Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) is a distinctive member of the serine–threonine protein AGC kinase family and an effective kinase for cAMP signal transduction. In recent years, scuticociliate has caused a lot of losses in domestic fishery farming, therefore, we have carried out morphological and molecular biological studies. In this study, diseased guppies (Poecilia reticulata) were collected from an ornamental fish market, and scuticociliate Philaster apodigitiformis Miao et al., 2009 was isolated. In our prior transcriptome sequencing research, we discovered significant expression of the β-PKA gene in P. apodigitiformis during its infection process, leading us to speculate its involvement in pathogenesis. A complete sequence of the β-PKA gene was cloned, and quantified by quantitative reverse transcription-polymerase chain reaction to analyse or to evaluate the functional characteristics of the β-PKA gene. Morphological identification and phylogenetic analysis based on small subunit rRNA sequence, infection experiments and haematoxylin–eosin staining method were also carried out, in order to study the pathological characteristics and infection mechanism of scuticociliate. The present results showed that: (1) our results revealed that β-PKA is a crucial gene involved in P. apodigitiformis infection in guppies, and the findings provide valuable insights for future studies on scuticociliatosis; (2) we characterized a complete gene, β-PKA, that is generally expressed in parasitic organisms during infection stage and (3) the present study indicates that PKA plays a critical role in scuticociliate when infection occurs by controlling essential steps such as cell growth, development and regulating the activity of the sensory body structures and the irritability system.

The strong adsorption to kaolinite of four polyaromatic, cationic dyes (9-aminoacridine, 3,6-diaminoacridine, azure A and safranin O), which adsorb much less to alumina or silica, was investigated by means of acid-base titrations, measurements of adsorption at varying pH and dye concentration, and by ATR-FTIR spectroscopy. The four dyes adsorb to kaolinite to similar extents, with little change over the pH range 3–10, but at higher pH (above the pKas of the dyes) the adsorption of 9-aminoacridine and 3,6-diaminoacridine decreases, that of azure A increases, and that of safranin O stays approximately constant. Although the dyes adsorb to kaolinite much more strongly than metal ions do, titration and spectroscopic data show that there is only limited chemical interaction between the adsorbed dyes and the kaolinite surface. The results indicate that electrostatic interaction between the dye molecules and the kaolinite surface is necessary for adsorption, but that hydrophobic interactions also contribute. It is proposed that the relatively hydrophobic silica faces of kaolinite, which carry low-density permanent negative charge, facilitate aggregation and adsorption of the positively charged, flat, aromatic dye molecules.

Retinomotor movements include elongation and contraction of rod and cone photoreceptors, and mass migration of melanin-containing pigment granules (melanosomes) of the retinal pigment epithelium (RPE) within the eyes of fish, frogs, and other lower vertebrates. Eyes of these animals do not contain dilatable pupils; therefore the repositioning of the rods and cones and a moveable curtain of pigment granules serve to modulate light intensity within the eye. RPE from sunfish (Lepomis spp.) can be isolated from the eye and dissociated into single cells, allowing in vitro studies of the cytoskeletal and regulatory mechanisms of organelle movement. Pigment granule aggregation from distal tips of apical projections into the cell body can be triggered by the application of underivatized cAMP, and dispersion is effected by cAMP washout in the presence of dopamine. While the phenomenon of cAMP-dependent pigment granule aggregation in isolated RPE was described many years ago, whether cAMP acts through the canonical cAMP-PKA pathway to stimulate motility has never been demonstrated. Here, we show that pharmacological inhibition of PKA blocks pigment granule aggregation, and microinjection of protein kinase A catalytic subunit triggers pigment granule aggregation. Treatment with a cAMP agonist that activates the Rap GEF, Epac (Effector protein activated by cAMP), had no effect on pigment granule position. Taken together, these results confirm that cAMP activates RPE pigment granule motility by the canonical cAMP-PKA pathway. Isolated RPE cells labeled with antibodies against PKA RIIα and against PKA-phosphorylated serine/threonine amino acids show diffuse, punctate labeling throughout the RPE cell body and apical projections. Immunoblotting of RPE lysates using the anti-PKA substrate antibody demonstrated seven prominent bands; two bands in particular at 27 and 64 kD showed increased levels of phosphorylation in the presence of cAMP, indicating their phosphorylation could contribute to the pigment granule aggregation mechanism.

Participation and relative importance of phosphatidylinositol-3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling, either alone or in combination, have been investigated during 17α,20β-dihydroxy-4-pregnen-3-one (DHP)-induced meiotic G2−M1 transition in denuded zebrafish oocyte. Results demonstrate that concomitant with rapid phosphorylation (activation) of Akt (Ser473) and MAPK (ERK1/2) at as early as 15 min of incubation, DHP stimulation promotes enhanced an GVBD response and histone H1 kinase activation between 1 and 5 h in full-grown oocytes in vitro. While p-Akt reaches its peak at 60 to 90 min and undergoes downregulation to the basal level by 240 min, ERK1/2 phosphorylation (activation) increases gradually until 120 min and remains high thereafter. Although, priming with MEK1/2 inhibitor U0126 is without effect, PI3K inhibitors, wortmannin or LY294002, delay the GVBD response significantly (P < 0.001) until 3 h but not at 5 h of incubation. Interestingly, blocking PI3K and MEK function together could abrogate steroid-induced oocyte maturation at all time points tested. While DHP stimulation promotes phospho-PKA catalytic (p-PKAc) dephosphorylation (inactivation) between 30–120 min of incubation, simultaneous inhibition of PI3K and MEK1/2 kinases abrogates DHP action. Conversely, elevated intra-oocyte cAMP, through priming with either adenylyl cyclase (AC) activator forskolin (FK) or dibutyryl cAMP (db-cAMP), abrogates steroid-induced Akt and ERK1/2 phosphorylation. Taken together, these results suggest that DHP-induced Akt and ERK activation precedes the onset of meiosis (GVBD response) in a cAMP-sensitive manner and PI3K/Akt and MEK/MAPK pathways together have a pivotal influence in the downregulation of PKA and resumption of meiotic maturation in zebrafish oocytes in vitro.

The dissociation constant for chlorsulfuron {2-chloro-N-[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino] carbonyl] benzenesulfonamide} in aqueous solution measured by spectrophotometric titration is 3.58 ± 0.05. Chlorsulfuron was more strongly adsorbed on IRA-400-Cl strong anion exchange resin than on IR-4B-OH weak anion exchange resin or Al2O3 anionotropic adsorbent. Hydrogen bonding was probably responsible for the adsorption observed on IR-120-Na(H) cation exchange resin. No chlorsulfuron was adsorbed on Al2O3 cationotropic absorbent, technical montmorillonite, illite, or kaolinite. Adsorption did occur on organic matter derived from a histosol. Chlorsulfuron was strongly adsorbed on activated charcoal but had little affinity for α-cellulose. Adsorption onto hydrophobic polymeric XAD-2 adsorbent at pH 5.2 was not significant for chlorsulfuron concentrations below 30 μM. No significant adsorption occurred on a variety of mineral soils low in organic matter. Adsorption on a Sharpsburg silty clay loam was inversely related to solution pH. Hydrogen bonding and charge transfer bonds were postulated as the major mechanisms responsible for chlorsulfuron adsorption in soil.

Laboratory experiments were conducted to evaluate soil adsorption and mobility of sulfentrazone. Sulfentrazone is a new phenyl triazolinone herbicide intended for use in soybean. Adsorption was evaluated through a soil solution technique, and mobility was evaluated with soil thin-layer chromatography. Experimental variables included soil, sulfentrazone concentration (adsorption study only), and pH. Adsorption was influenced by all experimental variables; however, pH had the greatest effect. Adsorption generally decreased in response to increasing pH. However, the greatest decrease occurred above the pKa of sulfentrazone (i.e., 6.56). Mobility generally reflected adsorption.

Dormant states of organisms are easier to store than the living state because they tolerate larger variations in temperature, light, storage space etc., making them attractive for laboratory culture stocks and also for experiments under special circumstances, especially space flight experiments. Like several other organisms, Euglena gracilis is capable of forming desiccation tolerant resting states in order to survive periods of unfavourable environmental conditions. In earlier experiments it was found that dormant Euglena cells must not become completely desiccated. Some residual moisture is required to ensure recovery of the resting states. To analyse the water demand in recovery of Euglena resting states, cells were transferred to a defined amount of cotton wool (0.5 g). Subsequently different volumes of medium (1, 2, 3, 4, 5, 8, 10 and 20 ml) were added in order to supply humidity; a control was set up without additional liquid. Samples were sealed in transparent 50 ml falcon tubes and stored for 9 months under three different conditions:

• • Constant low light conditions in a culture chamber at 20°C,

• • In a black box, illuminated with short light emitting diode-light pulses provided by joule thieves and

• • In darkness in a black box.

After 9 months, cells were transferred to fresh medium and cell number, photosynthetic efficiency and movement behavior was monitored over 3 weeks. It was found that cells recovered under all conditions except in the control, where no medium was supplied. Transcription levels of 21 genes were analysed with a Multiplex-polymerase chain reaction. One hour after rehydration five of these genes were found to be up-regulated: ubiquitin, heat shock proteins HSP70, HSP90, the calcium-sensor protein frequenin and a distinct protein kinase, which is involved in gravitaxis. The results indicate a transient general stress response of the cells.

The dissipation of trifloxysulfuron in solution has not been evaluated. The objectives of this study were to evaluate the effect of spray carrier pH (pH values of 5, 7, and 9) on dissipation of trifloxysulfuron during a 21-d period and to evaluate the resulting effects on efficacy on Palmer amaranth. ANOVA of half-life values indicated that hydrolysis occurred more rapidly under acidic spray carrier pH with no differences between neutral and alkaline pH levels. Trifloxysulfuron solutions that were applied to Palmer amaranth significantly decreased dry weights compared with nontreated at all pH levels and time intervals. These results indicated that trifloxysulfuron dissipation was most rapid when the spray tank pH was more acidic, but trifloxysulfuron efficacy was not adversely affected by chemical degradation on Palmer amaranth. Data suggest that pesticide applicators may reduce trifloxysulfuron degradation in spray tanks by using neutral or alkaline spray carriers.

The fate of imazamox, imazethapyr, and imazaquin in soil was evaluated at various soil moisture levels and at soil pH levels of 5 and 7. The percentage of each herbicide sorbed, desorbed, dissipated, and metabolized over time was compared. Soil was kept at its field pH (pH 7) or modified to a lower pH (pH 5) and equilibrated to field moisture contents ranging from 0.27 to 0.21 g water/ g soil, at and below the field capacity of the soil. Soil moisture in the range studied did not affect the fate of the herbicides. The percentage of applied herbicide found in soil solution was greatest for imazamox and least for imazaquin (imazamox > imazethapyr > imazaquin) and was greater at pH 7 than at pH 5 for all three herbicides. Over time, less herbicide was in the soil solution and less was desorbed. Metabolism followed the same pattern. Among the herbicides, metabolism followed the following sequence, imazamox > imazethapyr > imazaquin with metabolism greater at pH 7 than at pH 5 for all three herbicides. At pH 7, the half-life for imazamox was 1.4 wk and for imazethapyr was 16 wk, and the estimated half-life for imazaquin was 191 wk.

Sulfentrazone persistence in soil requires many crop rotational restrictions. The sorption and mobility of sulfentrazone play an important role in its soil persistence. Thus, a series of laboratory experiments were conducted to mimic the soil properties of cation and anion exchange with different intermediates. The molecular characterization and ionization shift of sulfentrazone from a neutral molecule to an anion were determined using a three-dimensional graphing technique and titration curve, respectively. Sorption and mobility of 2.6 × 10−5 M 14C-sulfentrazone were evaluated using a soil solution technique with ion exchange resins and polyacrylamide gel electrophoresis, respectively. Solution pH ranged from 4.0 to 7.4. As pH increased, sulfentrazone sorption to an anion resin increased and its sorption to a cation resin decreased. Percent sulfentrazone in solution was pH-dependent and ranged between 0 to 18% and 54 to 88% for the anion and cation resins, respectively. Mobility of sulfentrazone on a 20% polyacryalmide gel resulted in Rf values of +0.02 and +0.39 for pH of 4.0 and 7.4, respectively. A double peak for sulfentrazone was detected in the polyacrylamide gel when the pH (6.0 and 6.8) was near the reported pKa of 6.56. There was no clear interaction for the sorption of sulfentrazone at 1.0 mg kg−1 to Congaree loamy sand or Decatur silty clay loam saturated with either calcium or potassium. Sulfentrazone behavior with the polyacrylamide electrophoresis gels and ion resins indicate the potential for this herbicide to occur as a polar or Zwitter ion. Sulfentrazone was adsorbed by potassium, calcium, and sodium saturated resins and subsequently desorbed using variable pH solutions. The level of sulfentrazone adsorption will vary among soil types and the amount of desorption into solution may be soil cation-dependent.

Spray carrier pH affects the solubility of sulfonylurea herbicides and, therefore, could affect absorption and subsequent translocation of these compounds in weeds. Trifloxysulfuron is a sulfonylurea herbicide developed for POST weed control in cotton, sugarcane, and turfgrass with a pKa of 4.81. The objective of this study was to evaluate the absorption and translocation of foliar-applied 14C-trifloxysulfuron in Palmer amaranth and Texasweed at pH 5, 7, and 9 over a period of 4 to 72 h after treatment (HAT). For absorption, effects of time, species, and pH were significant. Absorption averaged over species and pH increased logarithmically from 4 to 72 HAT. Absorption was greater for Palmer amaranth (88%) than for Texasweed (29%) when averaged over time and pH. Absorption averaged over species and time increased in the order of pH 5 (52%) < pH 9 (60%) = pH 7 (61%). Consequently, this translated into greater translocation of 14C-trifloxysulfuron in Texasweed when sprayed with the higher pH spray solutions. These data indicate that absorption and translocation of trifloxysulfuron in some weed species may be enhanced by increasing the pH of the spray solution by 2 pH units above the pKa.

One of the most important public health problems in the world today is the emergence and dissemination of drug-resistant malaria parasites. Plasmodium falciparum is the causative agent of the most lethal form of human malaria. New anti-malarial strategies are urgently required, and their design and development require the identification of potential therapeutic targets. However, the molecular mechanisms controlling the life cycle of the malaria parasite are still poorly understood. The published genome sequence of P. falciparum and previous studies have revealed that several homologues of eukaryotic signalling proteins, such as protein kinases, are relatively conserved. Protein kinases are now widely recognized as important drug targets in protozoan parasites. Cyclic AMP-dependent protein kinase (PKA) is implicated in numerous processes in mammalian cells, and the regulatory mechanisms of the cAMP pathway have been characterized. P. falciparum cAMP-dependent protein kinase plays an important role in the parasite's life cycle and thus represents an attractive target for the development of anti-malarial drugs. In this review, we focus on the P. falciparum cAMP/PKA pathway to provide new insights and an improved understanding of this signalling cascade.

The acrosome reaction (AR) is a fundamental event for fertilization, which is induced in concert with acrosome reaction-inducing substance (ARIS) and asterosap, both of which are components of starfish egg jelly (EJ). During the AR, a spermatozoon undergoes a series of physiological changes, such as in intracellular cGMP concentration ([cGMP]i), pHi and intracellular Ca2+ concentration ([Ca2+]i). Affinity purification of cGMP-binding protein resulted in the isolation of a regulatory subunit of the cAMP-dependent protein kinase A (PKA), suggesting the involvement of a cAMP-dependent pathway in the AR. By using a cAMP enzyme immunoassay, [cAMP]i was found to increase in starfish spermatozoa when stimulated with ARIS and asterosap. ARIS could also increase the [cAMP]i in the presence of high pH seawater. Pretreatment of spermatozoa with two specific and cell-permeable PKA inhibitors, H89 and KT5720, prevented the induction of the AR in a concentration-dependent manner. These results suggest that PKA activity participates in the induction of the AR with ARIS and asterosap. To investigate this, we have cloned a gene that encodes a regulatory subunit of PKA that had been identified in starfish spermatozoa.

Much computational research aimed at understanding ionizable group interactions in proteins has focused on numerical solutions of the Poisson–Boltzmann (PB) equation, incorporating protein exclusion zones for solvent and counterions in a continuum model. Poor agreement with measured pKas and pH-dependent stabilities for a (protein, solvent) relative dielectric boundary of (4,80) has lead to the adoption of an intermediate (20,80) boundary. It is now shown that a simple Debye–Hückel (DH) calculation, removing both the low dielectric and counterion exclusion regions associated with protein, is equally effective in general pKa calculations. However, a broad-based discrepancy to measured pH-dependent stabilities is maintained in the absence of ionizable group interactions in the unfolded state. A simple model is introduced for these interactions, with a significantly improved match to experiment that suggests a potential utility in predicting and analyzing the acid pH-dependence of protein stability. The methods are applied to the relative pH-dependent stabilities of the pore-forming domains of colicins A and N. The results relate generally to the well-known preponderance of surface ionizable groups with solvent-mediated interactions. Although numerical PB solutions do not currently have a significant advantage for overall pKa estimations, development based on consideration of microscopic solvation energetics in tandem with the continuum model could combine the large ΔpKas of a subset of ionizable groups with the overall robustness of the DH model.

The aspartyl dyad of free HIV-1 protease has apparent pKas of ∼3 and ∼6, but recent NMR studies indicate that the aspartyl dyad is fixed in the doubly protonated form over a wide pH range when cyclic urea inhibitors are bound, and in the monoprotonated form when the inhibitor KNI-272 is bound. We present computations and measurements related to these changes in protonation and to the thermodynamic linkage between protonation and inhibition. The Poisson–Boltzmann model of electrostatics is used to compute the apparent pKas of the aspartyl dyad in the free enzyme and in complexes with four different inhibitors. The calculations are done with two parameter sets. One assigns ε = 4 to the solute interior and uses a detailed model of ionization; the other uses ε = 20 for the solute interior and a simplified representation of ionization. For the free enzyme, both parameter sets agree well with previously measured apparent pKas of ∼3 and ∼6. However, the calculations with an internal dielectric constant of 4 reproduce the large pKa shifts upon binding of inhibitors, but the calculations with an internal dielectric constant of 20 do not. This observation has implications for the accurate calculation of pKas in complex protein environments.

Because binding of a cyclic urea inhibitor shifts the pKas of the aspartyl dyad, changing the pH is expected to change its apparent binding affinity. However, we find experimentally that the affinity is independent of pH from 5.5 to 7.0. Possible explanations for this discrepancy are discussed.