ATPase Specific Activity Research Articles

Sublethal concentrations of conventional nematicides alter the physiological activities of Meloidogyne incognita and suppress parasitism

Reducing nematicide dose rates could be a useful strategy for mitigating their negative effects on health and the environment. In this study, enzymatic activities and the parasitic ability of Meloidogyne incognita after exposure to sub-lethal concentrations (0.25, 1, 2, and 5 ppm) of ethoprophos, fenamiphos, and oxamyl were investigated. Although the tested concentrations did not show nematicidal properties in vitro, they reduced root galls by at least 30% at 0.25 ppm and up to 67% at 5 ppm in pots, besides disrupting nematode fertility. For all three nematicides at 2 ppm, a chemotaxis assay showed that ≤ 11% of the nematode population was successfully oriented to the host roots, compared to 44% in the control. Ethoprophos and fenamiphos at 5 ppm showed poor inhibitory effects on acetylcholinesterase (AChE) activity (5.6% and 12.5%, respectively). In contrast, the same nematicides were shown to be strong ATPase inhibitors, causing 82.4% and 82.8% inhibition, respectively. At the same concentration, oxamyl moderately inhibited AChE and ATPase-specific activities, the inhibition being 22.5% and 35.2%, respectively. This study suggests that the use of very low nematicide concentrations could be a promising strategy for nematode management. Furthermore, it has also highlighted the role of ATPases as a possible target site for suppressing nematode activity in the development of future nematicides.

Abstract 5166: In silico identification of specific inhibitor(s) of tumor associated Vacuolar ATPase ‘a2' isoform: A critical target for anti-cancer drug development

Abstract The discovery of effective anti-cancer therapies has immense consequences on global health. Vacuolar (H+)-ATPases (V-ATPases) are multi-subunit nano-motors that acidify the lumen of intracellular organelles in normal physiology. In cancer cells, the plasma membrane V-ATPases acidify the extracellular space. This is critical in tumor invasion, metastasis, and chemo-resistance. The `a2` isoform of V-ATPase (a2V) is the major pH sensing unit of this complex and is distinctly overexpressed on the surface of cancer cells making it an excellent drug target. The chemical inhibitors such as bafilomycin/concanamycin target V-ATPase subunits also critical in normal cell function. Currently, there are no selective a2V inhibitors commercially available as anti-cancer agents. Here, we employed in silico molecular modeling to analyze specific a2V binding sites in order to design targeted small molecules a2V inhibitors. We have identified several natural compounds as a2V ligands which inhibit specifically a2V in cancer. Employing human ovarian and breast epithelial cells (normal or tumor), in vitro studies measured each a2V ligand(s) effects on cancer cell proliferation. We selected five a2V ligands showing the most potent anti-proliferative activity in cancer cells and were least toxic to normal cells. We further measured each ligand(s) effects on specific ATPase activity in vitro by determining the decrease in inorganic phosphate levels. Next, the two most potent a2v ligands (KDB001, KDB002) which belonged to alkaloid and mono-terpenoid phenol groups, were selected to measure the changes in cytosolic and extracellular pH- SNARF assay and pH of cell supernatants. KDB001 induced cytosolic pH acidification in a dose-dependent manner indicating inhibition of V-ATPase activity. Based on the above experiments, the best ligand with most prominent effect on a2V activity (KDB-001) was further validated in vivo for anti-ovarian cancer efficacy in an athymic nude mouse model. KDB001 administration resulted in a significant delay in in vivo ovarian tumor growth. Studies are underway to design and synthesize more efficient analogs for KDB001 molecular modelling, toxicity assessment and chemical synthesis approach. In conclusion, the study has discovered new inhibitors of V-ATPase-a2V, positioning these promising compounds to enter the anti-cancer drug development pipeline. Citation Format: Arpita Kulshrestha, Gajendra K. Katara, Awanish Kumar, Kenneth D. Beaman. In silico identification of specific inhibitor(s) of tumor associated Vacuolar ATPase ‘a2' isoform: A critical target for anti-cancer drug development [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5166.

Crystal structure of a human plasma membrane phospholipid flippase

ATP11C, a member of the P4-ATPase flippase, translocates phosphatidylserine from the outer to the inner plasma membrane leaflet, and maintains the asymmetric distribution of phosphatidylserine in the living cell. We present the crystal structures of a human plasma membrane flippase, ATP11C-CDC50A complex, in a stabilized E2P conformation. The structure revealed a deep longitudinal crevice along transmembrane helices continuing from the cell surface to the phospholipid occlusion site in the middle of the membrane. We observed that the extension of the crevice on the exoplasmic side is open, and the complex is therefore in an outward-open E2P state, similar to a recently reported cryo-EM structure of yeast flippase Drs2p-Cdc50p complex. We noted extra densities, most likely bound phosphatidylserines, in the crevice and in its extension to the extracellular side. One was close to the phosphatidylserine occlusion site as previously reported for the human ATP8A1-CDC50A complex, and the other in a cavity at the surface of the exoplasmic leaflet of the bilayer. Substitutions in either of the binding sites or along the path between them impaired specific ATPase and transport activities. These results provide evidence that the observed crevice is the conduit along that phosphatidylserine traverses from the outer leaflet to its occlusion site in the membrane and suggest that the exoplasmic cavity is important for phospholipid recognition. They also yield insights into how phosphatidylserine is incorporated from the outer leaflet of the plasma membrane into the transmembrane.

Candida albicans Pma1p Contributes to Growth, pH Homeostasis, and Hyphal Formation.

Candida albicans occupies diverse ecological niches within the host and must tolerate a wide range of environmental pH. The plasma membrane H+-ATPase Pma1p is the major regulator of cytosolic pH in fungi. Pma1p extrudes protons from the cytosol to maintain neutral-to-alkaline pH and is a potential drug target due to its essentiality and fungal specificity. We characterized mutants in which one allele of PMA1 has been deleted and the other truncated by 18–38 amino acids. Increasing C-terminal truncation caused corresponding decreases in plasma membrane ATPase-specific activity and cytosolic pH. Pma1p is regulated by glucose: glucose rapidly activates the ATPase, causing a sharp increase in cytosolic pH. Increasing Pma1p truncation severely impaired this glucose response. Pma1p truncation also altered cation responses, disrupted vacuolar morphology and pH, and reduced filamentation competence. Early studies of cytosolic pH and filamentation have described a rapid, transient alkalinization of the cytosol preceding germ tube formation; Pma1p has been proposed as a regulator of this process. We find Pma1p plays a role in the establishment of cell polarity, and distribution of Pma1p is non-homogenous in emerging hyphae. These findings suggest a role of PMA1 in cytosolic alkalinization and in the specialized form of polarized growth that is filamentation.

Structural and functional characterization of a maltose/maltodextrin ABC transporter comprising a single solute binding domain (MalE) fused to the transmembrane subunit MalF

Canonical ATP-binding cassette import systems rely on extracellular substrate binding proteins (SBP) for function. In gram-negative bacteria, SBPs are usually freely diffusible in the periplasm and, where studied, exist in excess over their cognate transporters. However, in vitro studies with the maltose transporter of Escherichia coli (MalFGK2) have demonstrated that mechanistically one copy of its SBP (MalE) per transport complex is sufficient for activity. To address whether such a condition is physiologically relevant, we have characterized a homolog of the E. coli system from the gram-negative bacterium Bdellovibrio bacteriovorus which has a single copy of a maltose binding domain fused to the MalF subunit. Both transporters share substrate specificity for maltose and linear maltodextrins. Specific ATPase and transport activities of the B. bacteriovorus transporter were comparable to those of the E. coli system assayed at a 1:1 M ratio of MalE to the transport complex. While MalEEc was able to additionally increase ATPase activity of MalFGK2Bb, the isolated MalE domain of B. bacteriovorus failed to stimulate the E. coli system. Strikingly, interactions of the MalE domain with the transmembrane subunits during the transport cycle as studied by site-specific cross-linking were found to differ from those observed for E. coli MalE-FGK2.

Osmoregulation in the Plotosidae Catfish: Role of the Salt Secreting Dendritic Organ.

Unlike other marine teleosts, the Plotosidae catfishes reportedly have an extra-branchial salt secreting dendritic organ (DO). Salinity acclimation [brackishwater (BW) 3aaa, seawater (SWcontrol) 34aaa, and hypersaline water (HSW) 60aaa] for 14 days was used to investigate the osmoregulatory abilities of Plotosus lineatus through measurements of blood chemistry, muscle water content (MWC), Na+/K+-ATPase (NKA) specific activity and ion transporter expression in gills, DO, kidney and intestine. Ion transporter expression was determined using immunoblotting, immunohistochemistry (IHC) and quantitative polymerase chain reaction (qPCR). HSW elevated mortality, plasma osmolality and ions, and hematocrit, and decreased MWC indicating an osmoregulatory challenge. NKA specific activity and protein levels were significantly higher in DO compared to gill, kidney and intestine at all salinities. NKA specific activity increased in kidney and posterior intestine with HSW but only kidney showed correspondingly higher NKA α-subunit protein levels. Since DO mass was greater in HSW, the total amount of DO NKA activity expressed per gram fish was greater indicating higher overall capacity. Gill NKA and V-ATPase protein levels were greater with HSW acclimation but this was not reflected in NKA activity, mRNA or ionocyte abundance. BW acclimation resulted in lower NKA activity in gill, kidney and DO. Cl- levels were better regulated and the resulting strong ion ratio in BW suggests a metabolic acidosis. Elevated DO heat shock protein 70 levels in HSW fish indicate a cellular stress. Strong NKA and NKCC1 (Na+:K+:2Cl- cotransporter1) co-localization was observed in DO parenchymal cells, which was rare in gill ionocytes. NKCC1 immunoblot expression was only detected in DO, which was highest at HSW. Cystic fibrosis transmembrane regulator Cl- channel (CFTR) localize apically to DO NKA immunoreactive cells. Taken together, the demonstration of high NKA activity in DO coexpressed with NKCC1 and CFTR indicates the presence of the conserved secondary active Cl- secretion mechanism found in other ion transporting epithelia suggesting a convergent evolution with other vertebrate salt secreting organs. However, the significant osmoregulatory challenge of HSW indicates that the DO may be of limited use under more extreme salinity conditions in contrast to the gill based ionoregulatory strategy of marine teleosts.

Short- and long-term salinity challenge, osmoregulatory ability, and (Na+, K+)-ATPase kinetics and α-subunit mRNA expression in the gills of the thinstripe hermit crab Clibanarius symmetricus (Anomura, Diogenidae)

The evolutionary history of the Crustacea reveals ample adaptive radiation and the subsequent occupation of many osmotic niches resulting from physiological plasticity in their osmoregulatory mechanisms. We evaluate osmoregulatory ability in the intertidal, thinstripe hermit crab Clibanarius symmetricus after short-term exposure (6 h) or long-term acclimation (10 days) to a wide salinity range, also analyzing kinetic behavior and α-subunit mRNA expression of the gill (Na+, K+)-ATPase. The crab strongly hyper-regulates its hemolymph at 5 and 15‰S (Salinity, g L−1) but weakly hyper-regulates up to ≈27‰S. After 6 h exposure to 35‰S and 45‰S, C. symmetricus slightly hypo-regulates its hemolymph, becoming isosmotic after 10 days acclimation to these salinities. (Na+, K+)-ATPase specific activity decreases with increasing salinity for both exposure periods, reflecting physiological adjustment to isosmoticity. At low salinities, the gill enzyme exhibits a single, low affinity ATP binding site. However, at elevated salinities, a second, high affinity, ATP binding site appears, independently of exposure time. (Na+, K+)-ATPase α-subunit mRNA expression increases only after 10 days acclimation to 5‰S. Our findings suggest that hemolymph hyper-regulation is effected by alterations in enzyme activity during short-term exposure, but is sustained by increased mRNA expression during long-term acclimation. The decrease in gill (Na+, K+)-ATPase activity seen as a consequence of increasing salinity appears to underlie biochemical adjustments to hemolymph isosmoticity as hypo-regulatory ability diminishes.

Reconstitution and functional studies of hamster P-glycoprotein in giant liposomes.

This paper describes the preparation of giant unilamellar vesicles with reconstituted hamster P-glycoprotein (Pgp, ABCB1) for studying the transport activity of this efflux pump in individual liposomes using optical microscopy. Pgp, a member of ABC (ATP-binding cassette) transporter family, is known to contribute to the cellular multidrug resistance (MDR) against variety of drugs. The efficacy of many therapeutics is, thus, hampered by this efflux pump, leading to a high demand for simple and effective strategies to monitor the interactions of candidate drugs with this protein. Here, we applied small Pgp proteoliposomes to prepare giant Pgp-bearing liposomes via modified electroformation techniques. The presence of Pgp in the membrane of giant proteoliposomes was confirmed using immunohistochemistry. Assessment of Pgp ATPase activity suggested that this transporter retained its activity upon reconstitution into giant liposomes, with an ATPase specific activity of 439 ± 103 nmol/mg protein/min. For further confirmation, we assessed the transport activity of Pgp in these proteoliposomes by monitoring the translocation of rhodamine 123 (Rho123) across the membrane using confocal microscopy at various ATP concentrations (0–2 mM) and in the presence of Pgp inhibitors. Rate of change in Rho123 concentration inside the liposomal lumen was used to estimate the Rho123 transport rates (1/s) for various ATP concentrations, which were then applied to retrieve the Michaelis-Menten constant (Km) of ATP in Rho123 transport (0.42 ± 0.75 mM). Similarly, inhibitory effects of verapamil, colchicine, and cyclosporin A on Pgp were studied in this system and the IC50 values for these Pgp inhibitors were found 26.6 ± 6.1 μM, 94.6 ± 47.6 μM, and 0.21 ± 0.07 μM, respectively. We further analyzed the transport data using a kinetic model that enabled dissecting the passive diffusion of Rho123 from its Pgp-mediated transport across the membrane. Based on this model, the permeability coefficient of Rho123 across the liposomal membrane was approximately 1.25×10−7 cm/s. Comparing the membrane permeability in liposomes with and without Pgp revealed that the presence of this protein did not have a significant impact on membrane integrity and permeability. Furthermore, we used this model to obtain transport rate constants for the Pgp-mediated transport of Rho123 (m3/mol/s) at various ATP and inhibitor concentrations, which were then applied to estimate values of 0.53 ± 0.66 mM for Km of ATP and 25.2 ± 5.0 μM for verapamil IC50, 61.8 ± 34.8 μM for colchicine IC50, and 0.23 ± 0.09 μM for cyclosporin A IC50. The kinetic parameters obtained from the two analyses were comparable, suggesting a minimal contribution from the passive Rho123 diffusion across the membrane. This approach may, therefore, be applied for screening the transport activity of Pgp against potential drug candidates.

Transcriptional, translational and systemic alterations during the time course of osmoregulatory acclimation in two palaemonid shrimps from distinct osmotic niches

Palaemonid shrimps exhibit numerous adaptive strategies, both in their life cycles and in biochemical, physiological, morphological and behavioral characteristics that reflect the wide variety of habitats in which they occur, including species that are of particular interest when analyzing adaptive osmoregulatory strategies. The present investigation evaluates the short- (hours) and long-term (days) time courses of responses of two palaemonid shrimps from separate yet overlapping osmotic niches, Palaemon northropi (marine) and Macrobrachium acanthurus (diadromous, fresh water), to differential salinity challenges at distinct levels of structural organization: (i) transcriptional, analyzing quantitative expression of gill mRNAs that encode for subunits of the Na+/K+-ATPase and V(H+)-ATPase ion transporters; (ii) translational, examining the kinetic behavior of gill Na+/K+-ATPase specific activity; and (iii) systemic, accompanying consequent adjustment of hemolymph osmolality. Palaemon northropi is an excellent hyper-hypo-osmoregulator in dilute and concentrated seawater, respectively. Macrobrachium acanthurus is a strong hyper-regulator in fresh water and hypo-regulates hemolymph osmolality and particularly [Cl−] in brackish water. Hemolymph hyper-regulation in fresh water (Macrobrachium acanthurus) and dilute seawater (Palaemon northropi) is underlain by augmented expression of both the gill Na+/K+-ATPase and V(H+)-ATPase. In contrast, in neither species is hypo-regulation sustained by changes in Na+/K+-ATPase mRNA expression levels, but rather by regulating enzyme specific activity. The integrated time course of Na+/K+- and V(H+)-ATPase expression and Na+/K+-ATPase activity in the gills of these palaemonid shrimps during acclimation to different salinities reveals versatility in their levels of regulation, and in the roles of these ion transporting pumps in sustaining processes of hyper- and hypo-osmotic and chloride regulation.

Toxicological implications of Margaritaria discoidea aqueous seed extract on Na+, K+-ATPase specific activity, glucose and serum electrolytes level in wistar rats

The effect of aqueous seed extract of Margaritaria discoidea on Na+ K+ ATPase activity and toxicity studies of its seed extract were investigated in albino rats. The study involved oral administration of different doses of the aqueous extract to groups of male albino rats at 25, 50,100, and 200mg/kg body weight resulted in significant (p<0.05) decrease in sodium, potassium and glucose levels while chloride and calcium ions showed a significant (p<0.05) increase relative to control. The seed extract also showed significant (p<0.05) decrease in Na+ K+ ATPase specific activity in a dose dependent manner relative to control. The median lethal dose (LD50) of this extract in mice was established at 316.2mg/kg body weight using probit of mortality. The results showed that the acute toxicity potency of the aqueous extract of the seeds was practically non-toxic. The study showed that seeds of Margaritaria discoidea could be toxic if not consumed in moderate quantities.

Multiple biomarkers response in Nile tilapia, Oreochromis niloticus (L.) exposed to sublethal concentrations of hexavalent chromium

The effect of two sublethal concentrations (15, 30 mg/l) of Cr6+ on Nile tilapia, Oreochromis niloticus has been studied using some hematological and biochemical parameters aiming to evaluate their possible potential use as biomarkers of stress and/or defense. Hemoglobin content decreased throughout the experiment period, compared to the control. Moreover, the plasma glucose content and uric acid increased in both Cr6+ concentrations. Plasma urea decreased during the first 24 hr of exposure, then it increased, thereafter decreased and finally fish recovered. Also, the brain AChE of the lower concentration showed significant fluctuations starting with inhibition of the enzyme then activation and final tendency to recover, while the higher concentration showed a significant increase throughout the whole experimentalperiod with no sign of recovery. Plasma AChE has been inhibited and no sign of recovery was detected. Gill Na+-K+-ATPase specific activity remained unchanged in fish exposed to both Cr6+ concentrations during the whole experimental time. Liver metallothionein (MT) content increased in both fish treated with Cr6+ concentrations.The present results indicated that blood Hb, plasma glucose followed by brain AChE are more sensitive indicators of general stress. However, MT, to a lesser extent, may be considered a valuable biomarker of defense. Finally, plasma AChE may also be considered as a biomarker of defense.

U18666A Treatment Results in Cholesterol Accumulation, Reduced Na(+), K(+)-ATPase Activity, and Increased Oxidative Stress in Rat Cortical Astrocytes.

The objective of this study was to determine the effect of U18666A, an inhibitor of cholesterol synthesis and its intracellular transport, on oxidative stress parameters in cortical astrocytes cultured from Wistar rats (0-3 days old). The cultures were incubated with U18666A (0.25 µg/mL) for 48 h, conditions that are considered ideal to mimic Niemann-Pick type C disease. A variety of indicators of oxidative stress were measured. U18666A treatment increased cholesterol 2-fold in treated compared to control astrocytes. Oxidative stress was significantly elevated in treated cells as demonstrated by a 1.7-fold increase in thiobarbituric acid reactive substances, a 60% decrease is sulfhydral groups, and a 3.7-fold increase in carbonyl groups, indicative of increased lipid and protein oxidation following U18666A treatment. Consistent with these changes, both catalase and superoxide dismutase activities were significantly reduced nearly 50% in treated compared to control astrocytes. Collectively, these change resulted in a 50% reduction in Na(+), K(+)-ATPase specific activity following U18666A treatment, suggesting a significant alteration in its plasma membrane environment. Overall, these changes indicate that U18666A treatment results in increased cholesterol levels and an increased level of oxidative stress in cortical astrocytes, consistent with what is observed in Niemann-Pick type C disease.

Assays, Surrogates, and Alternative Technologies for a TB Lead Identification Program Targeting DNA Gyrase ATPase

Mycobacterium tuberculosis (Mtb) DNA gyrase ATPase was the target of a tuberculosis drug discovery program. The low specific activity of the Mtb ATPase prompted the use of Mycobacterium smegmatis (Msm) enzyme as a surrogate for lead generation, since it had 20-fold higher activity. Addition of GyrA or DNA did not significantly increase the activity of the Msm GyrB ATPase, and an assay was developed using GyrB alone. Inhibition of the Msm ATPase correlated well with inhibition of Mtb DNA gyrase supercoiling across three chemical scaffolds, justifying its use. As the IC50 of compounds approached the enzyme concentration, surrogate assays were used to estimate potencies (e.g., the shift in thermal melt of Mtb GyrB, which correlated well with IC50s >10 nM). Analysis using the Morrison equation enabled determination of Kiapps in the sub-nanomolar range. Surface plasmon resonance was used to confirm these IC50s and measure the Kds of binding, but a fragment of Mtb GyrB had to be used. Across three scaffolds, the dissociation half life, t1/2, of the inhibitor-target complex was ≤8 min. This toolkit of assays was developed to track the potency of enzyme inhibition and guide the chemistry for progression of compounds in a lead identification program.

Reconstitution of Multidrug Resistance Efflux Pumps in Giant Liposomes

Multidrug resistance (MDR), a phenomenon in which cells become resistant to a number of drugs, is a growing obstacle for treatment of cancer and bacterial infections. A group of membrane transporters, known as MDR efflux pumps that bind to a wide range of foreign molecules and pump them out of the cell, are suspected to be the main contributors to MDR. P-glycoprotein (Pgp), which is the most well studied MDR efflux pump, has a critical role in the distribution of drugs in the body. As a result, screening the interactions between this protein and candidate drugs has recently become a required step in the FDA approval process. Biophysical characterization of Pgp and its transport activity may provide key information for the design and development of effective therapeutics. Giant unilamellar vesicles (GUV) are excellent models for natural cell membranes and present appealing platforms for biophysical studies of Pgp under well-defined conditions. Here, we reconstitute Pgp from Chinese hamster ovary B30 cells in giant unilamellar vesicles and investigate its ATPase activity as well as its transport activity.In this study, we applied small proteoliposomes with reconstituted Pgp for electroformation of Pgp-containing giant vesicles via our recently reported technique of hydrogel-assisted electroformation. These electroformed giant vesicles were characterized using fluorescence microscopy, immunohistochemistry, and ATPase assays. Fluorescence immunostaining confirmed the presence of Pgp in the membrane of GUVs. ATPase assay demonstrated that Pgp in GUVs retained its ATPase activity upon electroformtion. The specific ATPase activity of Pgp in GUVs was about 640 nmol/mg⋅min, comparable to that of this protein in small proteoliposomes, ∼660 nmol/mg⋅min. Moreover, we are investigating the transport activity of Pgp in these giant liposomes using fluorescence microscopy. The present study may provide valuable insight to the function of this MDR efflux pump.

The Contribution of Candida albicans Vacuolar ATPase Subunit V 1 B, Encoded by VMA2 , to Stress Response, Autophagy, and Virulence Is Independent of Environmental pH

Candida albicans vacuoles are central to many critical biological processes, including filamentation and in vivo virulence. The V-ATPase proton pump is a multisubunit complex responsible for organellar acidification and is essential for vacuolar biogenesis and function. To study the function of the V₁B subunit of C. albicans V-ATPase, we constructed a tetracycline-regulatable VMA2 mutant, tetR-VMA2. Inhibition of VMA2 expression resulted in the inability to grow at alkaline pH and altered resistance to calcium, cold temperature, antifungal drugs, and growth on nonfermentable carbon sources. Furthermore, V-ATPase was unable to fully assemble at the vacuolar membrane and was impaired in proton transport and ATPase-specific activity. VMA2 repression led to vacuolar alkalinization in addition to abnormal vacuolar morphology and biogenesis. Key virulence-related traits, including filamentation and secretion of degradative enzymes, were markedly inhibited. These results are consistent with previous studies of C. albicans V-ATPase; however, differential contributions of the V-ATPase Vo and V₁ subunits to filamentation and secretion are observed. We also make the novel observation that inhibition of C. albicans V-ATPase results in increased susceptibility to osmotic stress. Notably, V-ATPase inhibition under conditions of nitrogen starvation results in defects in autophagy. Lastly, we show the first evidence that V-ATPase contributes to virulence in an acidic in vivo system by demonstrating that the tetR-VMA2 mutant is avirulent in a Caenorhabditis elegans infection model. This study illustrates the fundamental requirement of V-ATPase for numerous key virulence-related traits in C. albicans and demonstrates that the contribution of V-ATPase to virulence is independent of host pH.

Expressiom of calcium ATPase in different tissues of protein deficient rats supplemented with Moringa olifera leaves (809.6)

Ca2+‐ATPases are key proteins for the maintenance of calcium homeostasis and essential to the correct functioning of cell machinery. Reports indicates that exposure of cell to oxidative injury will affect Ca2+‐ATPase. In this study, we investigated the effect of Protein energy malnutrition (PEM) on Ca2+‐ATPases and the ameliorating effect of Moringa oliefera leaves. Eighteen Wistar rats randomized into groups A and B were used for the study. Group A (12 rats) were placed on protein deficient diet while group B (6 rats) were placed on protein sufficient diet. They were maintained on respective diets for 6 weeks after which all rats in group B and 6 rats in group A were sacrificed. The remaining rats in group A (relabeled as C) were maintained on protein deficient diet supplemented with Moringa oliefera leaves for another 6 weeks and then sacrificed. The skeletal muscle, brain and liver were removed and homogenized. Ca2+‐ATPase specific activities in the selected tissues and the enzyme’s kinetic constants were then determined and compared. Our result indicates that Ca2+‐ATPases activity (for all the organs) was highest in group C and lowest in the protein deficient rats not supplemented (A). The activity was also highest in the skeletal muscle and lowest in the liver (for all the groups). The Km and Vmax (for all the organs) was lowest in group C and highest in group A. Our result suggest that PEM reduces the activity of Ca2+‐ATPases and that Moringa oliefera is more efficacious than fish in regulating the enzyme activity.

Euryhalinity of the estuarine copepod Pseudodiaptomus richardi and its high potential to be employed as live food in aquaculture

Abstract The use of copepods in aquaculture has been raising great interest in the last years, especially as live food for larviculture of marine/estuarine fish. The copepod Pseudodiaptomus richardi is a euryhaline species, extremely promising for culture in a vast array of salinities. The survival of P. richardi was thus here evaluated 24 h after its abrupt transfer to higher salinities (15 and 30), from control salinity 5. Besides survival, body fluid osmolality and body hydration were also evaluated upon these same hyper-osmotic shocks, but after 5 and 30 min, 1 and 24 h. The whole body activity of the Na + /K + -ATPase was evaluated after 48 h of acclimation to these same salinities. Adult copepods displayed 100% survival upon both hyper-osmotic shocks after 24 h; copepodites and nauplii had some mortality in salinity 30 (16–22%). Body hydration decreased in both salinities (78 to 66–70%). In salinity 15 body hydration was maintained after 5 min, and recovered after 24 h. In the highest salinity (30) the hyper-osmotic shock induced immediate (5 min) water loss, coherent with their loss of swimming activity, and there was no trend of recovery, even after 24 h. The reduction in body hydration does not necessarily reflect volume reduction, but increases in dry mass, from salt uptake. Copepods transiently lose water when submitted to salinity 30, quickly showing a decrease in body hydration – given the strong outward osmotic gradient – but probably recover volume after intra-and extra-cellular solute (inorganic and organic) concentrations increase, and water uptake. This result is consistent with their recovery of normal swimming behavior after a few hours in salinity 30. Body fluid osmolality at all salinities was always ~ 200–300 mOsm/kg H 2 O above water osmolality. Na + /K + -ATPase specific activity of adult copepods acclimated to the salinities of 5, 15, or 30 was stable, of ~ 2 nmol ADP/μg protein·h. P. richardi is thus a hyper-conformer which keeps a constant rate of salt uptake to remain hyper-osmotic at all salinities. In conclusion, it can be safely used as live food for fish larvae grown in salinities 5–30. In salinities approaching full-strength seawater, it shows a transient osmoregulatory disturbance when abruptly exposed, but it is fully able to acclimate, survive and reproduce even in these higher salinities.

Mutant LV476-7AA of A-subunit of Enterococcus hirae V1-ATPase: High affinity of A3B3 complex to DF axis and low ATPase activity

Vacuolar ATPase (V-ATPase) of Enterococcus hirae is composed of a soluble functional domain V1 (A3B3DF) and an integral membrane domain Vo (ac), where V1 and Vo domains are connected by a central stalk, composed of D-, F-, and d-subunits; and two peripheral stalks (E- and G-subunits). We identified 120 interacting residues of A3B3 heterohexamer with D-subunit in DF heterodimer in the crystal structures of A3B3 and A3B3DF. In our previous study, we reported 10 mutants of E. hirae V1-ATPase, which showed lower binding affinities of DF with A3B3 complex leading to higher initial specific ATPase activities compared to the wild-type. In this study, we identified a mutation of A-subunit (LV476-7AA) at its C-terminal domain resulting in the A3B3 complex with higher binding affinities for wild-type or mutant DF heterodimers and lower initial ATPase activities compared to the wild-type A3B3 complex, consistent with our previous proposal of reciprocal relationship between the ATPase activity and the protein-protein binding affinity of DF axis to the A3B3 catalytic domain of E. hirae V-ATPase. These observations suggest that the binding of DF axis at the contact region of A3B3 rotary ring is relevant to its rotation activity.

Candida albicans VMA3 Is Necessary for V-ATPase Assembly and Function and Contributes to Secretion and Filamentation

The vacuolar membrane ATPase (V-ATPase) is a protein complex that utilizes ATP hydrolysis to drive protons from the cytosol into the vacuolar lumen, acidifying the vacuole and modulating several key cellular response systems in Saccharomyces cerevisiae. To study the contribution of V-ATPase to the biology and virulence attributes of the opportunistic fungal pathogen Candida albicans, we created a conditional mutant in which VMA3 was placed under the control of a tetracycline-regulated promoter (tetR-VMA3 strain). Repression of VMA3 in the tetR-VMA3 strain prevents V-ATPase assembly at the vacuolar membrane and reduces concanamycin A-sensitive ATPase-specific activity and proton transport by more than 90%. Loss of C. albicans V-ATPase activity alkalinizes the vacuolar lumen and has pleiotropic effects, including pH-dependent growth, calcium sensitivity, and cold sensitivity. The tetR-VMA3 strain also displays abnormal vacuolar morphology, indicative of defective vacuolar membrane fission. The tetR-VMA3 strain has impaired aspartyl protease and lipase secretion, as well as attenuated virulence in an in vitro macrophage killing model. Repression of VMA3 suppresses filamentation, and V-ATPase-dependent filamentation defects are not rescued by overexpression of RIM8, MDS3, EFG1, CST20, or UME6, which encode positive regulators of filamentation. Specific chemical inhibition of Vma3p function also results in defective filamentation. These findings suggest either that V-ATPase functions downstream of these transcriptional regulators or that V-ATPase function during filamentation involves independent mechanisms and alternative signaling pathways. Taken together, these data indicate that V-ATPase activity is a fundamental requirement for several key virulence-associated traits in C. albicans.

Kinetic Analysis of Gill (Na+,K+)-ATPase Activity in Selected Ontogenetic Stages of the Amazon River Shrimp, Macrobrachium amazonicum (Decapoda, Palaemonidae): Interactions at ATP- and Cation-Binding Sites

We investigated modulation by ATP, Mg²⁺, Na⁺, K⁺ and NH₄⁺ and inhibition by ouabain of (Na⁺,K⁺)-ATPase activity in microsomal homogenates of whole zoeae I and decapodid III (formerly zoea IX) and whole-body and gill homogenates of juvenile and adult Amazon River shrimps, Macrobrachium amazonicum. (Na⁺,K⁺)-ATPase-specific activity was increased twofold in decapodid III compared to zoea I, juveniles and adults, suggesting an important role in this ontogenetic stage. The apparent affinity for ATP (K(M) = 0.09 ± 0.01 mmol L⁻¹) of the decapodid III (Na⁺,K⁺)-ATPase, about twofold greater than the other stages, further highlights this relevance. Modulation of (Na⁺,K⁺-ATPase activity by K⁺ also revealed a threefold greater affinity for K⁺ (K₀.₅ = 0.91 ± 0.04 mmol L⁻¹) in decapodid III than in other stages; NH₄⁺ had no modulatory effect. The affinity for Na⁺ (K₀.₅ = 13.2 ± 0.6 mmol L⁻¹) of zoea I (Na⁺,K⁺)-ATPase was fourfold less than other stages. Modulation by Na⁺, Mg²⁺ and NH₄⁺ obeyed cooperative kinetics, while K⁺ modulation exhibited Michaelis-Menten behavior. Rates of maximal Mg²⁺ stimulation of ouabain-insensitive ATPase activity differed in each ontogenetic stage, suggesting that Mg²⁺-stimulated ATPases other than (Na⁺,K⁺)-ATPase are present. Ouabain inhibition suggests that, among the various ATPase activities present in the different stages, Na⁺-ATPase may be involved in the ontogeny of osmoregulation in larval M. amazonicum. The NH₄⁺-stimulated, ouabain-insensitive ATPase activity seen in zoea I and decapodid III may reflect a stage-specific means of ammonia excretion since functional gills are absent in the early larval stages.