Inhibition of acetylcholinesterase by anisomycin

The antibiotic anisomycin, an inhibitor of protein synthesis in eucaryotic cells, which blocks long-term memory in mice, is shown to interact with the cholinergic system by inhibiting reversibly the acetylcholinesterase. The inhibition is a competitive one, the inhibition constant K_i being 5.0 × 10^?3 for human brain acetylcholinesterase and 1.7 × 10^?3 for acetylcholinesterase of bovine erythrocytes. The anisomycin effect on acetylcholinesterase is compared with the puromycin and cycloheximide-inhibition of the enzyme. The significance of the cholinergic effect of anisomycin in addition to its inhibitory effect on protein synthesis for the interpretation of memory experiments is discussed.     

The influence of temperature, relative humidity and host factors on the attachment and survival of Boophilus microplus (Canestrini) larvae to skin slices

Attachment of B. microplus larvae was examined using slices (0.5 mm) of bovine skin stretched over a suitable medium. Optimal temperature for attachment lay between 31 and 38°C. At 38°C, 70 to 80% had attached by 4 h but this was followed 6–7 h after the release of the larvae by a significant decrease in the percentage attached. By 8 h, the percentage attached had risen again and remained at 70–80% for the subsequent 16 h. Attachment was not influenced by ambient relative humidity within the range 20–75 % on the time scale studied (16 h). Most larvae denied access to the skin surface at 20 or 45 % R.H. (38°C) died within 24 h. Those allowed to feed survived at 45% but most died at 20% R.H. There was no difference in attachment when bovine or rabbit serum or phosphate buffered saline was used, nor when skin was taken from Zebu × European cattle or Herefords. Attachment was no different when skin was taken from neck, rib or rump, or from cattle with different levels of resistance to B. microplus but there was reduced attachment with mouse skin. Attachment was reduced on skin which had been stored at ? 15°C for 1 day but storage at 10°C for 16 h had no effect. These results are related to problems of tick water balance, host specificity and stimuli for tick attachment.     

Modified Adamek's medium renders high yields of Metarhizium robertsii blastospores that are desiccation tolerant and infective to cattle-tick larvae

Blastospores are yeast-like cells produced by entomopathogenic fungi that are infective to arthropods. The economical feasible production of blastospores of the insect killing fungus Metarhizium spp. must be optimized to increase yields. Moreover, stabilization process is imperative for blastospore formulation as a final product. In this sense, our goal was to increase blastospore production of two Metarhizium isolates (ESALQ1426 and ESALQ4676) in submerged liquid cultures. A modified Adamek's medium was supplemented with increased glucose concentrations and the fermentation time was accelerated by using a blastospore pre-culture as inoculum. Virulence of air-dried stable blastospores was compared with conidia toward larvae of the cattle tick, Rhipicephalus microplus. Our results revealed that blastospore production of Metarhizium is isolate- and species-dependent. Glucose-enriched cultures (140 g glucose/L) inoculated with pre-cultures improved yields with optimal growth conditions attained for Metarhizium robertsii ESALQ1426 that rendered as high as 5.9 × 10⁸ blastospores/mL within 2 d. Resultant air-dried blastospores of ESALQ1426 were firstly proved to infect and quickly kill cattle tick larvae with comparable efficiency to conidia. Altogether, we argue that both osmotic pressure, induced by high glucose titers, and isolate selection are critical to produce high yields of blastospores that hold promise to control cattle-tick larvae.     

Enzymatic synthesis of [U-14C] ribose-labeled inosine.

A very potent anticholinesterase compound, 7-(diethoxyphosphinyloxy)-N-methylquinolinium fluorosulfate, has been used to determine the normality of acetylcholinesterase solutions. The inhibitor reacts rapidly and completely with acetylcholinesterase. The bimolecular rate constant is 2.5 × 10⁸m^?1 min^?1 and the equilibrium constant is about 10⁶. The reaction produces an inactive diethylphosphoryl enzyme in which the active serine is phosphorylated. The reaction produces the highly fluorescent 1-methyl-7-hydroxyquinolinium dipolar ion as a leaving group. The inhibited enzyme is quite stable and hydrolyzes to produce active enzyme only at the rate of 0.04%/min. The inhibitor was used in two ways for measuring the normality of acetylcholinesterase solutions: (1) The very fast reaction of the inhibitor with cholinesterase makes it convenient to determine the normality of enzyme solutions by measuring the decrease in enzyme activity caused by the addition of an accurately known quantity of the inhibitor. (2) The highly fluorescent nature of the leaving group makes it possible to measure the low concentration that is produced by the reaction of excess inhibitor with the enzyme. The two methods yielded activities per site of 6.9 × 10⁵ min^?1 and 7.3 × 10⁵ min^?1 using enzyme normalities of 1–2 × 10^?8m and 1–5 × 10^?m, respectively, using a commercial 11 S enzyme preparation from electric eel and acetylthiocholine as the enzyme substrate.     

The carbon dioxide hydration activity of brush-border carbonic anhydrase from the dog kidney

The steady-state kinetic parameters for the hydration of CO₂ catalyzed by membrane-bound carbonic anhydrase from the renal brush-border of the dog are compared with the same parameters for water-soluble bovine erythrocyte carbonic anhydrase. For the membrane-bound enzyme, the turnover number k_cat is 6.5 × 10⁵ s^?1 and the Michaelis constant is 7.5 mm for CO₂ hydration at pH 7.4 and 25 °C. The corresponding constants for bovine carbonic anhydrase under these conditions are 6.3 × 10⁵ s^?1 and 15 mm (Y. Pocker and D.W. Bjorkquist (1977)Biochemistry16, 5698–5707). The rate constant for the transfer of a proton between carbonic anhydrase and buffer was determined from the dependence of the catalytic rate on the concentration of the buffers imidazole and N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (Hepes); the value of 2 × 10⁸m^?1s^?1 describes this constant for both forms of carbonic anhydrase at pH 7.4. Furthermore, the pH dependence of the initial velocity of hydration of CO₂ in the range of pH 6.5 to 8.0 is identical for the membrane-bound and soluble enzyme at low buffer concentration (1–2 mm imidazole). We conclude that the membrane plays no detectable role in affecting the CO₂ hydration activity and that the active site of the renal, membrane-bound carbonic anhydrase is exposed to the aqueous phase.     

Toxicity of Khaya senegalensis (Meliaceae) Seed Oil Against the Larvae of Boophilus decoloratus: A One-Host Tick in Cattle

The in vitro toxicity of seed oil of Khaya senegalensis (family: Meliaceae) was tested against the larvae of a one host tick, Boophilus decoloratus (family: Ixodidae or hard tick) known as ‘blue tick’ parasitic mainly to cattle commonly found in savannas of tropical equatorial Africa. The 20, 40, 60, 80, and 100 % concentrations of seed oil were found to kill all (100 % mortality) the larvae after 28, 28, 24, 20 and 20 h respectively.     

A laboratory evaluation of the pathogenicity of Bacillus popilliae var. rhopaea, the agent of milky disease in Rhopaea verreauxi (Coleoptera: Scarabaeidae)

Two methods of infection, i.e., feeding known numbers of spores and rearing larvae in contaminated peat, were used to bioassay the susceptibility of Rhopaea verreauxi to Bacillus popilliae var. rhopaea at 23°C. The susceptibility of the three larval instars was similar as measured by the ID₅₀ and IC₅₀ values. However, within an instar, newly molted larvae were less susceptible than mature larvae when infected by the contaminated peat method. It is suggested that this was due to reduced food intake. The range of ID₅₀ values for all bioassays with R. verreauxi larvae were 1.1 × 10⁷ to 4.0 × 10⁷ spores per larva, and IC₅₀ values were 3.4 × 10⁶ to 5.0 × 10⁷ spores per g of contaminated peat. The slope of the probit line was always low (0.6 to 1.8) except for young first-instar larvae infected by contaminated peat when the slope was 4.0. Disease per se did not affect food intake, though intake was reduced at high doses of contaminated peat. Young larvae often died without developing symptoms but, with increasing age, infected larvae were more likely to develop symptoms. Bioassays with Othnonius batesi and Rhopaea morbillosa indicated a much lower susceptibility per os than for R. verreauxi. It is concluded that the potential for using B. popilliae var. rhopaea to control R. verreauxi is high, but the bacillus is unlikely to be of value in control of O. batesi or R. morbillosa.     

The effect of density on the survival of BoophilusMicroplus on previously unexposed cattle

Previously unexposed ‘naive’ cattle were infested with between 500 and 20,000 larvae of the tick Boophilus microplus, to observe whether density dependent mortality of the tick occurs on cattle which have not acquired resistance. Similar proportions of larvae matured at different densities, which contrasts with previous findings of increased mortality at higher densities on cattle with acquired resistance to B. microplus. Major differences were observed between the proportions of larvae maturing in different experiments.     

The in vivo production of Bacillus popilliae var. rhopaea spores

The effect of various factors on the yield of Bacillus popilliae var. rhopaea spores formed in Rhopaea verreauxi larvae have been studied. Lack of adequate food, temperatures above and below 23°C, and infecting doses above 10⁶ spore larva, all significantly lowered spore yield per larva. Larval age had a pronounced effect; second-instar and young third-instar larvae produ ed about 1 × 10¹⁰ spores while old third-instar larvae produced about 4 × 10¹⁰ spores. Incubation of larvae for longer than 4 weeks did not increase spore yield per larva. Yields were similar whether larvae were infected by injection or per os. Three other host species could be used to mass-produce B. popilliae var. rhopaea spores but all were less efficient than R. verreauxi. Milky third-instar R. verreauxi larvae, which were field collected, yielded 1.57 × 10¹⁰ spores per larva.     

Heliothis zea larval mortality time from topical and per os dosages of Nomuraea rileyi conidia

Heliothis zea larval mortality time was established for topical applications and per os (feeding) with fungal conidia of Nomuraea rileyi. The 6-to 7-day-old larvae received per os dosages ranging from 6 × 10⁶ to 9 × 10⁹ and a topical dosage of ca. 1 × 10⁹. These spore loads initiated fungal infections in larvae resulting in 1.5–100% accumulated mortality during the 30-day testing periods. The data indicated that regardless of the larval treatment method (topical or per os), or conidial dosage rates, the larval mortality time was never shorter than 5 days post-treatment, nor longer than 22 days. The highest percentage of larval deaths occurred from 7 to 9 days with a maximum mortality at 8 days.     

A comparison of the effects of ionic strength on three preparations of acetylcholinesterase in the presence and absence of gallamine

The kinetic behaviour of three forms of acetylcholinesterase as a function of ionic strength of the medium was investigated. The forms of enzyme were that bound to human erythrocyte membranes, acetylcholinesterase solubilized from these by Triton X-100, and a commercial preparation of the enzyme from bovine erythrocytes. The properties investigated were hydrolysis of the substrate acetylthiocholine, decarbamylation of dimethylcarbamyl-acetylcholinesterase and ageing of isopropylmethylphosphonyl-acetylcholinesterase. The effect of 10^?5 M gallamine triethiodide on these properties was also examined as a function of ionic strength.Detailed results for the variation of kinetic behaviour with ionic strength and the presence of gallamine are presented. No unified theory to predict the influence of these variables on all three forms of the enzyme could be formulated. Thus, the enzyme conformation stabilized by gallamine at low ionic strength was not necessarily similar to that of the gallamine-free enzyme at physiological ionic strength. Nor was it useful to consider the free enzyme at low ionic strength to be a model of the membrane-bound enzyme in vivo (Crone, 1973).It was concluded that kinetic results for solubilized and partially or wholly purified acetylcholinesterase cannot be extrapolated to the membrane-bound enzyme. Prediction of the effect of drugs on the system in vivo requires the use of the membrane-bound enzyme.     

Glucose metabolism in the cattle tick, Boophilus microplus

The contributions of the glucose catabolic pathways in different stages of the life cycle of the cattle tick, Boophilus microplus, were estimated on the basis of rates of ¹⁴CO₂ production from specifically labelled glucose. In free-living larvae the pentose cycle accounted for 20 per cent of the total glucose catabolism. In female ticks in the final 24 hr of parasitic feeding and in the pre-oviposition period the pentose cycle proportion rose to over 50 per cent and returned in the final stage of oviposition to 20 per cent. These changes are attributed to the importance of the pentose cycle in biosynthetic processes which must reach their peak during vitellogenesis in the pre-oviposition period.     

There is an energy cost for catalytic turnover which arises due to enzyme degradation

A method for calculating the energy cost per catalytic turnover which results from enzyme synthesis and degradation is presented. Applying the method to published data on 20 rat liver enzymes yields values ranging from 9.48 × 10^?8 to 5.56 ATP per catalytic turnover. The majority of the values are between 10^?5 and 10^?2 ATP per turnover. A possible role for the expenditure of this energy in regulating enzyme activity in particular cases is discussed.     

Inhibition of AChE by malathion and some structurally similar compounds

Inhibition of bovine erythrocyte acetylcholinesterase (free and immobilized on controlled pore glass) by separate and simultaneous exposure to malathion and malathion transformation products which are generally formed during storage or through natural or photochemical degradation was investigated. Increasing concentrations of malathion, its oxidation product malaoxon, and its isomerisation product isomalathion inhibited free and immobilized AChE in a concentration-dependent manner. K_I, the dissociation constant for the initial reversible enzyme inhibitor-complex, and k₃, the first order rate constant for the conversion of the reversible complex into the irreversibly inhibited enzyme, were determined from the progressive development of inhibition produced by reaction of native AChE with malathion, malaoxon and isomalathion. K_I values of 1.3 × 10^{? 4} M^{? 1}, 5.6 × 10^{? 6} M^{? 1} and 7.2 × 10^{? 6} M^{? 1} were obtained for malathion, malaoxon and isomalathion, respectively. The IC₅₀ values for free/immobilized AChE, (3.7 ± 0.2) × 10^{? 4} M/(1.6 ± 0.1) × 10^{? 4}, (2.4 ± 0.3) × 10^{? 6}/(3.4 ± 0.1) × 10^{? 6} M and (3.2 ± 0.3) × 10^{? 6} M/(2.7 ± 0.2) × 10^{? 6} M, were obtained from the inhibition curves induced by malathion, malaoxon and isomalathion, respectively. However, the products formed due to photoinduced degradation, phosphorodithioic O,O,S-trimethyl ester and O,O-dimethyl thiophosphate, did not noticeably affect enzymatic activity, while diethyl maleate inhibited AChE activity at concentrations > 10 mM. Inhibition of acetylcholinesterase increased with the time of exposure to malathion and its inhibiting by-products within the interval from 0 to 5 minutes. Through simultaneous exposure of the enzyme to malaoxon and isomalathion, an additive effect was achieved for lower concentrations of the inhibitors (in the presence of malaoxon/isomalathion at concentrations 2 × 10^{? 7} M/2 × 10^{? 7} M, 2 × 10^{? 7} M/3 × 10^{? 7} M and 2 × 10^{? 7} M/4.5 × 10^{? 7} M), while an antagonistic effect was obtained for all higher concentrations of inhibitors. The presence of a non-inhibitory degradation product (phosphorodithioic O,O,S-trimethyl ester) did not affect the inhibition efficiencies of the malathion by-products, malaoxon and isomalathion.     

The reproduction potentials of four entomopathogenic nematode strains related to cost-effective production for biological control

Bioassays to evaluate the mortality, virulence and reproduction potentials of four indigenous EPN strains, S-PQ16, S-BM12, H-KT3987 and H-CB3452 on insect larvae of mealworm (Tenebrio molitor) and greater wax moth (Galleria mellonella) revealed the highest mortality rates of two insect larvae at the highest inoculation dose of 100 IJs to range from 89 to 100 percent and 94.3–100 percent at 48 h after inoculation, respectively. Virulence was high for all nematode strains, with LC₅₀ values between 29.6 and 47.3 IJs/insect host. The highest IJ yields were different between nematode strains and insect host, from 66.8 × 10³ IJs (S-PQ16) to 118.6 × 10³ IJs (H-KT3987) on T. molitor, and from 54.2 × 10³ IJs (S-BM12) to 163.3 × 10³ IJs (H-KT3987) on G. mellonella. The culturing cost in terms of food expenditure for rearing insect larvae varied between insect larvae and nematode strains, from 6.76 to 26.63 USD per billion IJs for nematode strains cultured on T. molitor larvae and from 3.54 to 7.81 USD per billion IJs for nematode strains cultured on G. mellonella larvae. The full cost for a nematode product of 2.5 × 10⁹ IJs per hectare, produced through in vivo mass culturing, of the most efficient nematode strain, H-KT3987, was 191.3 USD, slightly cheaper than 199.4 USD for the same nematode product produced through in vitro mass culturing.     

Microbial control of the fruit tree leafroller,Archips argyrospila [Lep.: Tortricidae] in California

Field populations of larvae of the fruit tree leafrollerArchips argyrospila (Wlk). were practically eliminated following spray application ofBacillus thuringiensis Berliner serotype III at 192 and 80×10⁶ I.U. per litre on the host trees,Cercis occidentalis. Spray applications of lower rates ofB. thurienginsis serotype III at 18.9 and 32.1×10⁶ I.U. per litre and mist application ofB. thuringiensis serotype 1 at 8.0 and 16.0×10⁶ I.U. per litre gave partial control of populations ofA. argyrospila larvae. A granulosis typeBaculovirus, applied by hand sprayer at 1.4×10⁹ granules/ml produced approximately 50% reduction of 5th instarA. argyrospila larvae onC. occidentalis trees. It was concluded thatB. thuringiensis and the granulosis typeBaculovirus are promising control agents forA. argyrospila larvae.     

Observations on the morphology and infectivity for cattle of Babesia bovis parasites in unfed Boophilus microplus larvae after incubation at various temperatures

Dalgliesh R. J. and Stewart N. P. 1979. Observations on the morphology and infectivity for cattle of Babesia bovis parasites in unfed Boophilus microplus larvae after incubation at various temperatures. International Journal for Parasitology9: 115–120. The temperature of incubation of unfed Boophilus microplus larvae infected with Babesia bovis influenced the morphology and infectivity of the Babesia within the tick. Incubation at 37°C for 1–3 days stimulated the development of parasites morphologically similar to those usually observed in fed larvae harvested from cattle; similar forms appeared more slowly in larvae incubated at 31°C or 25°C. Extracts prepared from larvae after incubation at 37°C for 3–5 days or 30°C for 8 days were consistently infective for cattle. Prior storage of larvae at 14°C for up to 28 days enhanced the development of infectivity at 37°C; infectivity could still be produced after 65 days storage at 14°C but not after 76 days. Larvae released on a host transmitted B. bovis sooner if they had been incubated at 37°C for 4 days. It was concluded that the development of B. bovis to an infective stage in B. microplus is temperature dependent and does not require the stimulus of feeding by the host.     

Abundance estimation of <Emphasis Type="Italic">Ixodes</Emphasis> ticks (Acari: Ixodidae) on roe deer (<Emphasis Type="Italic">Capreolus capreolus</Emphasis>)

Despite the importance of roe deer as a host for Ixodes ticks in central Europe, estimates of total tick burden on roe deer are not available to date. We aimed at providing (1) estimates of life stage and sex specific (larvae, nymphs, males and females, hereafter referred to as tick life stages) total Ixodes burden and (2) equations which can be used to predict the total life stage burden by counting the life stage on a selected body area. Within a period of 1½ years, we conducted whole body counts of ticks from 80 hunter-killed roe deer originating from a beech dominated forest area in central Germany. Averaged over the entire study period (winter 2007–summer 2009), the mean tick burden per roe deer was 64.5 (SE ± 10.6). Nymphs were the most numerous tick life stage per roe deer (23.9 ± 3.2), followed by females (21.4 ± 3.5), larvae (10.8 ± 4.2) and males (8.4 ± 1.5). The individual tick burden was highly aggregated (k = 0.46); levels of aggregation were highest in larvae (k = 0.08), followed by males (k = 0.40), females (k = 0.49) and nymphs (k = 0.71). To predict total life stage specific burdens based on counts on selected body parts, we provide linear equations. For estimating larvae abundance on the entire roe deer, counts can be restricted to the front legs. Tick counts restricted to the head are sufficient to estimate total nymph burden and counts on the neck are appropriate for estimating adult ticks (females and males). In order to estimate the combined tick burden, tick counts on the head can be used for extrapolation. The presented linear models are highly significant and explain 84.1, 77.3, 90.5, 91.3, and 65.3% (adjusted R ²) of the observed variance, respectively. Thus, these models offer a robust basis for rapid tick abundance assessment. This can be useful for studies aiming at estimating effects of abiotic and biotic factors on tick abundance, modelling tick population dynamics, modelling tick-borne pathogen transmission dynamics or assessing the efficacy of acaricides.     

Neutron scattering studies of escherichia coli tyrosyl-trna synthetase and of its interaction with trna tyr

Small-angle neutron scattering studies of Escherichia coli tyrosyl-tRNA synthetase indicate that in solution this enzyme is a dimer of M_r, 91 (±6) × 10³ with a radius of gyration R_G of 37.8 ± 1.1 Å.The increase in the scattering mass of the enzyme upon binding tRNA^Tyr has been followed in 20 mm-imidazole · HCl (pH 7.6), 10 mm-MgCl₂, 0.1 mm-EDTA, 10 mm-2-mercaptoethanol, 150 mm-KCl. A stoichiometry of one bound tRNA per dimeric enzyme molecule was found. The R_G of the complex is equal to 41 ± 1 Å. Titration experiments in 74% ²H₂O, close to the matching point of tRNA, show an R_G of 38.5 ± 1 Å for the enzyme moiety in the complex. From these values, a minimum distance of 49 Å between the centre of mass of the bound tRNA and that of the enzyme was calculated.In low ionic strength conditions (20 mm-imidazole-HCl (pH 7.6), 10 mm-MgCl₂, 0.1 mm-EDTA, 10 mm-2-mercaptoethanol) and at limiting tRNA concentrations with respect to the enzyme, titrations of the enzyme by tRNA^Tyr are characterized by the appearance of aggregates, with a maximum scattered intensity at a stoichiometry of one tRNA per two enzyme molecules. At this point, the measured M_r and R_G values are compatible with a compact 1:2, tRNA: enzyme complex. This complex forms with a remarkably high stability constant: (enzyme:tRNA:enzyme)/(enzyme:tRNA)(enzyme) of 0.1 to 0.3(× 10⁶) m^?1 (at 20 °C). Upon addition of more tRNA, the complex dissociates in favour of the 1:1, enzyme:tRNA complex, which has a higher stability constant (1 to 3 (× 10⁶) m^?1).     

Factors affecting pathogenicity of NPV preparations to the corn earworm,Heliothis armigera

Pathogenicity ofHeliothis nuclear polyhedrosis virus (HSNPV) to the corn earworm,Heliothis armigera, was studied using 3 different inoculative methods. The LD50 values of 4^th-instar larvae inoculated with corn-fed, diet-fed and inoculum-imbiding method were 1.85×10⁶, 2.55×10⁵ and 1,22×10³ PIBs/larva, respectively. The inoculum-imbiding is more sensitive and convenient for inoculatingH. armigera with HSNPV. The HSNPV product, Elcar^®, was highly pathogenic toH. armigera, the LD50 values of 2^nd-, 3^rd- and 4^th-instar larvae being 27, 83 and 1,221 PIBs/larva, respectively, as measured by the inoculum-imbiding method. The mortality of 4^th-instar larvae caused by HSNPV was increased, but the incubation period was shortened with higher incubation temperatures. However, the high temperature at 35°C caused a lower mortality, and a prolongation of the median lethal time (LT50). Stability and persistence of HSNPV preparations were better in January–February and April–May than in June–July and October–November periods when sprayed on corn silks under field conditions. The HSNPV was inactivated by weak alkaline dew (pH 8.1) collected from soybean leaves, but it remained active on those from corn, tomato and asparagus with pH 7.2–7.3. The artificial heavy rainfall of 242 mm/h for 30 min did not wash off HSNPV preparations sprayed on the corn silks.