forming units (CFU) of B‐30488 in rhizosphere, resulting in amelioration of drought stress by positively influencing the dehydration‐induced physiological responses. The whole study reflects a prospective role of sodium alginate and CaCl₂ in influencing the biofilm formation of B‐30488, and depicts the assistance of seed coating supplements in stress adaptation and protection of plants by alleviation of drought stress effects in chickpea without causing any major changes in the functional diversity of soil micro‐organisms.