STATISTICAL ASPECTS OF THE INDEPENDENT JOINT ACTION OF POISONS, PARTICULARLY INSECTICIDES

Methods are described for discovering whether a mixture of two poisons is as toxic as predicted on the hypothesis of independent joint action. These include X²tests and a procedure for finding the maximum likelihood estimate of the coefficient of correlation in resistance to the poisons. The methods are illustrated using data from insecticidal tests.
In the insecticidal tests, flour beetles, Tribolium castaneum Herbst, were sprayed with, or exposed to films of, different insecticides in solution in Shell oil P 31. The insecticides were pyrethrins, D.D.T., and B.H.C., singly and in pairs. The statistical analysis of the results showed that pyrethrins and D.D.T. could have acted independently both as films and as direct sprays; D.D.T. and B.H.C. could have acted independently as films but not as direct sprays; and B.H.C. and pyrethrins could not have acted independently either as films or as direct sprays.
These findings are discussed, and it is concluded that independent action should be regarded as a special case of a more general type of joint action for which the term dissimilar is proposed. A general method of approach is suggested for the conception and development of hypotheses of the joint action of poisons.     

A note on the efficiency of a pyrethrum spray in controlling Ephestia elutella Hb. moths in a granary

A stowage in a London granary containing 500 tons of Manitoba wheat was sprayed with 0.8 % w/v pyrethrins in Shell oil P31, in an attempt to control an Ephestia elutella infestation. The mean deposit on the surface of the wheat was 1-5 mg./sq.cm., of which one-sixth was due to the settling of mist.
All the moths in the stowage at the time of spraying, estimated at 68,900, were killed or fatally affected within 24 hr., and those which emerged later, estimated at 7500, were killed by the insecticidal film.
The spray was not observed to have any marked effect on the size of the migrating larval population.     

THE FORMATION OF INSECTICIDAL FILMS ON BUILDING MATERIALS: II. TESTS OF THE EFFICIENCY OF VARIOUS TYPES OF PRETREATMENT

Thirty different pretreatments have been tested, in addition to those mentioned in a previous contribution, for their efficiency in prolonging the effective toxic life of a film of pyrethrins in Shell Oil P 31 deposited on brick. Reasons for the success or failure of the various types of pretreatment are considered. Seven substances selected for further trial were tested on four more building materials—limewashed brick, wood, cement, and cement-sand mixture. The five pretreatments which gave good results on all or most of these substrates were then tested to determine how long they would continue to support a toxic film on cement. Solutions of size and gelatin caused the greatest persistence of toxicity of the pyrethrum films. The flour beetle, Tribolium castaneum Herbst, was used throughout as the test insect.     

The preparation of a standard pyrethrum extract in heavy mineral oil, with observations on the relative toxicities of the pyrethrins in oil and aqueous media

The preparation of a pyrethrum extract in highly refined heavy mineral oil, suitable for use as a basis of reference in the biological evaluation of commercial pyrethrum-heavy oil preparations, is described. The solution was standardized with respect to colour, resin content and equal proportions of the pyrethrins. A standard solution containing pyrocatechol remained stable, when judged by the chemical determination of the pyrethrins, over a period of months.
When applied in a heavy mineral oil medium, pyrethrin II was shown to possess a toxicity to Tribolium castaneum Hbst. approaching, if not equal to, that of pyrethrin 1. When applied in an aqueous medium the toxicity of pyrethrin II was very much lower than that of pyrethrin I.     

The toxicities of three petroleum oils to the grain weevils

The toxicities of three petroleum oils to Calandra granaria L. and C. oryzae L. have been determined. The oils were Shell oil P31, Odourless Distillate (O.D.), and Pool burning oil (P.B.O.). At 20 and 25° C. C. oryzae was more resistant than C. granaria to a direct spray of P31. At 20° C. C. oryzae was more resistant than C. granaria to direct sprays of O.D. and P.B.O. The relative toxicities to both species of direct sprays of the three oils could be expressed as: P.B.O. < P31 ≥ O.D. ≥ P.B.O. C. oryzae was the species more resistant to films of P31 on Whatman no. 544 filter-paper.
Films of P31 or a P31/water emulsion on brick, and films of P31 on sacking and cement, were non-toxic to C. granaria , but films of P31 on cement pretreated with gelatin were highly toxic to this species.
Beetles that received doses of P31 a little in excess of those sufficient to knock them down rarely recovered completely. P31 probably suffocates the beetles by blocking their spiracles and/or tracheae.
P31 appears to be the most useful of the three oils for the control of C. granaria. In practice it should be effective as a direct spray, though not as a film, and could therefore be used to control infestations in which beetles were exposed. Films of P31 on surfaces suitably pretreated might, however, be used for control.     

BIOASSAY SYSTEMS FOR THE PYRETHRINS

The special properties of pyrethrum assays with crawling insects by the film technique lend themselves to the twin cross-over design, hitherto restricted to assays with graded responses. The conditions under which this design is appropriate, and the treatment of control responses, are discussed. The design is particularly efficient with laboratory cultures of insects with fairly stable tolerance levels, and is also useful with heterogeneous populations collected in the field. Examples of the increased precision afforded are considered. The reversibility of the primary responses of grain weevils to the pyrethrins is also demonstrated.     

A MICRO-DROP APPLICATOR AND ITS USE FOR THE TREATMENT OF CERTAIN SMALL INSECTS WITH LIQUID INSECTICIDE

An instrument has been constructed for applying minute drops of liquid insecticide to selected parts of individual insects. The principle of the instrument, which is not original, is to express minute amounts of liquid from a hypodermic syringe, and blow the drop from the needle tip by a puff of air. This model is considered to have advantages over previous models working on the same principle. In particular, a very convenient form of fitting was developed for blowing the drop from the needle tip, and fittings of this type could be incorporated in existing similar applicators with little modification of the latter. The high directional accuracy of dropfire necessary for treating selected parts of small insects was achieved.
An immersion method was developed for determining the sizes of individual drops of Shell oil P₃₁ delivered, and hence the uncontrolled variability of drop-volume. It showed that the standard deviation in the volumes of drops intended to be equal in size was about 9% of a mean volume of 0.017 μ1. falling progressively to about 2% as the mean volume increased to 0.33 μl. These standard deviations, however, are of limited value, because the sizes of successive drops delivered appeared frequently to be correlated. A method in which the drops are delivered on to an oleophobic surface for measurement of the diameters of the oil lenses formed was found insufficiently accurate for determining the relative variability of drops of P_31.
A suction method was devised for manipulating, without anaesthetization or chilling, small beetles such as Calandra and Tribolium. This enabled large numbers to be individually treated with the applicator in a reasonable time. A similar but slower method was used successfully with the larvae of Ephestia. The biological results achieved have been very satisfactory.
The effects of uncontrolled variation in drop size on dose-response data are discussed.     

Physical factors affecting the toxicity of sprays to stored product insects; the quantity of carrier in which a given amount of active ingredient is applied

The problem of whether a given amount of active ingredient is more effectively applied in concentrated or dilute form is discussed. If y is percentage mortality and x ₁ and x ₂ are respectively log-concentration of active ingredient and log-volume (or deposit) of insecticide applied, y may be regarded as a uniform, continuous function of x ₁ and x ₂. The amount of active ingredient is constant, i.e. x ₁+ x ₂= k , so that
dx/dx₁=−dy/dx₂=δy/δx₁−δy/δx₂
Probit mortality, Y , can be substituted for y in (4). Thus, whether an active ingredient is better applied in concentrated or dilute form depends on the relative magnitudes of ∂ y /∂ x ₁ and ∂ y /∂ x ₂, or of ∂ Y /∂ x ₁ and ∂ Y /∂ x ₂. Equation (4) is true whenever an insecticide consists of an active ingredient in a diluent, whatever the dosage-mortality relationship. Previous work is discussed in the light of (4) and its probit form, and it appears that the concentration at which an active ingredient is best applied can depend upon the nature and quantity of the active ingredient, and the method of application of the spray. There may be other facto*** The probit form of (4) is applied to the probit plane and confirmed experimentally. Flour beetles, Tribolium castaneum Herbst were sprayed with pyrethrins in Shell oil P 31, and it was found that ∂ Y /∂ x ₁ > ∂ Y /∂ x ₂, so that a given quantity of pyrethrins was more toxic in concentrated solution than in dilute.     

检查Epstein-Barr病毒IgA/EA抗体的ELISA法

建立了检查鼻咽癌病人血清中IgA/EA抗体的、改进的ELISA法,用巴豆油,正丁酸钠和阿糖胞苷激活并处理HRIK细胞株,使之表达EA抗原,提取抗原时加蛋白酶抑制剂,以增加产量和稳定性;用鼠抗人IgA单克隆抗体和兔抗鼠IgG抗血清的三层夹心法。提高了敏感性,使阳性检出率达到97％,而免疫酶法的阳性率仅60％,所用抗体工作浓度的几何平均稀释度为免疫酶法的8倍,两法抗体滴度的分布呈平行关系,本法适用于大规模现场普查和鼻咽癌的早期诊断,具有快速、特异和敏感等优点。     

Phosphorylation of Parkin at Serine65 is essential for activation: elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity

Mutations in PINK1 and Parkin are associated with early-onset Parkinson''s disease. We recently discovered that PINK1 phosphorylates Parkin at serine65 (Ser⁶⁵) within its Ubl domain, leading to its activation in a substrate-free activity assay. We now demonstrate the critical requirement of Ser⁶⁵ phosphorylation for substrate ubiquitylation through elaboration of a novel in vitro E3 ligase activity assay using full-length untagged Parkin and its putative substrate, the mitochondrial GTPase Miro1. We observe that Parkin efficiently ubiquitylates Miro1 at highly conserved lysine residues, 153, 230, 235, 330 and 572, upon phosphorylation by PINK1. We have further established an E2-ubiquitin discharge assay to assess Parkin activity and observe robust discharge of ubiquitin-loaded UbcH7 E2 ligase upon phosphorylation of Parkin at Ser⁶⁵ by wild-type, but not kinase-inactive PINK1 or a Parkin Ser65Ala mutant, suggesting a possible mechanism of how Ser⁶⁵ phosphorylation may activate Parkin E3 ligase activity. For the first time, to the best of our knowledge, we report the effect of Parkin disease-associated mutations in substrate-based assays using full-length untagged recombinant Parkin. Our mutation analysis indicates an essential role for the catalytic cysteine Cys431 and reveals fundamental new knowledge on how mutations may confer pathogenicity via disruption of Miro1 ubiquitylation, free ubiquitin chain formation or by impacting Parkin''s ability to discharge ubiquitin from a loaded E2. This study provides further evidence that phosphorylation of Parkin at Ser⁶⁵ is critical for its activation. It also provides evidence that Miro1 is a direct Parkin substrate. The assays and reagents developed in this study will be important to uncover new insights into Parkin biology as well as aid in the development of screens to identify small molecule Parkin activators for the treatment of Parkinson''s disease.     

STUDIES ON THE SYNERGISTIC EFFECT OF PIPERONYL BUTOXIDE AND ISOBUTYLUNDECYLENEAMIDE ON PYRETHRINS AND ALLETHRIN

Experiments are described to compare the toxicity of natural pyrethrins and allethrin, the completely synthetic homologue of cinerin I, and to show the effect of the two synergists, piperonyl butoxide and iaobutyl undecyleneamide (IN 930) on both these active ingredients, using a measured-drop technique with Mucca domestiur L. the housefly, and a residual-film technique with Cimex lectularius L. the bed bug. In the conditions of the experiment, pyrethrins were shown to be twice as toxic as allethrin to flies, and 5.5 times as toxic as allethrin to bugs. The two synergists were tested at several ratios to the two insecticides, ranging from 1 : 1 to 20 : 1. The results were plotted as series of log. concentration/probit regression linea. These were parallel for the bug tests; but in the fly tests, the slope of the line increased with a rise in the proportion of Synergist to insecticide. The estimated median lethal concentrations indicated, in all cases, that the toxicity increased with a rise in the ratio of synergist to insecticide, at least up to 20 : 1. However, the enhancement of toxicity was greatest for the smaller ratios and fell off as the ratio increased. Piperonyl butoxide was the more powerful synergist, increasing the potency of pyrethrins 5 times and allethrin 4 times to flies, and pyrethrins twice and allethrin 3 times to bugs, whereas IN 930 did not increase the potency of either ingredient more than twice to either test insect.
The addition of piperonyl butoxide to residual films of pyrethrins greatly prolonged their effectiveness; but an experiment designed to investigate the effect of the synergist on the stability of this insecticide showed that this action, if it exists, must be slight.     

Bioassay systems for the pyrethrins; water-base sprays against Aëdes aegypti L. and other flying insects

A bioassay system for the pyrethrins is discussed in which the quantity of toxicant that flying insects accumulate is measured, as well as the responses of the insects. The determination of spray pick-up from water-base sprays is described, and the use of primary responses, such as activation and paralysis, in biological assays is advocated. The test system is discussed in detail for Aëdes aegypti L. and the special characteristics of some other species of flying insects noted. It is considered that the location of the site of action of the pyrethrins under these conditions is at the peripheral nervous system of the test insects.     

Efficacy and nontarget effects of reduced-risk insecticides on Aphis glycines (Hemiptera: Aphididae) and its biological control agent Harmonia axyridis (Coleoptera: Coccinellidae)

The efficacy of three reduced-risk insecticides (pyrethrins, insecticidal soap, and narrow-range mineral oil) was determined for nymphs and adults of the soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), an exotic pest of North American soybean, Glycine max (L.) Merr. These insecticides also were evaluated for nontarget effects on one of the aphid's key biological control agents, multicolored Asian lady beetle, Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae), including first and third instars, pupae, and adults. A Potter Spray Tower was used to conduct direct spray laboratory bioassays. Results indicated that although pyrethrins and narrow-range mineral oil caused 100% mortality to A. glycines nymphs and adults at 72 h posttreatment, insecticidal soap caused equivalent mortality to only the nymphs during the same time period. However, A. glycines adult mortality due to the insecticidal soap (83.3%) was significantly greater than the control. Pyrethrins were highly toxic to first instars of H. axyridis (98% mortality), but they had no effect on third instars, pupae, or adults. Mineral oil and insecticidal soap were moderately lethal to first (48.9 and 40% mortality, respectively) and third (31.9 and 38.8% mortality, respectively) instars of H. axyridis, but they had no effect on pupae and adults. Our results suggest that pyrethrins, insecticidal soap, and narrow-range mineral oil may prove useful for soybean aphid management in organic soybean due to efficacy against the aphid with differential nontarget effects on select stages of H. axyridis. Additional studies will be necessary to elucidate the efficacy of these insecticides under field conditions.     

A simple cell based assay to measure Parkin activity

Parkin is an ubiquitin-protein ligase mutated in Autosomal Recessive - Juvenile Parkinsonism. Here, we describe a cell-based assay to measure Parkin's ubiquitin-protein ligase activity. It relies on the ability of Parkin to recognise depolarised mitochondria and exploits a cell line where Parkin expression is inducible. In these cells, Parkin expression promotes mitophagy and accelerates cell death in response to mitochondrial depolarisers. Time-lapse imaging confirmed cell death and revealed increased perinuclear mitochondrial clustering following induction of Parkin expression in cells exposed to carbonyl cyanide m-chlorophenylhydrazone. Similar effects were not observed with α-synuclein or DJ-1, other proteins associated with the development of Parkinson's disease, confirming the specificity of the assay. We have used this assay to demonstrate that ligase-defective Parkin mutants are inactive, and cellular proteasomal activity (using the proteasomal inhibitors MG132, clasto-lactacystin β-lactone and epoxomicin) is essential for the Parkin mediated effect. As the assay is suitable for high-throughput screening, it has the potential to identify novel proteostasis compounds that stimulate the activity of Parkin mutants for therapeutic purposes, to identify modulators of kinase activities that impact on Parkin function, and to act as a functional read-out in reverse genetics screens aimed at identifying modifiers of Parkin function during mitophagy.     

Solid-Phase Radioimmunoassay for Substance P

The solid-phase immunoassay for quantification of substance P has been developed. The assay is based on the repartition of anti-substance P antibodies between the insoluble phase-immobilized substance P and the free peptide. The immobilized substance P-antibody complex is then quantified with 125I-protein A. The method allowed detection of 10 pg of substance P. The values of substance P concentration obtained by the present method in different regions of the rat brain were comparable to those obtained by standard radioimmunoassay with 125I-tyr-8-substance P as tracer. The described solid-phase radioimmunoassay is a simple, sensitive, and reliable technique for quantification of substance P-like immunoreactivity in biological samples.     

生物破乳剂产生菌的筛选及其方法研究

针对生物破乳剂产生菌筛选难的问题,采用显色法、溶血细胞测试法、表面张力测定法和排油圈法从6种不同菌源对生物破乳菌产生茵进行了筛选.通过试验筛选得到了17株生物破乳剂产生茵,其中24h内破乳率高于70%的破乳菌有5株;油田含油污泥、采油废水生物处理污泥和污水处理厂剩余污泥是筛选破乳菌的较好的菌源:显色法、溶血圈法存在检测范围的局限性;表面张力测定法和排油圈法是最为简易和准确的生物表面活性剂产生茵的筛选方法,采用模型乳状液对生物破乳剂产生菌进行筛选最为直接和准确,但工作量大、所需时间长,因此在筛选高效破乳菌时,建议采用表面张力、排油圈法进行初筛,而后通过模型乳状液破乳进行验证.     

生物破乳剂产生菌的筛选及其方法研究

针对生物破乳剂产生菌筛选难的问题, 采用显色法、溶血细胞测试法、表面张力测定法和排油圈法从6种不同菌源对生物破乳菌产生菌进行了筛选。通过试验筛选得到了17株生物破乳剂产生菌, 其中24h内破乳率高于70%的破乳菌有5株; 油田含油污泥、采油废水生物处理污泥和污水处理厂剩余污泥是筛选破乳菌的较好的菌源; 显色法、溶血圈法存在检测范围的局限性; 表面张力测定法和排油圈法是最为简易和准确的生物表面活性剂产生菌的筛选方法, 采用模型乳状液对生物破乳剂产生菌进行筛选最为直接和准确, 但工作量大、所需时间长, 因此在筛选高效破乳菌时, 建议采用表面张力、排油圈法进行初筛, 而后通过模型乳状液破乳进行验证。     

A method for identification of inhibitors of the phosphorylation reactions of bacterial response regulator proteins using (31)P nuclear magnetic resonance spectroscopy.

Bacterial response regulators are attractive targets for antibacterial drug development, yet random screening against these targets has failed as yet to identify chemicals that constitute viable leads. Alternative methods to provide leads for drug development based on identification and optimization of low affinity ligands from NMR screens have been described. However, leads from these processes still require verification in a bioassay, which is often problematic if compounds have unfavorable optical and solubility properties. A simple method, based on using NMR to observe the activity of the target, is described. It has the advantages of being able to characterize both low affinity leads and a wider selection of compounds in a structure activity relationships series, without the problems affecting a fluorescence assay. In this example we use (31)P to monitor the turnover of a bacterial response regulator, but the generic approach could be applied to other nuclei and thus a range of biological systems.     

High-performance liquid chromatographic determination of quinine in rat biological fluids

A high-performance liquid chromatographic (HPLC) method with ultraviolet detection for the determination of quinine in rat biological fluids is described. Due to its selectivity and sensitivity, the proposed method can be used in the case of such rat biological fluids as cerebrospinal fluid (CSF) and perilymph for which the accessible volumes are limited to 100 μl and 10 μl, respectively. Consequently, the assay method has been applied to the measurements of quinine concentration in rat plasma, CSF and perilymph samples.     

A comparative kinetic study on human pancreatic and Thermomyces lanuginosa lipases: Inhibitory effects of tetrahydrolipstatin in the presence of lipid substrates

The inhibitory effects of tetrahydrolipstatin (THL) on the hydrolytic activity of human pancreatic lipase (HPL) and T. lanuginosa lipase (TLL) on various lipidic substrates ‘poisoned’ with THL as previously described was studied, using either the pH-stat, monomolecular film or oil drop technique.Prior to adding lipase (method C), an ethanolic solution of THL was injected in a tributyrin (TC4) or a purified soybean oil (PSO) emulsion prepared in a pH-stat vessel. Under these conditions, THL was found to be a potent HPL inhibitor. After being dissolved in the pure triglyceride phase (method D), THL also strongly inhibited HPL. However, with TC4 as substrate TLL was efficiently inhibited by THL only when method C was used and not method D. The very different inhibitory effects on HPL and TLL recorded with method D and PSO as substrate were confirmed using the monomolecular film and oil drop techniques.With a monomolecular film of dicaprin (di-C10) as substrate, 1 molecule of THL embedded in 400 000 molecules of di-C10 sufficed to reduce the HPL activity to half of its initial value.HPL was therefore efficiently inhibited by THL with all the methods and substrates tested here. Paradoxically, TLL was inhibited by THL molecules transiently present in the aqueous phase and not by the THL molecules present at the triglyceride/water interface. It should therefore be stressed that the inhibitory effects of THL on each lipase depend strongly on the method and the substrate used.