Evaluations on the Lethal, Choice and Secondary Effects of Four Insecticidal Baits against the German Cockroach (Blattaria, Blattellidae)

ABSTRACT The laboratory studies were carried out for evaluating control effects of four commercial insecticidal baits such as two different hydramethylnon [2.0%(AI)] products (DBK^® and Combat‐Gold^®), fipronil [0.05 %(AI)] (Combat‐Power^®) and (0.6% chlorpyrifos [0.6%(AI)] (Raid‐Roachbait^®) against German cockroaches (Blattella germanica). The control rates of four kinds of toxic baits were all 100.0% mortality of German cockroaches in 5 days after treatment. The results of chlorpyrifos and fipronil brought 100.0% mortalities in 2 to 3 days after treatment, respectively. After 3 day treatment, there was no significant difference of control effect among the four toxic baits. As a result of this study, fipnonil and chlorpyrifos showed faster killing action against German cockroaches than the two hydramethylnon formulation products. In the choice test, DBK^® (hydramethylnon) (average 17.0 ind.) significantly attracted more German cockroaches than Combat‐Gold^® (hydramethylnon) (avg. 7.0 ind.), Combat‐Power^® (fipronil) (avg. 5.3 ind.) and Raid‐Roachbait^® (chlorpyrifos) (avg. 3.3 ind.). The difference in attraction effects came 10 minutes after treatment. In order to evaluate the secondary killing effect of toxic baits by coprophagy against adult Blattella germanica, the comparative test was carried out. The adult mortality rates were significantly different among the four toxic baits showing 86.7% mortality for fipronil, 60.0% for hydramethylnon (DBK^®), 30.0% for chlorpyrifos, and below 13.3% for hydramethylnon (Combat‐Gold^®) in 6 days. During the first 12 days, fipronil showed the highest mortality rate (90.0%), followed by hydramethylnon (DBK^®) (60.0%). The secondary killing effect of toxic baits by coprophagy appeared in all of the baits against adult Blattella germanica.     

Effect of five diatomaceous earth formulations against Tribofium castaneum （Coleoptera： Tenebrionidae）, Oryzaephflus surinamensis （Coleoptera： Silvanidae） and Rhyzopertha dominica （Coleoptera： Bostrychidae）

Laboratory bioassays were conducted to determine the effect of food source on the survival of Tribolium castaneum Herbst, Oryzaephilus surinamensis L. and Rhyzopertha dominica F., after exposure to five diatomaceous earth （DE） formulations： Protect-It, Insecto, Perma-GuardTM, Dryacide and SilicoSec. Adults of these species were exposed to DEs at the rate of 0.5 mg/cm^2 for 1 day on filter paper inside plastic Petri dishes. After exposure, the initial mortality was counted and live individuals of the three species were held for a week in glass vials containing 50 mg wheat flour, rice and whole wheat, respectively. In the second experiment, after 1 day exposure to DEs, beetles were transferred to Petri dishes without food and held for a week to determine if the presence of food source would decrease the mortality of beetles. Experiments were carried out at 27℃ and 55% RH in the dark. The initial mortality in both of the experiments reached 100% for the three species exposed to Protect-It and in the case ofR. dominica and O. surinamensis exposed to Dryacide. In contrast, low level of mortality （〈 10%） was observed for T. castaneum exposed to Perma-GuardTM and Insecto. The mortality after the post-treatment period on food was decreased for the three species exposed to Perma-GuardTM and in the case of T. castaneum and R. dominica exposed to Insecto and SilicoSec. Adults of O. surinamensis were the most susceptible followed by R. dominica and 100% adult mortality was obtained, whereas T. castaneum were the least susceptible beetles to DEs. Protect-It and Dryacide were the most efficient DE formulations and can be used effectively in a stored grain integrated pest management program.     

Irritability and repellency of synthetic pyrethroids on an Aedes aegypti population from Thailand

The main objective of this study was to find the optimal dosage of deltamethrin, cyphenothrin, d‐tetramethrin, and tetramethrin that would elicit repellency and irritability responses of Aedes aegypti. The F1‐F3 generations of field mosquitoes collected from Pu Teuy Village, Sai‐Yok District, Kanchanaburi Province, Thailand, were tested with four pyrethroids to determine the LC₂₅, LC₅₀, and LC₉₉. These concentrations were 0.010%, 0.020%, and 0.055%, respectively, for deltamethrin; 0.113%, 0.167%, and 0.353%, respectively, for cyphenothrin; 2.091%, 2.770%, and 5.114%, respectively, for d‐tetramethrin; and 2.377%, 4.251%, and 10.715%, respectively, for tetramethrin. All dosages were tested in the excito‐repellency system. Survival analysis was used to compare each chamber of the test. It was found that cyphenothrin had a stronger repellent effect than the other pyrethroids, while the contact irritant effect was similar among compounds tested. The LC₅₀ of each pyrethroid was found to be the optimal dose for repelling Ae. aegypti. There was no significant difference in LC₉₉ values for either non‐contact or contact trials for each pyrethroid.     

KINETICS OF AMINO ACID INFLUX INTO NITELLA FLEXILIS1

The uptake of amino acids by Nitella flexilis has been investigated. Influx of glycine, alanine, and valine appears to be a diffusive process. Influx ranged from 0.14 to 0.06 and 0.04 pmoles/(cm)(sec), respectively. Aspartic acid uptake is an active transport mechanism. The V_max is 2.8 pmoles/(cm)(sec); the transport constant (Michaelis constant) K_m, 7.8 × 10^?3 M. The uptake of arginine is apparently due to 2 transport systems, one with a V_max and K_m of 3.1 pmoles/(cm)(sec) and 3.2 × 10^?3M, respectively. The second system has a V_max of 1.4 pmoles/(cm)(sec) and a K_m of 2.1 × 10^?4 M. The possibility that the second system is diffusive has been considered.     

The electrical changes accompanying fertilization and cortical vesicle secretion in the medaka egg

The electrical properties of the egg of the medaka, Oryzias latipes, were studied before, during, and after fertilization. The resting potential of the unfertilized egg averaged ?39 ± 9 mV in Yamamoto's Ringers (Y. Ringers), but 20% of the values were between ?50 and ?60 mV. Fertilization triggers a small depolarization of 4 ± 3 mV in 10% Y. Ringers with an average duration of 20 ± 10 sec. The amplitude of this depolarization is independent of [Na⁺]_o, [Ca²⁺]_o, and [Cl^?]_o, so it appears to be due to a nonspecific leak triggered by sperm-egg fusion. The depolarization is followed by a longer hyperpolarizing phase with an average amplitude of 31 ± 12 mV. Recovery from this hyperpolarization has a fast phase lasting 155 ± 18 sec, followed by a slower phase which reaches a steady average membrane potential of ?19 ± 1 mV by 9 min after fertilization. The membrane resistance falls 10-fold during the first 2 min after fertilization, from 40 (1520 kΩ-cm²) to 3 MΩ. This is largely due to an increase in the K⁺ conductance. At the peak of the hyperpolarization, the membrane potential exhibits a 28 mV/decade [K⁺]_o dependence and a 6 mV/decade [Na⁺]_o dependence. The membrane resistance slowly recovers over the next 8 min to a value about 30% larger than before fertilization. The relation of current vs voltage was linear before, during, and after fertilization and indicated a reversal potential of ?98 ± 20 mV for the hyperpolarization peak. The egg's capacitance averaged 0.04 ± 0.01 μF (0.9 μF/cm²) before fertilization and approximately doubles within 90 sec after fertilization. It then decreases over a 9-min period, reaching a value 25% smaller than before fertilization.     

The second component of the fertilization potential in sea urchin (Pseudocentrotus depressus) eggs involves both Na+ and K+ permeability

The fertilization potential of the Pseudocentrotus depressus egg involved three transiently depolarizing components which had a different time course and a peak value. Three peaks were at less than 10 sec, 43 ± 4 sec (mean ± SD), and 182 ± 22 sec after the onset of the fertilization potential. Their peak values (mean ± SD) were 37 ± 4, 17 ± 3, and −31 ± 5 mV in standard artificial sea water. The effect of external ions on the membrane potential at the peak of the second component was measured with a conventional voltage-recording microelectrode. The peak value changed 51 mV with a 10-fold change in external Na⁺ concentration. However, it was about 65 mV more negative than the equilibrium potential of Na⁺, assuming that the internal Na⁺ concentration was 13.5 mM. H⁺, Ca²⁺, Mg²⁺, and Cl⁻ did not contribute to the peak value. The peak value was sensitive to the external K⁺ concentration. These data fitted a theoretical line obtained from the Goldman-Hodgkin-Katz equation, using a ratio of P_Na:P_K:P_Cl = 1.1:1.0:0. This means that the permeability to both Na⁺ and K⁺ is responsible for the second component of the fertilization potential. The fertilization potential was also measured in the artificial sea water containing Li⁺ or Cs⁺. The egg at the second component of the fertilization potential was almost equally permeable to Li⁺ as well as Na⁺ or K⁺ and somewhat permeable to Cs⁺. By contrast, the resting membrane potential before fertilization depended to a large extent upon K⁺ permeability.     

Effects of glufosinate‐ammonium herbicide and pod sealant on spider Pardosa agrestis

Herbicides based upon glufosinate‐ammonium (GLA) are among the world's most widely used. They also are applied on the most prominent oil crops as desiccants in combination with pod sealants to prevent pod shatter and seed loss close to harvest. Even though these crops occupy a significant part of the world's agroecosystems, the effects of GLA herbicides on non‐target arthropods, and in particular natural enemies of pests, have been studied very rarely, and such effects of pod sealants have never been studied. We studied in our laboratory mortality as well as prey capture efficiency of the common GLA herbicide and desiccant Basta 15^®, pod sealant Arrest^®, and a mixture of both on the wolf spider Pardosa agrestis. We found that Basta 15^® and the mixture had lethal effect on spiders. We also found significant, short‐term effect on predatory activity of spiders after all treatments. Basta 15^® significantly influenced the amount of captured prey also in the long term. This is the first study showing lethal effect on spiders of the herbicide and herbicide plus pod sealant mixture. This is also the first study examining the effects of pod sealant on the mortality and predatory activity of a pest antagonist. More studies regarding the effects of agricultural chemical mixes are needed to uncover their effects on beneficial organisms existing within agroecosystems.     

Kinetic constants for the inhibition of camel retinal acetylcholinesterase by the carbamate insecticide lannate

We have designed this study to determine various kinetic parameters of camel retinal membrane‐bound acetylcholinesterase (AChE; EC 3.1.1.7) inhibition by carbamate insecticide lannate [methyl N‐{{(methylamino)carbonyl}oxy} ethanimidothioate]. All these kinetic constants were derived by simple graphical methods. The value of kinetic parameters was estimated as follows: 0.061 (μM)⁻¹, 1.14 (μM)⁻¹, 0.216 μM, 0.016 min⁻¹, 0.0741 (μM min)⁻¹, 0.746 μM, and 4.42 μM for velocity constant (K_v), new inhibition constant (K_nic), dissociation constant (K_d), carbamylation rate constant (k_2c), overall carbamylation rate constant (k′2 ), 50% inhibition constant (K_I50), and 99% inhibition constant (K_I99), respectively. These unique methods may be used to estimate such kinetic parameters for time‐dependent inhibition of enzymes by variety of chemicals, insecticides, herbicides, and drugs. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 41–46, 1999     

Glutathione S‐transferase π complexes with and stimulates Na+,K+‐ATPase

Glutathione S‐transferase (GST) was found to complex with the Na⁺,K⁺‐ATPase as shown by binding assay using quartz crystal microbalance. The complexation was obstructed by the addition of antiserum to the α‐subunit of the Na⁺,K⁺‐ATPase, suggesting the specificity of complexation between GST and the Na⁺,K⁺‐ATPase. Co‐immunoprecipitation experiments, using the anti‐α‐subunit antiserum to precipitate the GST‐Na⁺,K⁺‐ATPase complex and then using antibodies specific to an isoform of GST to identify the co‐precipitated proteins, revealed that GSTπ was complexed with the Na⁺,K⁺‐ATPase. GST stimulated the Na⁺,K⁺‐ATPase activity up to 1.4‐fold. The level of stimulation exhibited a saturable dose–response relationship with the amount of GST added, although the level of stimulation varied depending on the content of GSTπ in the lots of GST received from supplier. The stimulation was also obtained when recombinant GSTπ was used, confirming the results. When GST was treated with reduced glutathione, GST activity was greatly stimulated, whereas the level of stimulation of the Na⁺,K⁺‐ATPase activity was similar to that when untreated GST was added. When GST was treated with H₂O₂, GST activity was greatly diminished while the stimulation of the Na⁺,K⁺‐ATPase activity was preserved. The results suggest that GSTπ complexes with the Na⁺,K⁺‐ATPase and stimulates the latter independent of its GST activity. Copyright © 2012 John Wiley & Sons, Ltd.     

Abscisic acid,gibberellins and brassinosteroids in Kelpak®, a commercial seaweed extract made from Ecklonia maxima

The seaweed extract Kelpak^® made from the kelp Ecklonia maxima is registered as a biostimulant for use in agriculture. It elicits many beneficial responses including improved root and shoot growth, higher yields and greater resistance to abiotic and biotic stresses. Previously, cytokinins, auxins and polyamines were identified in Kelpak^®. The aim of the present study was to quantify other groups of plant growth regulators (PGRs)—abscisic acid (ABA), gibberellins (GAs) and brassinosteroids—that may be present in E. maxima and Kelpak^®. Kelpak^® samples harvested between 2008 and 2010 and stored for up to 26 months were analysed using ultra performance liquid chromatography tandem mass spectrometry. ABA levels were below the limits of detection in E. maxima but were detected in low concentrations in Kelpak^®, ranging from 0.31 to 20.70 pg mL^?1 Kelpak^®. Eighteen GAs were found in E. maxima and Kelpak^® with concentrations from 187.54 to 565.96 pg mL^?1 Kelpak^®. The biologically active GAs (GA₁, GA₃, GA₄, GA₅, GA₆ and GA₇) comprised less than 3 % in Kelpak^®. Although GA₁₃ (a final product in the metabolic pathway) was present in low concentrations in E. maxima, very high concentrations were present in Kelpak^®. The brassinosteroids brassinolide (BL) and castasterone (CS) were identified in E. maxima and Kelpak^®. Concentrations varied with harvest and storage time, ranging from 384.72 to 793.23 pg BL mL^?1 Kelpak^® and 62.84 to 567.51 pg CS mL^?1 Kelpak^®. It is likely that this cocktail of natural PGRs present in Kelpak^® may act individually or in concert and thus contribute to the numerous favourable physiological responses elicited by Kelpak^® application to plants.     

Enhancement of docosahexaenoic acid production by Schizochytrium sp. using a two-stage oxygen supply control strategy based on oxygen transfer coefficient

Aims: To improve the yield and productivity of docosahexaenoic acid (DHA) by Schizochytrium sp. in terms of the analysis of microbial physiology. Methods and Results: A two‐stage oxygen supply control strategy, aimed at achieving high concentration and high productivity of DHA, was proposed. At the first 40 h, K_La was controlled at 150·1 h^?1 to obtain high μ for cell growth, subsequently K_La was controlled at 88·5 h^?1 to maintain high q_p for high DHA accumulation. Finally, the maximum lipid, DHA content and DHA productivity reached 46·6, 17·7 g l^?1 and 111 mg l^?1 h^?1, which were 43·83%, 63·88% and 32·14% over the best results controlled by constant K_La. Conclusions: This paper described a two‐stage oxygen supply control strategy based on the kinetic analysis for efficient DHA fermentation by Schizochytrium sp. Significance and Impact of the study: This study showed the advantage of two‐stage control strategy in terms of microbial physiology. As K_La is a scaling‐up parameter, the idea developed in this paper could be scaled‐up to industrial process and applied to other industrial biotechnological processes to achieve both high product concentration and high productivity.     

Subcellular Localization and Kinetic Characterization of a Gill (Na+, K+)-ATPase from the Giant Freshwater Prawn Macrobrachium rosenbergii

The stimulation by Mg²⁺, Na⁺, K⁺, NH₄ ⁺, and ATP of (Na⁺, K⁺)-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na⁺, K⁺)-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M _r. Under saturating Mg²⁺, Na⁺, and K⁺ concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V _M = 115.0 ± 2.3 U mg^?1, K _0.5 = 0.10 ± 0.01 mmol L^?1. Stimulation by Na⁺ (V _M = 110.0 ± 3.3 U mg^?1, K _0.5 = 1.30 ± 0.03 mmol L^?1), Mg²⁺ (V _M = 115.0 ± 4.6 U mg^?1, K _0.5 = 0.96 ± 0.03 mmol L^?1), NH₄ ⁺ (V _M = 141.0 ± 5.6 U mg^?1, K _0.5 = 1.90 ± 0.04 mmol L^?1), and K⁺ (V _M = 120.0 ± 2.4 U mg^?1, K _M = 2.74 ± 0.08 mmol L^?1) followed single saturation curves and, except for K⁺, exhibited site–site interaction kinetics. Ouabain inhibited ATPase activity by around 73 % with K _I = 12.4 ± 1.3 mol L^?1. Complementary inhibition studies suggest the presence of F₀F₁–, Na⁺-, or K⁺-ATPases, but not V(H⁺)- or Ca²⁺-ATPases, in the gill microsomal preparation. K⁺ and NH₄ ⁺ synergistically stimulated enzyme activity (≈25 %), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH₄ ⁺, and K⁺ of the gill enzyme.     

Anaesthesia and transport of juvenile tambaqui Colossoma macropomum (Cuvier, 1818) with tricaine methane‐sulphonate: Implications on secondary and oxidative stress responses

The aim of this study was to evaluate the anaesthetic efficacy and the effect of sedation with tricaine (TMS^®), also known as MS‐222, on secondary and oxidative stress parameters in juvenile tambaqui, Colossoma macropomum transported in hyperoxia for 2, 6 and 10 hr. Juveniles were placed in aquaria containing six different concentrations of buffered tricaine (150, 180, 210, 240, 270 and 300 mg/L) and the times for anaesthesia induction and recovery determined. Fish transported in hyperoxic conditions were investigated for glycemia, ionic concentration (K⁺, Ca⁺⁺, Na⁺) and osmolality, haematocrit (Ht) and haemoglobin concentration (Hb), partial pressure of gases (pCO₂ and pO₂), pH and bicarbonate concentration () in whole blood collected from caudal vasculature. Total antioxidant capacity against peroxyl radicals (ACAP), glutathione‐S‐transferase (GST) activity and lipid peroxidation levels (TBARS) were investigated in gills, brain and liver. All concentrations of TMS^® induced deep anaesthesia in juvenile tambaqui in this study. A concentration of 240 mg/L of TMS^® was sufficient to induce rapid anaesthesia (<3 min) with uneventful recovery (<5 min). In light of the secondary and oxidative stress responses of fish transported using TMS^®, which were generally not significantly different compared to responses of fish transported in anaesthetic‐free water, sedation with 20 mg/L is not advantageous and therefore is dispensable for the transport of this species for up to 10 hr.     

<Emphasis Type="Italic">Lecanicillium attenuatum</Emphasis> isolates affecting the invasive cypress aphid (<Emphasis Type="Italic">Cinara cupressi</Emphasis>) in Chile

The cypress aphid (Cinara cupressi) is listed among the hundred most important invasive pests in the world. In Chile, it was first detected in 2003 and currently is present throughout the country. In the course of a survey of their natural enemies in Chile, three strains of entomopathogenic fungi were isolated. The isolates were identified and tested against the aphid in laboratory experiments. Two further entomopathogenic fungi (ARSEF 5126 and 5128), formulated in the mycoinsecticides Vertalec^® and Mycotal^®, were used as reference strains. The three Chilean isolates were identified genomically as Lecanicillium attenuatum and were pathogenic to third-instar nymphs. The isolate ARSEF 13279 yielded the lowest overall lethal concentration (LC₅₀), 3 × 10⁵ conidia ml^?1 at four days post-inoculation, and the shortest lethal time (LT₅₀), 3.7 days after inoculation with 10⁶ conidia ml^?1. The results indicate that the isolates have considerable potential as microbial control agents of the invasive cypress aphid.     

Properties and secondary structure of tannase from <Emphasis Type="Italic">Penicillium herquei</Emphasis>

A tannase with a molecular mass of 72 kDa was obtained from Penicillium herquei isolated from valonia acorns following fermentation in a 5 L bioreactor. This tannase showed optimum activity at pH 6.0 and 30°C. The enzyme was inhibited by Fe³⁺, Zn²⁺, dithiothrietol (DTT), β-mercaptoethanol, formaldehyde, and ethanol, and induced by K⁺, Mn²⁺, Tween 80, and Triton X-100. The Michaelis constant (K _m) and the second-order constant (k _cat/K _m) values of the tannase for propyl gallate (PG) were 0.62 mM and 174.1 mM/sec. The circular dichroism (CD) spectra indicated that the secondary structure of the tannase contained 14% α helix, 32.4% anti-parallel β-sheet, 4.8% β-sheet, 18.8% β-turn, and 30% random coil. Native tannase in ultrapure water manifested as spherical nano-particle aggregates with diameters ranging from 50 to 300 nm determined by atomic force microscopy (AFM).     

Trypanosoma lewisi: alterations in membrane function in the rat.

DEAE-cellulose-purified Trypanosoma lewisi from 4-day (dividing trypanosomes) and 7-day (non-dividing trypanosomes) infections in rats were compared for initial uptake of glucose, leucine, and potassium. Glucose entered the parasitic cells by mediated (saturable) processes, whereas leucine and K⁺ entered by mediated processes and diffusion. Glucose entry was significantly elevated in 4-day cells (V_max 4.00 ± 1.02 nmoles/ 1 × 10⁸ cells/min) with respect to 7-day cells (V_max 1.83 ± 0.62 nmoles 1 × 10⁸ cells/min). Likewise, the affinity of the glucose carrier was significantly greater in 4-day cells (K_m = 0.30 ± 0.02 mM) than in 7-day cells (K_m = 0.59 ± 0.11 mM). When leucine and K⁺ transport were compared in 4- and 7-day populations, significant elevations in the rate of entry (V_max) of both substrates were observed for 4-day cells; K_m values for leucine and K⁺ were not altered by the stage of infection. For leucine, the V_max and K_m for 4-day cells were 2.40 ± 0.50 nmoles/1 × 10⁸ cells/30 sec and 78 ± 7 μM, respectively; corresponding values in 7-day cells were 1.06 ± 0.02 nmoles/1 × 10⁸ cells/30 sec and 66 ± 11 μM. For K⁺, the V_max and K_m for 4-day cells were 15.97 ± 0.38 nmoles/1 × 10⁸ cells/min and 1.2 mM, respectively; corresponding values in 7-day cells were 4.76 ± 1.82 nmoles/1 × 10⁸ cells/min and 1.05 mM. The observed increase in the rate of K⁺ entry into 4-day cells was attributable to enhanced influx; no significant difference in the rate of K⁺ efflux was noted when 4- and 7-day cells were compared ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of K⁺ leak for 4- and 7-day cells were 68.1 ± 9.3 and 67.9 ± 15.2 min, respectively). Potassium influx was ouabain insensitive. Membrane function in 7-day cells was not uniformly inhibited. No significant difference in the activity of the membrane-bound enzyme, 5′-nucleotidase, was observed when 4- and 7-day cells were compared.     

The biotechnological potential of subtilisin‐like fibrinolytic enzyme from a newly isolated Lactobacillus plantarum KSK‐II in blood destaining and antimicrobials

An antimicrobial oxidative‐ and SDS‐stable fibrinolytic alkaline protease designated as KSK‐II was produced by Lactobacillus plantarum KSK‐II isolated from kishk, a traditional Egyptian food. Maximum enzyme productivity was obtained in medium containing 1% lactose and 0.5% soybean flour as carbon and nitrogen sources, respectively. Purification of enzyme increased its specific activity to 1,140‐fold with a recovery of 33% and molecular weight of 43.6 kDa. Enzyme activity was totally lost in the presence of ethylenediaminetetraacetic acid and was restored after addition of Fe²⁺ suggesting that KSK‐II is a metalloprotease and Fe²⁺ acts as cofactor. Enzyme hydrolyzed not only the natural proteins but also synthetic substrates, particularly Suc‐Ala‐Ala‐Pro‐Phe‐pNA. KSK‐II can hydrolyze the Lys‐X easier than Arg‐X; thus, it was considered as a subtilisin‐family protease. Its apparent K_m, V_max, and K_cat were 0.41 mM, 6.4 µmol mg^?1 min^?1, and 28.0 s^?1, respectively. KSK‐II is industrially important from the perspectives of its maximal activity at 50°C (stable up to 70°C), ability to function at alkaline pH (10.0), stability at broad pH ranges (7.5–12.0) in addition to its stability toward SDS, H₂O₂, organic solvents, and detergents. We emphasize for the first time the potential of fibrinolytic activity for alkaline proteases used in detergents especially in blood destaining. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:316–324, 2015     

Prostaglandin E2 modulates Na+,K+-ATPase activity in rat hippocampus: implications for neurological diseases

Prostaglandin E₂ (PGE₂) is quantitatively one of the major prostaglandins synthesized in mammalian brain, and there is evidence that it facilitates seizures and neuronal death. However, little is known about the molecular mechanisms involved in such excitatory effects. Na⁺,K⁺‐ATPase is a membrane protein which plays a key role in electrolyte homeostasis maintenance and, therefore, regulates neuronal excitability. In this study, we tested the hypothesis that PGE₂ decreases Na⁺,K⁺‐ATPase activity, in order to shed some light on the mechanisms underlying the excitatory action of PGE₂. Na⁺,K⁺‐ATPase activity was determined by assessing ouabain‐sensitive ATP hydrolysis. We found that incubation of adult rat hippocampal slices with PGE₂ (0.1–10 μM) for 30 min decreased Na⁺,K⁺‐ATPase activity in a concentration‐dependent manner. However, PGE₂ did not alter Na⁺,K⁺‐ATPase activity if added to hippocampal homogenates. The inhibitory effect of PGE₂ on Na⁺,K⁺‐ATPase activity was not related to a decrease in the total or plasma membrane immunocontent of the catalytic α subunit of Na⁺,K⁺‐ATPase. We found that the inhibitory effect of PGE₂ (1 μM) on Na⁺,K⁺‐ATPase activity was receptor‐mediated, as incubation with selective antagonists for EP1 (SC‐19220, 10 μM), EP3 (L‐826266, 1 μM) or EP4 (L‐161982, 1 μM) receptors prevented the PGE₂‐induced decrease of Na⁺,K⁺‐ATPase activity. On the other hand, incubation with the selective EP2 agonist (butaprost, 0.1–10 μM) increased enzyme activity per se in a concentration‐dependent manner, but did not prevent the inhibitory effect of PGE₂. Incubation with a protein kinase A (PKA) inhibitor (H‐89, 1 μM) and a protein kinase C (PKC) inhibitor (GF‐109203X, 300 nM) also prevented PGE₂‐induced decrease of Na⁺,K⁺‐ATPase activity. Accordingly, PGE₂ increased phosphorylation of Ser943 at the α subunit, a critical residue for regulation of enzyme activity. Importantly, we also found that PGE₂ decreases Na⁺,K⁺‐ATPase activity in vivo. The results presented here imply Na⁺,K⁺‐ATPase as a target for PGE₂‐mediated signaling, which may underlie PGE₂‐induced increase of brain excitability.     

Inter‐subunit interaction of gastric H+,K+‐ATPase prevents reverse reaction of the transport cycle

The gastric H⁺,K⁺‐ATPase is an ATP‐driven proton pump responsible for generating a million‐fold proton gradient across the gastric membrane. We present the structure of gastric H⁺,K⁺‐ATPase at 6.5 Å resolution as determined by electron crystallography of two‐dimensional crystals. The structure shows the catalytic α‐subunit and the non‐catalytic β‐subunit in a pseudo‐E₂P conformation. Different from Na⁺,K⁺‐ATPase, the N‐terminal tail of the β‐subunit is in direct contact with the phosphorylation domain of the α‐subunit. This interaction may hold the phosphorylation domain in place, thus stabilizing the enzyme conformation and preventing the reverse reaction of the transport cycle. Indeed, truncation of the β‐subunit N‐terminus allowed the reverse reaction to occur. These results suggest that the β‐subunit N‐terminus prevents the reverse reaction from E₂P to E₁P, which is likely to be relevant for the generation of a large H⁺ gradient in vivo situation.     

Optimization of ammonium acquisition and metabolism by potassium in rice (Oryza sativa L. cv. IR-72)

We present the first characterization of K⁺ optimization of N uptake and metabolism in an NH₄⁺‐tolerant species, tropical lowland rice (cv. IR‐72). ¹³N radiotracing showed that increased K⁺ supply reduces futile NH₄⁺ cycling at the plasma membrane, diminishing the excessive rates of both unidirectional influx and efflux. Pharmacological testing showed that low‐affinity NH₄⁺ influx may be mediated by both K⁺ and non‐selective cation channels. Suppression of NH₄⁺ influx by K⁺ occurred within minutes of increasing K⁺ supply. Increased K⁺ reduced free [NH₄⁺] in roots and shoots by 50–75%. Plant biomass was maximized on 10 mm NH₄⁺ and 5 mm K⁺, with growth 160% higher than 10 mm NO₃^‐‐grown plants, and 220% higher than plants grown at 10 mm NH₄⁺ and 0.1 mm K⁺. Unlike in NH₄⁺‐sensitive barley, growth optimization was not attributed to a reduced energy cost of futile NH₄⁺ cycling at the plasma membrane. Activities of the key enzymes glutamine synthetase and phosphoenolpyruvate carboxylase (PEPC) were strongly stimulated by elevated K⁺, mirroring plant growth and protein content. Improved plant performance through optimization of K⁺ and NH₄⁺ is likely to be of substantial agronomic significance in the world's foremost crop species.