Biocontrol of the shore fly Scatella tenuicosta with Hypoaspis miles and H. aculeifer in peat pots

Hypoaspis miles Berlese and H. aculeiferCanestrini (Acari: Laelapidae) were usedagainst Scatella tenuicosta Collin(Diptera: Ephydridae) at the rates of 7, 14and 36 mites per 0.3-l peat pot (1000, 2000 and5000 per net-m² of peat) with mint (Mentha piperita L.) as the crop. The predatorswere applied either one day (preventivecontrol) or nine days after (curative control)exposing the pots to the flies in thegreenhouse. Based on the number of flies thatemerged from the pots, the control efficacy of preventive H. aculeifer treatments at therate of 36 mites per pot was 84–100% and 92–97% (range over four replicate blocks)after two and three weeks, respectively, fromthe application. The control efficacy ofcurative treatments was 93–100% and 83–94% after two and three weeks, respectively,from the application. Preventive treatmentswith H. miles applied at the rate of 36per pot controlled the flies by 92–100% and 68–97% two and three weeks, respectively, afterthe application. Curative treatments with 36H. miles per pot were less effective (–53–68% and –16–65%, respectively). All treatments with lower mite rates with eitherspecies were ineffective. Due to moreconsistent reduction of fly numbers by Hypoaspis aculeifer, the species was concluded to be a better biocontrol agent ofshore flies in short-term greenhouse cropsgrown in peat pots, probably due to its higherpredation capacity in comparison to H. miles.     

S-Nitrosation of Glutathione Transferase P1-1 Is Controlled by the Conformation of a Dynamic Active Site Helix

S-Nitrosation is a post-translational modification of protein cysteine residues, which occurs in response to cellular oxidative stress. Although it is increasingly being linked to physiologically important processes, the molecular basis for protein regulation by this modification remains poorly understood. We used transient kinetic methods to determine a minimal mechanism for spontaneous S-nitrosoglutathione (GSNO)-mediated transnitrosation of human glutathione transferase (GST) P1-1, a major detoxification enzyme and key regulator of cell proliferation. Cys⁴⁷ of GSTP1-1 is S-nitrosated in two steps, with the chemical step limited by a pre-equilibrium between the open and closed conformations of helix α2 at the active site. Cys¹⁰¹, in contrast, is S-nitrosated in a single step but is subject to negative cooperativity due to steric hindrance at the dimer interface. Despite the presence of a GSNO binding site at the active site of GSTP1-1, isothermal titration calorimetry as well as nitrosation experiments using S-nitrosocysteine demonstrate that GSNO binding does not precede S-nitrosation of GSTP1-1. Kinetics experiments using the cellular reductant glutathione show that Cys¹⁰¹-NO is substantially more resistant to denitrosation than Cys⁴⁷-NO, suggesting a potential role for Cys¹⁰¹ in long term nitric oxide storage or transfer. These results constitute the first report of the molecular mechanism of spontaneous protein transnitrosation, providing insight into the post-translational control of GSTP1-1 as well as the process of protein transnitrosation in general.     

Apical and basolateral effects of PTH in OK cells: Transport inhibition,messenger production,effects of pertussis toxin,and interaction with a PTH analog

Summary The cellular distribution (apicalvs. basolateral) of parathyroid hormone (PTH) signal transduction systems in opossum kidney (OK) cells was evaluated by measuring the action of PTH on apically located transport processes (Na/P_i cotransport and Na/H exchange) and on the generation of intracellular messengers (cAMP and IP₃).PTH application led to immediate inhibition of Na/H-exchange without a difference in dose/response relationships between apical and basolateral cell-surface hormone addition (halfmaximal inhibition at 5×10^–10 m). PTH required 2–3 hr for maximal inhibition of Na/P_i cotransport with a half-maximal inhibition occurring at ×10^–12 m for apical application. PTH addition to either side of the monolayer produced a dose-dependent production of both cAMP and IP₃. Half-maximal activation of IP₃ was at about 7×10^–12 m PTH and displayed no differences between apical and basolateral hormone addition, while cAMP was produced with a half maximal concentration of 7×10^–9 m for apical PTH application and 10^–9 m for basolateral administration.The PTH analog [nle^8.18, tyr³⁴]PTH(3-34), (nlePTH), produced partial inhibition of Na/P_i cotransport (agonism) with no difference between apical and basolateral application. When applied as a PTH antagonist, nlePTH displayed dose-dependent antagonism of PTH inhibition of Na/P_i cotransport on the apical surface, failing to have an effect on the basolateral surface. Independent of addition to the apical or basolateral cell surface, nlePTH had only weak stimulatory effect on production of cAMP, whereas high levels of IP₃ could be measured after addition of this PTH analog to either cell surface. Also an antagonistic action of nlePTH on PTH-dependent generation of the internal messengers, cAMP and IP₃, was observed; at the apical and basolateral cell surface nlePTH reduced PTH-dependent generation of cAMP, while PTH-dependent generation of IP₃ was only reduced by nlePTH at the apical surface.Pertussis toxin (PT) preincubation produced an attenuation of both PTH-dependent inhibition of Na/P_i cotransport and IP₃ generation while producing an enhancement of PTH-dependent cAMP generation; these effects displayed no cell surface polarity, suggesting that PTH action through either adenylate cyclase or phospholipase C was transduced through similar sets of G-proteins at each cell surface.It is concluded that apparent receptor activities with high and low affinity for PTH exist on both cell surfaces; those with apparent high affinity seem to be coupled preferentially to phospholipase C and those with apparent low affinity to adenylate cyclase. The differences in apparent affinity of receptor events coupled to adenylate cyclase and the differences in PTH/nlePTH interaction on the two cell surfaces are suggestive of the existence of differences in apparent PTH-receptor activities on the two cell surfaces.     

Na+/H+-exchange in A6 cells: Polarity and vasopressin regulation

Summary We have analyzed the mechanism of Na⁺-dependent pH_i; recovery from an acid load in A6 cells (an amphibian distal nephron cell line) by using the intracellular pH indicator 27-bis(2-carboxyethyl)5, 6 carboxyfluorescein (BCECF) and single cell microspectrofluorometry. A6 cells were found to express Na⁺/H⁺-exchange activity only on the basolateral membrane: Na⁺/H⁺-exchange activity follows simple saturation kinetics with an apparent K _mfor Na⁺ of approximately 11 mm; it is inhibited in a competitive manner by ethylisopropylamiloride (EIPA). This Na⁺/H⁺-exchange activity is inhibited by pharmacological activation of protein kinase A (PKA) as well as of protein kinase C (PKC). Addition of arginine vasopressin (AVP) either at low (subnanomolar) or at high (micromolar) concentrations inhibits Na⁺/H⁺-exchange activity; AVP stimulates IP₃ production at low concentrations, whereas much higher concentrations are required to stimualte cAMP formation. These findings suggest that in A6 cells (i) Na⁺/H⁺-exchange is located in the basolateral membrane and (ii) PKC activation (heralded by IP₃ turnover) is likely to be the mediator of AVP action at low AVP concentrations.This work was supported by the Swiss National Science Foundation (Grant No. 32-30785.91), the Stiftung für wissenschaftliche Forschung an der Universität Zürich, the Hartmann-Müller Stiftung, the Sandoz-Stiftung, the Roche Research Foundation, and the Geigy Jubiläumsstiftung. Prof. Dr. V. Casavola and Dr. R. Guerra were supported by a research grant, No. 91.02470.CT14 of the Consiglio Nazionale della Ricerche (C.N.R.) We are grateful to Prof. Dr. B.C. Rossier of the Institute of Pharmacology of Lausanne (Switzerland) for the gift of the A6 cells, to H.P. Gaeggeler for the supply of the necessary culture media and to Jutka Forgo for her excellent help in the day-to-day culturing of the A6 cells. The secretarial assistance of D. Rossi is gratefully acknowledged.     

Sodium-cotransport systems in intestine and kidney of the winter flounder

Summary Brush-border membrane vesicles were isolated from the intestine and kidney of the winter flounder,Pseudopleuronectes americanus, and the transport ofd-glucose,l-alanine and sodium was examined by a rapid filtration technique.d-glucose,l-alanine, and sodium entered the same osmotically reactive space suggesting that uptake into vesicles represents transport across rather than binding to the membrane. d-glucose andl-alanine uptake by intestinal and renal brush-border membrane vesicles was stimulated by sodium as compared to potassium or choline. In the presence of a sodium chloride gradient, overshooting uptake was observed indicating a transient intravesicular accumulation ofd-glucose andl-alanine. The sodium-dependentd-glucose uptake was inhibited by phlorizin andd-galactose while the transport ofl-alanine was inhibited byl-phenylalanine. The sodium-dependent transport ofd-glucose andl-alanine was affected by the electrical potential difference across the vesicle membrane; the addition of valinomycin in the presence of an inwardly directed potassium chloride gradient inhibited sodium-dependent solute uptake, whereas replacing chloride or gluconate with more permeant anions, such as SCN^–, stimulated uptake. Similar results were obtained with intestinal and renal membranes; they document the presence of sodium/d-glucose and sodium/l-alanine cotransport systems in the brush-border membrane of intestine and kidney.Sodium uptake into brush border membrane vesicles from the flounder intestine and kidney was saturable (tracer replacement) and trans-stimulated (tracer coupling), indicating transport via facilitated diffusion systems. Additionally, sodium uptake was only slightly affected by superimposing diffusion potentials demonstrating that the majority of sodium transport was by electroneutral coupled processes. In both the intestinal and kidney brush-border membrane vesicles sodium uptake was inhibited by an inwardly directed proton gradient suggesting the presence of a sodium/proton exchange mechanism. In intestinal, but not in renal membrane preparations, sodium uptake was stimulated by chloride. Chloride stimulation was abolished after preincubation with furosemide indicating the presence of an additional coupled sodium-chloride transport in the intestinal brush-border membranes.The experiments were carried out at the Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672, USAAddress effective February 1, 1980: Albert Einstein College of Medicine, Department of Physiology, 1300 Morris Park Avenue, Bronx, New York 10461, USA     

Identification and localization of sodium-phosphate cotransporters in hepatocytes and cholangiocytes of rat liver

Hepatocytes and cholangiocytes release ATP into bile, where it is rapidly degraded into adenosine and P(i). In rat, biliary P(i) concentration (0.01 mM) is approximately 100-fold and 200-fold lower than in hepatocytes and plasma, respectively, indicating active reabsorption of biliary P(i). We aimed to functionally characterize canalicular P(i) reabsorption in rat liver and to identify the involved P(i) transport system(s). P(i) transport was determined in isolated rat canalicular liver plasma membrane (LPM) vesicles using a rapid membrane filtration technique. Identification of putative P(i) transporters was performed with RT-PCR from liver mRNA. Phosphate transporter protein expression was confirmed by Western blotting in basolateral and canalicular LPM and by immunofluorescence in intact liver. Transport studies in canalicular LPM vesicles demonstrated sodium-dependent P(i) uptake. Initial P(i) uptake rates were saturable with increasing P(i) concentrations, exhibiting an apparent K(m) value of approximately 11 muM. P(i) transport was stimulated by an acidic extravesicular pH and by an intravesicular negative membrane potential. These data are compatible with transport characteristics of sodium-phosphate cotransporters NaPi-IIb, PiT-1, and PiT-2, of which the mRNAs were detected in rat liver. On the protein level, NaPi-IIb was detected at the canalicular membrane of hepatocytes and at the brush-border membrane of cholangiocytes. In contrast, PiT-1 and PiT-2 were detected at the basolateral membrane of hepatocytes. We conclude that NaPi-IIb is most probably involved in the reabsorption of P(i) from primary hepatic bile and thus might play an important role in the regulation of biliary P(i) concentration.     

Regulation of intestinal phosphate transport. II. Metabolic acidosis stimulates Na(+)-dependent phosphate absorption and expression of the Na(+)-P(i) cotransporter NaPi-IIb in small intestine

During metabolic acidosis, P(i) serves as an important buffer to remove protons from the body. P(i) is released from bone together with carbonate buffering protons in blood. In addition, in the kidney, the fractional excretion of phosphate is increased allowing for the excretion of more acid equivalents in urine. The role of intestinal P(i) absorption in providing P(i) to buffer protons and compensating for loss from bone during metabolic acidosis has not been clarified yet. Inducing metabolic acidosis (NH(4)Cl in drinking water) for 2 or 7 days in mice increased urinary fractional P(i) excretion twofold, whereas serum P(i) levels were not altered. Na(+)-dependent P(i) transport in the small intestine, however, was stimulated from 1.89 +/- 3.22 to 40.72 +/- 11.98 pmol/mg protein (2 days of NH(4)Cl) in brush-border membrane vesicles prepared from total small intestine. Similarly, the protein abundance of the Na(+)-dependent phosphate cotransporter NaPi-IIb in the brush-border membrane was increased 5.3-fold, whereas mRNA levels remained stable. According to immunohistochemistry and real-time PCR NaPi-IIb expression was found to be mainly confined to the ileum in the small intestine, and this distribution was not altered during metabolic acidosis. These results suggest that the stimulation of intestinal P(i) absorption during metabolic acidosis may contribute to the buffering of acid equivalents by providing phosphate and may also help to prevent excessive liberation of phosphate from bone.     

Parathyroid hormone treatment induces dissociation of type IIa Na+-P(i) cotransporter-Na+/H+ exchanger regulatory factor-1 complexes

The type IIa Na⁺-P_i cotransporter (NaP_i-IIa) and the Na⁺/H⁺ exchanger regulatory factor-1 (NHERF1) colocalize in the apical membrane of proximal tubular cells. Both proteins interact in vitro. Herein the interaction between NaP_i-IIa and NHERF1 is further documented on the basis of coimmunoprecipitation and co-pull-down assays. NaP_i-IIa is endocytosed and degraded in lysosomes upon parathyroid hormone (PTH) treatment. To investigate the effect of PTH on the NaP_i-IIa-NHERF1 association, we first compared the localization of both proteins after PTH treatment. In mouse proximal tubules and OK cells, NaP_i-IIa was removed from the apical membrane after hormonal treatment; however, NHERF1 remained at the membrane. Moreover, PTH treatment led to degradation of NaP_i-IIa without changes in the amount of NHERF1. The effect of PTH on the NaP_i-IIa-NHERF1 interaction was further studied using coimmunoprecipitation. PTH treatment reduced the amount of NaP_i-IIa coimmunoprecipitated with NHERF antibodies. PTH-induced internalization of NaP_i-IIa requires PKA and PKC; therefore, we next analyzed whether PTH induces changes in the phosphorylation state of either partner. NHERF1 was constitutively phosphorylated. Moreover, in mouse kidney slices, PTH induced an increase in NHERF1 phosphorylation; independent activation of PKA or PKC also resulted in increased phosphorylation of NHERF1 in kidney slices. However, NaP_i-IIa was not phosphorylated either basally or after exposure to PTH. Our study supports an interaction between NHERF1 and NaP_i-IIa on the basis of their brush-border membrane colocalization and in vitro coimmunoprecipitation/co-pull-down assays. Furthermore, PTH weakens this interaction as evidenced by different in situ and in vivo behavior. The PTH effect takes place in the presence of increased phosphorylation of NHERF1. proximal tubule; opossum kidney cells; phosphorylation; endocytosis     

Structure-function relations of the first and fourth predicted extracellular linkers of the type IIa Na+/Pi cotransporter: I. Cysteine scanning mutagenesis

The putative first intracellular and third extracellular linkers are known to play important roles in defining the transport properties of the type IIa Na+-coupled phosphate cotransporter (Kohler, K., I.C. Forster, G. Stange, J. Biber, and H. Murer. 2002b. J. Gen. Physiol. 120:693-705). To investigate whether other stretches that link predicted transmembrane domains are also involved, the substituted cysteine accessibility method (SCAM) was applied to sites in the predicted first and fourth extracellular linkers (ECL-1 and ECL-4). Mutants based on the wild-type (WT) backbone, with substituted novel cysteines, were expressed in Xenopus oocytes, and their function was assayed by isotope uptake and electrophysiology. Functionally important sites were identified in both linkers by exposing cells to membrane permeant and impermeant methanethiosulfonate (MTS) reagents. The cysteine modification reaction rates for sites in ECL-1 were faster than those in ECL-4, which suggested that the latter were less accessible from the extracellular medium. Generally, a finite cotransport activity remained at the end of the modification reaction. The change in activity was due to altered voltage-dependent kinetics of the Pi-dependent current. For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V < or = -80 mV, whereas the WT exhibited a linear voltage dependency. After cys modification, this mutant displayed a supralinear voltage dependency in the same voltage range. The opposite behavior was documented for cys substitution at Met-533 in ECL-4. Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials. Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport.