[Show abstract][Hide abstract] ABSTRACT: Osteoporosis is a bone disease that commonly results in a 30% incidence of fracture in hens used to produce eggs for human consumption. One of the causes of osteoporosis is the lack of mechanical strain placed on weight-bearing bones. In conventionally-caged hens, there is inadequate space for chickens to exercise and induce mechanical strain on their bones. One approach is to encourage mechanical stress on bones by the addition of perches to conventional cages. Our study focuses on the molecular mechanism of bone remodeling in end-of-lay hens (71 weeks) with access to perches. We examined bone-specific transcripts that are actively involved during development and remodeling. Using real-time quantitative PCR, we examined seven transcripts (COL2A1 (collagen, type II, alpha 1), RANKL (receptor activator of nuclear factor kappa-B ligand), OPG (osteoprotegerin), PTHLH (PTH-like hormone), PTH1R (PTH/PTHLH type-1 receptor), PTH3R (PTH/PTHLH type-3 receptor), and SOX9 (Sry-related high mobility group box)) in phalange, tibia and femur. Our results indicate that the only significant effect was a difference among bones for COL2A1 (femur > phalange). Therefore, we conclude that access to a perch did not alter transcript expression. Furthermore, because hens have been used as a model for human bone metabolism and osteoporosis, the results indicate that bone remodeling due to mechanical loading in chickens may be a product of different pathways than those involved in the mammalian model.
International Journal of Molecular Sciences 02/2015; 16(2):2663-77. DOI:10.3390/ijms16022663 · 2.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Na/K pump hydrolyzes ATP to export three intracellular Na (Nai) as it imports two extracellular K (Ko) across animal plasma membranes. Within the protein, two ion-binding sites (sites I and II) can reciprocally bind Na or K, but a third site (site III) exclusively binds Na in a voltage-dependent fashion. In the absence of Nao and Ko, the pump passively imports protons, generating an inward current (IH). To elucidate the mechanisms of IH, we used voltage-clamp techniques to investigate the [H]o, [Na]o, and voltage dependence of IH in Na/K pumps from ventricular myocytes and in ouabain-resistant pumps expressed in Xenopus oocytes. Lowering pHo revealed that Ho both activates IH (in a voltage-dependent manner) and inhibits it (in a voltage-independent manner) by binding to different sites. Nao effects depend on pHo; at pHo where no Ho inhibition is observed, Nao inhibits IH at all concentrations, but when applied at pHo that inhibits pump-mediated current, low [Na]o activates IH and high [Na]o inhibits it. Our results demonstrate that IH is a property inherent to Na/K pumps, not linked to the oocyte expression environment, explains differences in the characteristics of IH previously reported in the literature, and supports a model in which 1), protons leak through site III; 2), binding of two Na or two protons to sites I and II inhibits proton transport; and 3), pumps with mixed Na/proton occupancy of sites I and II remain permeable to protons.
[Show abstract][Hide abstract] ABSTRACT: Schistosomiasis affects over 200 million people worldwide, with over 200,000 deaths annually. Currently, praziquantel is the
only drug available against schistosomiasis. We report here that Schistosoma mansoni farnesyl diphosphate synthase (SmFPPS) and geranylgeranyl diphosphate synthase (SmGGPPS) are potential drug targets for the treatment of schistosomiasis. We expressed active, recombinant SmFPPS and SmGGPPS for subsequent kinetic characterization and testing against a variety of bisphosphonate inhibitors. Recombinant SmFPPS was found to be a soluble 44.2-kDa protein, while SmGGPPS was a soluble 38.3-kDa protein. Characterization of the substrate utilization of the two enzymes indicates that they
have overlapping substrate specificities. Against SmFPPS, several bisphosphonates had 50% inhibitory concentrations (IC50s) in the low micromolar to nanomolar range; these inhibitors had significantly less activity against SmGGPPS. Several lipophilic bisphosphonates were active against ex vivo adult worms, with worm death occurring over 4 to 6 days. These results indicate that FPPS and GGPPS could be of interest
in the context of the emerging resistance to praziquantel in schistosomiasis therapy.
[Show abstract][Hide abstract] ABSTRACT: Ca(2+)-ATPases are integral membrane proteins that actively transport Ca(2+) against substantial concentration gradients in eukaryotic cells. This active transport is energized by coupling ion translocation with ATP hydrolysis. In order to better understand this coupling mechanism, we studied the nucleotide specificities of isolated ATP binding domains (ABDs) of Solanum lycopersicon Ca(2+)-ATPase (LCA), a type IIA non-calmodulin regulated P-type pump found in tomato plants that is very similar to mammalian sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), and Arabidopsis Ca(2+)-ATPase, isoform 2 (ACA2), a type IIB calmodulin regulated P-type ATPase found in the endoplasmic reticulum of Arabidopsis cells. We used nucleotide protection against FITC labeling as a measure of binding since both LCA and ACA contained the KGAP(S,V,F)E motif, which has been shown to be modified by fluorescein isothiocyanate (FITC) in P-type pumps from animal cells. We demonstrated that the heterologously expressed GST-tagged ABDs from both LCA and ACA2 were modified by FITC and that ATP protects against this modification. Moreover, GTP was able to reduce, but not eliminate, the level of FITC labeling in both ABD constructs, suggesting that these plant pumps may also bind GTP with low affinity, which is in contrast to mammalian SERCA and PMCA type pumps which do not bind GTP.
[Show abstract][Hide abstract] ABSTRACT: Na,K-ATPase, an integral membrane protein, has been studied for over a half century with respect to its transporter function in the plasma membrane where it expels 3 Na(+) ions from the cell in exchange for two K(+) ions. In this study, we demonstrate a functioning Na,K-ATPase within HEK 293 cell nuclei. This subcellular localization was confirmed by western blotting, ouabain-sensitive ATPase activity of the nuclear membrane fraction, immunocytochemistry, and delivery of fluorescently-tagged Na,K-ATPase α- and β- subunits. In addition, we observed an overlap between nuclear Na,K-ATPase and Na/Ca-exchanger (NCX) when nuclei were immunostained with commercially available Na,K-ATPase and NCX antibodies suggesting a concerted physiological coupling between these transporters. In keeping with this, we observed an ATP-dependent, strophanthidin-sensitive Na(+) flux into the nuclear envelope (NE) lumen loaded with the Na-sensitive dye, CoroNa-Green. Analogous experiments using Fluo-5N, a low affinity Ca(2+) indicator, demonstrated a similar ATP-dependent and strophanthidin-sensitive Ca(2+) flux into the NE lumen. Our results reveal an intracellular physiological role for the coordinated efforts of the Na,K-ATPase and NCX to actively remove Ca(2+) from the nucleoplasm into the NE lumen (i.e. the nucleoplasmic reticulum).
[Show abstract][Hide abstract] ABSTRACT: Blocking efficacy of whole soymilk, nonfat soymilk, SuperBlock, and nonfat milk was evaluated by performing standard protein immunoblotting procedures on both purified protein and crude nuclear extracts from HEK 293 cells. Nonfat soymilk was found to have superior blocking efficacy compared with other blocking agents in terms of high signal-to-noise ratio with the shortest blocking times. In addition, the presence of low concentrations of the detergent Tween 20 (0.05-0.1%, v/v) in the wash buffer as well as antibody incubations significantly lessened the background compared with including only the detergent during wash steps.
[Show abstract][Hide abstract] ABSTRACT: The Na/K pump is a P-type ATPase that exchanges three intracellular Na(+) ions for two extracellular K(+) ions through the plasmalemma of nearly all animal cells. The mechanisms involved in cation selection by the pump's ion-binding sites (site I and site II bind either Na(+) or K(+); site III binds only Na(+)) are poorly understood. We studied cation selectivity by outward-facing sites (high K(+) affinity) of Na/K pumps expressed in Xenopus oocytes, under voltage clamp. Guanidinium(+), methylguanidinium(+), and aminoguanidinium(+) produced two phenomena possibly reflecting actions at site III: (i) voltage-dependent inhibition (VDI) of outwardly directed pump current at saturating K(+), and (ii) induction of pump-mediated, guanidinium-derivative-carried inward current at negative potentials without Na(+) and K(+). In contrast, formamidinium(+) and acetamidinium(+) induced K(+)-like outward currents. Measurement of ouabain-sensitive ATPase activity and radiolabeled cation uptake confirmed that these cations are external K(+) congeners. Molecular dynamics simulations indicate that bound organic cations induce minor distortion of the binding sites. Among tested metals, only Li(+) induced Na(+)-like VDI, whereas all metals tested except Na(+) induced K(+)-like outward currents. Pump-mediated K(+)-like organic cation transport challenges the concept of rigid structural models in which ion specificity at site I and site II arises from a precise and unique arrangement of coordinating ligands. Furthermore, actions by guanidinium(+) derivatives suggest that Na(+) binds to site III in a hydrated form and that the inward current observed without external Na(+) and K(+) represents cation transport when normal occlusion at sites I and II is impaired. These results provide insights on external ion selectivity at the three binding sites.
Proceedings of the National Academy of Sciences 10/2010; 107(43):18718-23. DOI:10.1073/pnas.1004214107 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Listeria monocytogenes is a food-borne pathogen that grows at refrigeration temperatures and increases its content of anteiso-C15:0 fatty acid, which is believed to be a homeoviscous adaptation to ensure membrane fluidity, at these temperatures. As a possible
novel approach for control of the growth of the organism, the influences of various fatty acid precursors, including branched-chain
amino acids and branched- and straight-chain carboxylic acids, some of which are also well-established food preservatives,
on the growth and fatty acid composition of the organism at 37°C and 10°C were studied in order to investigate whether the
organism could be made to synthesize fatty acids that would result in impaired growth at low temperatures. The results indicate
that the fatty acid composition of L. monocytogenes could be modulated by the feeding of branched-chain amino acid, C4, C5, and C6 branched-chain carboxylic acid, and C3 and C4 straight-chain carboxylic acid fatty acid precursors, but the growth-inhibitory effects of several preservatives were independent
of effects on fatty acid composition, which were minor in the case of preservatives metabolized via acetyl coenzyme A. The
ability of a precursor to modify fatty acid composition was probably a reflection of the substrate specificities of the first
enzyme, FabH, in the condensation of primers of fatty acid biosynthesis with malonyl acyl carrier protein.
[Show abstract][Hide abstract] ABSTRACT: Gram-positive bacteria, including Listeria monocytogenes, adjust membrane fluidity by shortening the fatty acid chain length and increasing the proportional production of anteiso fatty acids at lower growth temperatures. The first condensation reaction in fatty acid biosynthesis is carried out by beta-ketoacyl-acyl carrier protein synthase III (FabH), which determines the type of fatty acid produced in bacteria. Here, we measured the initial rates of FabH-catalyzed condensation of malonyl-acyl carrier protein and alternate branched-chain precursor acyl-CoAs utilizing affinity-purified His-tagged L. monocytogenes FabH heterologously expressed in Escherichia coli. Listeria monocytogenes FabH showed a preference for 2-methylbutyryl-CoA, the precursor of odd-numbered anteiso fatty acids, at 30 degrees C, which was further increased at a low temperature (10 degrees C), suggesting that temperature-dependent substrate selectivity of FabH underlies the increased formation of anteiso branched-chain fatty acids during low-temperature adaptation. The increased FabH preferential condensation of 2-methylbutyryl-CoA could not be attributed to a significantly higher availability of this fatty acid precursor as acyl-CoA pool levels were reduced similarly for all fatty acid precursors at low temperatures.
[Show abstract][Hide abstract] ABSTRACT: The Na/K pump actively exports 3 Na(+) in exchange for 2 K(+) across the plasmalemma of animal cells. As in other P-type ATPases, pump function is more effective when the relative affinity for transported ions is altered as the ion binding sites alternate between opposite sides of the membrane. Deletion of the five C-terminal residues from the alpha-subunit diminishes internal Na(+) (Na(i)(+)) affinity approximately 25-fold [Morth et al. (2007) Nature 450:1043-1049]. Because external Na(+) (Na(o)(+)) binding is voltage-dependent, we studied the reactions involving this process by using two-electrode and inside-out patch voltage clamp in normal and truncated (DeltaKESYY) Xenopus-alpha1 pumps expressed in oocytes. We observed that DeltaKESYY (i) decreased both Na(o)(+) and Na(i)(+) apparent affinities in the absence of K(o)(+), and (ii) did not affect apparent Na(o)(+) affinity at high K(o)(+). These results support a model of strict sequential external release of Na(+) ions, where the Na(+)-exclusive site releases Na(+) before the sites shared with K(+) and the DeltaKESYY deletion only reduces Na(o)(+) affinity at the shared sites. Moreover, at nonsaturating K(o)(+), DeltaKESYY induced an inward flow of Na(+) through Na/K pumps at negative potentials. Guanidinium(+) can also permeate truncated pumps, whereas N-methyl-D-glucamine cannot. Because guanidinium(o)(+) can also traverse normal Na/K pumps in the absence of both Na(o)(+) and K(o)(+) and can also inhibit Na/K pump currents in a Na(+)-like voltage-dependent manner, we conclude that the normal pathway transited by the first externally released Na(+) is large enough to accommodate guanidinium(+).
Proceedings of the National Academy of Sciences 09/2009; 106(36):15507-12. DOI:10.1073/pnas.0903752106 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The red blood cell membrane is specialized to exchange chloride and bicarbonate; usually the pH gradient, the chloride ratio, and the membrane potential are tightly coupled. We review the evidence that led to the ability to separately vary inside and outside pH in red cells. The effect of pH on Na pump activity and on the selectivity of the inside and the outside transport sites is reviewed. In red blood cells, at high pH, the outside site is not selective. An increase in protons leads to an increase in K(+) affinity, thus making the site more selective. The pK for this site is different in rats and humans; because of the high conservation of residues in these two species, there are only a few possible residues that can account for this difference. On the inside, work from unsided preparations suggests that, at high pH, the transport site is highly selective for Na(+). Once again, an increase in protons leads to an increase in K(+) affinity, but now the result is a less selective site. During their maturation, reticulocytes lose many membrane proteins. The type and fractional loss is species dependent. For example, most reticulocytes lose most of their Na pumps, retaining about 100 pumps per cell, but animals from the order Carnivora lose all their pumps. We review some of the evidence that PKC phosphorylation of N-terminus serines is responsible for endocytosis in other cell types and species variation in this region.
[Show abstract][Hide abstract] ABSTRACT: Recombinant Ca(2+)-ATPase from tomato (i.e. LCA1 for Lycopersicon esculentum [Since the identification and naming of LCA1, the scientific name for the tomato has been changed to Solanum lycopersicum.] Ca-ATPase) was heterologously expressed in yeast for structure-function characterization. We investigate the differences between plant and animal Ca pumps utilizing comparisons between chicken and rabbit SERCA-type pumps with Arabidopsis (ECA1) and tomato plant (LCA1) Ca(2+)-ATPases. Enzyme function was confirmed by the ability of each Ca(2+)-ATPase to rescue K616 growth on EGTA-containing agar and directly via in vitro ATP hydrolysis. We found LCA1 to be approximately 300-fold less sensitive to thapsigargin than animal SERCAs, whereas ECA1 was thapsigargin-resistant. LCA1 showed typical pharmacological sensitivities to cyclopiazonic acid, vanadate, and eosin, consistent with it being a P(IIA)-type Ca(2+)-ATPase. Possible amino acid changes responsible for the reduced plant thapsigargin-sensitivity are discussed. We found that LCA1 also complemented K616 yeast growth in the presence of Mn(2+), consistent with moving Mn(2+) into the secretory pathway and functionally compensating for the lack of secretory pathway Ca-ATPases (SPCAs) in plants.
Archives of Biochemistry and Biophysics 12/2008; 481(2):157-68. DOI:10.1016/j.abb.2008.11.010 · 3.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Peroxynitrite is a reactive nitrogen species produced when nitric oxide and superoxide react. In vivo studies suggest that reactive oxygen species and, perhaps, peroxynitrite can influence Na-K-ATPase function. However, the direct effects of peroxynitrite on Na-K-ATPase function remain unknown. We show that a single bolus addition of peroxynitrite inhibited purified renal Na-K-ATPase activity, with IC50 of 107+/-9 microM. To mimic cellular/physiological production of peroxynitrite, a syringe pump was used to slowly release (approximately 0.85 microM/s) peroxynitrite. The inhibition of Na-K-ATPase activity induced by this treatment was similar to that induced by a single bolus addition of equal cumulative concentration. Peroxynitrite produced 3-nitrotyrosine residues on the alpha, beta, and FXYD subunits of the Na pump. Interestingly, the flavonoid epicatechin, which prevented tyrosine nitration, was unable to blunt peroxynitrite-induced ATPase inhibition, suggesting that tyrosine nitration is not required for inhibition. Peroxynitrite led to a decrease in iodoacetamidofluorescein labeling, implying that cysteine modifications were induced. Glutathione was unable to reverse ATPase inhibition. The presence of Na+ and low MgATP during peroxynitrite treatment increased the IC50 to 145+/-10 microM, while the presence of K+ and low MgATP increased the IC50 to 255+/-13 microM. This result suggests that the EPNa conformation of the pump is slightly more sensitive to peroxynitrite than the E(K) conformation. Taken together, these results show that peroxynitrite is a potent inhibitor of Na-K-ATPase activity and that peroxynitrite can induce amino acid modifications to the pump.
American journal of physiology. Renal physiology 09/2008; 295(4):F1191-8. DOI:10.1152/ajprenal.90296.2008 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We examined the effect of extracellular terbium (Tb(3+)) and divalent metal cations (Ca(2+), Sr(2+), and Ba(2+)) on (86)Rb(+) influx into rabbit and human red blood cells. We found that Tb(3+) at 15 and 25 microM was a non-competitive inhibitor of (86)Rb(+) influx suggesting that Tb(3+) is not binding to the transport site. This result reduces the usefulness of Tb(3+) as a potential probe for the E(out) conformation (the conformation with the transport site facing extracellularly). Ba(2+), Sr(2+) and Ca(2+), at concentrations >50 mM, had minimal effects on Rb(+) influx into red blood cells (1 mM Rb-out). This suggests that the outside transport site is very specific for monovalent cations over divalent cations, in contrast to the inside transport site. We also found that chrysoidine (4-phenylazo-m-phenylenediamine) competes with Na(+) for ATPase activity and K(+) for pNPPase activity suggesting it is binding to the E(in) conformation. Chrysoidine and similar compounds may be useful as optical probes of the E(in) conformation.