Enzymatic bioremediation of organophosphorus insecticides by recombinant organophosphorous hydrolase

The gene for organophosphorous hydrolase (OPH) (GenBank accession no. M20392) was chemically synthesised with a codon bias toward E. coli, followed by its cloning and heterologous over-expression in E. coli under induction with 0.1 M isopropyl-d-thiogalactopyranoside (IPTG). The protein was localised in the membrane fraction and no amount of the expressed protein was soluble, thus hindering its purification and further downstream utility. The expressed enzyme was solubilised by mild treatment with ionic detergent [sodium dodecyl sulphate (SDS 1.0% (w/v))]. An innovative step of incubation at 4 °C was used to precipitate SDS, resulting in catalytically active OPH in the supernatant and detergent at the bottom. This solubilised, SDS-free, recombinant OPH was able to detoxify parathion and methylparathion ranging between 10-80% and 3.6-45% in enzyme reaction cycles after immobilization on Ca-alginate and agar-agar, respectively.     

Determination of L-carnitine by flow injection analysis with NADH fluorescence detection

A flow injection analysis method for determining L-carnitine is reported. The system uses the enzyme L-carnitine dehydrogenase covalently immobilized to Eupergit C. The NADH produced by the action of the enzyme, which is proportional to the L-carnitine concentration, is quantified using fluorescence detection. The system response was rapid and had a wide range of linearity. At a flow rate of 0.2 ml/min, a detection limit of 1 microM (20 pmol) was obtained for L-carnitine, peak areas were linear up to 100 microM, and samples could be injected every 4 min. The method performed well as a routine assay, showing high sensitivity (54,000 AU/microM), a precision of 0.96%, and the ability to carry out 144 consecutive assays with an RSD of 1.47% (good stability). Comparisons were made with other known methods for L-carnitine determination. Presence of D-carnitine had no effect on L-carnitine assay. The analysis was valid for determining L-carnitine concentrations in commercial pharmaceutical preparations.     

Determination of oxytetracycline by flow injection with chemiluminescence detection.

A flow-injection chemiluminescent method for the determination of oxytetracycline was developed. The method is based on an enhancement by oxytetracycline of the chemiluminescence light emission of tris(2,2'-bipyridine) ruthenium (II), generated by the continuous oxidation of tris(2,2'-bipyridine) ruthenium (II) by cerium (IV) sulphate in sulphuric acid. Under the optimum conditions, the calibration curve was linear over the range 1.0 x 10(-7)-1.0 x 10(-5) g/mL for oxytetracycline with the linear equation: DeltaINT = 148.77 x C + 0.6637 (R2 = 0.9994). The detection limit was 4.52 x 10(-8) g/mL. The proposed method was also successfully used to determine oxytetracycline in pharmaceutical formulations. The mean recovery of determination of oxytetracycline was 92.73%. A mechanism for the chemiluminescence enhancement by oxytetracycline of tris(2,2'-bipyridine)-ruthenium (II) and cerium (IV) sulphate system is also proposed.     

Biosensors and flow injection analysis.

Combining flow injection analysis with a biosensor is a novel biosensing process which has allowed speedy and accurate analysis. Diagnostic analysis is the most important application for biosensing flow injection analysis, but other applications include bioprocess monitoring, analysis of food and agricultural products, as well as environmental analysis. In addition, the analysis of compounds, such as explosives and abused drugs, and monitoring of Salmonella, the microorganism that causes food poisoning, have been reported.     

Mechanism of inhibition of cyclo-oxygenase in human blood platelets by carbamate insecticides.

Carbamates are a widely used class of insecticides and herbicides. They were tested for their ability to affect human blood platelet aggregation and arachidonic acid metabolism in platelets. (1) The herbicides of the carbamate type have no, or only little, influence up to a concentration of 100 microM; the carbamate insecticides, however, inhibit both aggregation and arachidonic acid metabolism in a dose- and time-dependent manner. (2) Carbaryl, the most effective compound, inhibits platelet aggregation and cyclo-oxygenase activity completely at 10 microM. The liberation of arachidonic acid from phospholipids and the lipoxygenase pathway are not affected, whereas the products of the cyclo-oxygenase pathway are drastically decreased. (3) By using [14C]carbaryl labelled in the carbamyl or in the ring moiety, it could be proved that the carbamyl residue binds covalently to platelet proteins. In contrast with acetylsalicylic acid, which acetylates only one protein, carbaryl carbamylates a multitude of platelet proteins. (4) One of the carbamylated proteins was found to be the platelet cyclo-oxygenase, indicating that carbaryl resembles in this respect acetylsalicylic acid, which is known to inhibit this enzyme specifically by acetylation.     

Fluorometric determination of urea by flow injection analysis.

Urea was determined using fluorometry with flow injection analysis. O-phthalaldehyde (OPA) reacts with enzymatically generated ammonia and sulfite in alkaline medium to give a highly fluorescent compound that has an excitation wavelength of 372 nm and an emission wavelength of about 430 nm. The method is more selective to ammonia than the one which uses mercaptoethanol in place of the sulfite. Urease was immobilized to a Pall Immunodyne membrane which is commercially available. The immobilization occurs through covalent bonding which results in a highly stable enzyme preparation. The enzymatic membrane was fitted in a 5 cm long, 0.125 inch o.d. Teflon tubing which served as the enzymatic reactor. The system is difficult to use for the analysis of urea in serum because some compounds normally present in serum fluoresce at the same wavelength. This results in higher values for urea. If the reaction system is to be used for the evaluation of urea in serum, a blank should be run so that urea concentration can be calculated by difference.     

Determination of ammonium and L-Glutamine in hybridoma cell cultures by sequential flow injection analysis

A flow injection anlytical system based on a gas diffusion membrane module for ammonia and an ammonium flow-through potentiometric detector has been set up for measurement of L-glutamine and ammonium ions in hybridoma cell cultures. The main feature of the system is that the same basic analytical concept and equipment is used in both measurements, the only difference being for the determination of L-glutamine, in which the sample flows through an immobilized glutaminase cartridge. The conditions to enable the performance of both analysis consecutively, avoiding potential interferences by unwanted deamination of other compounds in the samples, have been determined. Finally, the proposed system has been compared with reference analytical methods for batch hybridoma cell culture experiments.     

Determination of beta-D-glucose using flow injection analysis and composite-type amperometric tubular biosensors

An amperometric tubular cell involving composite biosensors for the determination of beta-d-glucose in a flow injection analysis (FIA) system is proposed. Diverse configurations and parameters are evaluated to improve the system's response. The configuration producing less noise resulted when the biosensor was located closer to the auxiliary electrode, which also required coupling both electrodes within the system under a continuous flow regime. Further, we report on the influence of the active area of the biosensor and of the flow rate used. Statistical analyses of the data revealed two regions with a linear response range for the determination of beta-d-glucose, with a detection limit of 4.7 x 10(-4) M and in the low concentration region a sensitivity of 17.46 +/- 0.12 microAM(-1). At the beta-d-glucose concentrations studied there was no evidence of enzymatic saturation. An increment on the ionic strength of the sample and carrier passing through the analysis system decreases its sensitivity. The reproducibility of the analytical system in terms of its standard deviation was 2.9% with a 95% confidence level, having a lifetime that lasted at least 100 days. beta-d-Glucose was determined in different commercial medical glucose-containing solutions, the experimental results are in good agreement with those reported by the manufacturer.     

有机磷农药对韭菜虫害的防治效果及农药的微生物降解

采用3．00kg(a．i)·hm^-2辛硫磷和2．63kg(a．i)·hm^-2甲基对硫磷来防治韭菜蓟马。3d后与不施农药的对照相比,虫口减退率分别达到98．28％和98．39％;20d后虫口减退率分别达到89．94％和94．04％．用浓度分别为15．00、18．00和21．00kg(a．i)·hm^-2的辛硫磷来防治韭蛆,3d后与不施农药的对照相比,虫口减退率分别达到了80．77％、93．10％和96．98％;35d后虫口减退率分别达到了92．44％、95．0596和96．81％．施用降解菌剂对防治蓟马和韭蛆没有不良影响,但可显著降低韭菜中的农药含量．蓟马施药防治后3d喷施45．00L·hm^-2的降解菌剂,3d后与不施菌对照相比,辛硫磷和甲基对硫磷的降解率分别为99．52％和98．83％;20d后韭菜(苔)中农药含量均检测不出．韭蛆施药防治后3d灌施75．00L·hm^-2降解菌剂,35d后不同辛硫磷施用量的降解率分别为100％、100％和99．69％．     

Determination of hydrogen peroxide in exhaled breath condensate by flow injection analysis with fluorescence detection

An improved and simplified high-performance liquid chromatographic (HPLC) method at UV detection 265 nm is presented for the determination of d4T in rat plasma. The mobile phase consists of methanol-distilled water-acetic acid in the 23:77:0.2 (v/v) ratio. Quantification is achieved by the peak-area ratio method with reference to the internal standard. This paper presents linearity, accuracy, precision, limit of quantification and limit of detection, specificity-selectivity and sample stability data. Based on the intra and inter-day validation, all coefficients of variation (CV) were found less than 15%. The assay is sufficiently rapid and sensitive and was applied in a pharmacokinetic study in rats.     

Comparative study of cases of poisoning by organophosphorous insecticides in various regions of Costa Rica

336 cases of organophosphorous poisoning from case records of hospitals in Costa Rica between 1972 and 1978 were classified in four categories according to signs and symptoms: 8% latent; 25.9% mild; 43.8% moderate and 22.3% severe. Eighteen of the total patients died during hospitalization. Suicidal ingestion was the principal cause of severe and latent poisoning, especially in urban areas, followed by occupational poisoning in agricultural workers in mild and moderate cases. Occupational poisoning was common in men; in women it was attempted suicide and in children accidental poisoning. Under 40 years of age, the main cause was suicidal ingestion among both men and women, and occupational poisoning after 40 years of age. After discharge from the hospital, 19.6% of the total poisoned patients were referred to psychiatric treatment; 22.7% and 18.2% had had mental and alcoholic problems, respectively, prior to poisoning. Attempted suicide was the principal cause of poisoning in 86.4% of these patients, indicating emotional instability. In this study, the incidence in psychiatric consultation after discharge from the hospital is not indicative of a relationship between mental disorders and acute insecticide poisoning. In 42.6% and in 22% of the deceased patients pralidoxime (PAM) and atropine respectively, were not administered as antidotes in the emergency room. Aminophylline and skeletal muscle relaxants among other contraindicated drugs were administered as part of the treatment in these deceased patients. The consequences of this misguided treatment are questioned.     

Mutations in acetylcholinesterase genes of Rhopalosiphum padi resistant to organophosphate and carbamate insecticides.

Apple grain aphid, Rhopalosiphum padi (Linnaeus), is an important wheat pest. In China, it has been reported that R. padi has developed high resistance to carbamate and organophosphate insecticides. Previous work cloned from this aphid 2 different genes encoding acetylcholinesterase (AChE), which is the target enzyme for carbamate and organophosphate insecticides, and its insensitive alteration has been proven to be an important mechanism for insecticide resistance in other insects. In this study, both resistant and susceptible strains of R, padi were developed, and their AChEs were compared to determine whether resistance resulted from this mechanism and whether these 2 genes both play a role in resistance. Bioassays showed that the resistant strain used was highly or moderately resistant to pirimicarb, omethoate, and monocrotophos (resistance ratio, 263.8, 53.8, and 17.5, respectively), and showed little resistance to deltamethrin or thiodicarb (resistance ratio, 5.2 and 3.4, respectively). Correspondingly, biochemistry analysis found that AChE from resistant aphids was very insensitive to the first 3 insecticides (I50 increased 43.0-, 15.2-, and 8.8-fold, respectively), but not to thiodicarb (I50 increased 1.1-fold). Enzyme kinetics tests showed that resistant and susceptible strains had different AChEs. Sequence analysis of the 2 AChE genes cloned from resistant and susceptible aphids revealed that 2 mutations in Ace2 and 1 in Ace1 were consistently associated with resistance. Mutation F368(290)L in Ace2 localized at the same position as a previously proven resistance mutation site in other insects. The other 2 mutations, S329(228)P in Ace1 and V435(356)A in Ace2, were also found to affect the enzyme structure. These findings indicate that resistance in this aphid is mainly the result of insensistive AChE alteration, that the 3 mutations found might contribute to resistance, and that the AChEs encoded by both genes could serve as targets of insecticides.     

Quantitation of DNA and RNA in crude tissue extracts by flow injection analysis.

An automated two-dye flow injection analysis system to quantitate DNA and RNA in crude extracts of tissues is described. The method uses the fluorochrome dyes ethidium bromide and Hoechst 33258. DNA concentration is determined directly from its fluorescence in Hoechst dye. RNA is estimated from fluorescence in ethidium bromide after subtraction of the fluorescence due to DNA. This method has several advantages: a simple extraction procedure, a low detection limit (0.01 micrograms DNA and 0.10 micrograms RNA), automation, and a high sample throughput.     

Prerequisites for the on-line control of microbial processes by flow injection analysis.

Problems associated with the use of biosensors in process control, e.g. difficulties of sterilization and sensor fouling, are shortly displayed, and possibilities to overcome them are outlined. The advantages of flow injection analysis (FIA) are demonstrated and examples for efficient sampling systems connected with this method are reviewed. Special emphasis is given to problem-orientated sample pretreatments, preventing fast inactivation of immobilized enzymes in the analysis system. Examples of problem-orientated sample pretreatment units are given. A proposal for a computer-controlled self-calibrating FIA system is given.