Advantages of using development models of the carrion beetles Thanatophilus micans (Fabricius) and T. mutilatus (Castelneau) (Coleoptera: Silphidae) for estimating minimum post mortem intervals, verified with case data.

Some beetles are as useful as blow flies for estimating the minimum post mortem interval (PMImin) or time since death. Examples include Thanatophilus micans (Fabricius) and Thanatophilus mutilatus (Castelneau), two geographically and ecologically overlapping African beetles. Molecular means of identifying these species, descriptions of their natural history, thermal summation models for the development of each species, and a case in which T. micans was recovered are presented. These beetles colonise bodies soon after death, their development spans more time than that of flies, and they may be little affected by maggot-generated heat. From an experimental perspective, they can be reared individually, which allows the identification of sick individuals and has analytical advantages relative to fly larvae that must be reared in groups. Estimating minimum post mortem intervals for both species using the case data strongly suggests that developmental models parameterised for one species should not be used to make forensic estimates for closely related species for which no specific model is available and emphasises the need for correct identifications.

Baited traps may be an alternative to conventional pesticides in integrated crop management of chicory (Compositae) in South Africa.

Chicory, Chicorium intybus L. (Compositae), is a major field crop in the Eastern Cape Province of South Africa. Several pests feed on the leaves of the plant, resulting in reduced yield. The most important of these are the noctuid moths Helicoverpa armigera (Hübner), Chrysodeixis acuta (Walker), and Trichoplusia orichalcea (F.). The use of attract-and-kill traps offers an alternative to broad-based insecticides in the control of these species. Three fields were treated with normal insecticides and three fields with yellow-baited traps. Eight additional traps were placed in each field, with half of the traps containing the insecticide 2,2-dichlorovinyl dimethyl phosphate (dichlorvos) and half without dichlorvos; and half yellow and half green. Total moth numbers and nonphytophage diversity were measured from these eight traps. Although no differences in H. armigera or T. orichalcea catches were observed between insecticide- and trap-treated fields, numbers of C. acuta and the total number of moths were significantly higher in insecticide-treated fields. Yellow traps containing dichlorvos contained more moths than yellow traps without dichlorvos, or green traps with dichlorvos, or green traps without dichlorvos; but they also contained more nonphytophagous insects. Yellow traps also enhanced the catches of thrips on card traps associated with them. These results offer an opportunity for the South African chicory industry to reduce pesticide applications and thus mitigate environmental impacts.

The effect of inducing agents on the metabolism of ethidium bromide by isolated rat hepatocytes.

The metabolism of ethidium bromide by isolated rat hepatocytes is significantly enhanced by pre-treatment of animals with phenobarbitone (PB) and 3-methylcholanthrene (3-MC). Pre-treatment with PB and 3-MC results in a 2.5- and 1.5-fold increase, respectively in the amount of the principal metabolite, ethidium 8-N-glucuronide, compared with that formed by hepatocytes from untreated rats. The formation of ethidium 3-N-glucuronide is not enhanced by pre-treatment with either PB or 3-MC. Two new metabolites, hydroxyethidium glucuronide and a transient unidentified species, have been detected by HPLC and are formed only by hepatocytes from animals pre-treated with 3-MC.

Intracellular localization and metabolism of the phenanthridinium trypanocide, ethidium bromide, by isolated rat hepatocytes.

1. Confocal laser scanning microscopy (CLSM) has shown that ethidium (3 ,8-diamino-5-ethyl-6-phenylphenanthridinium) bromide, an aromatic phenanthridinium trypanocide, is taken up rapidly into the nucleoli and nuclear membranes of isolated rat hepatocytes. 2. It is biotransformed by the hepatocytes and at least five metabolites have been detected by high-performance liquid chromatography (HPLC). 3. Two new metabolites, 3- and 8-N-glucuronosylethidium, have been identified by HPLC-electrospray mass spectrometry and they represent the major pathway of metabolism, accounting for 6.4 +/- 0.7 and 19.5 +/- 1.2% respectively of total recovered drug after incubation. A third metabolite, 3,8-diacetylethidium, is formed in trace quantities. 4. The other two metabolites, 3-acetylethidium and 8-acetylethidium, have been reported previously.

Effect of oral administration of vitamin C on human aqueous humor ascorbate concentration.

AIM: To study oral administration of vitamin C on human aqueous humour ascorbate concentration. METHODS: High performance liquid chromatography (HPLC) coupled with electrochemical detector (ECD) was used. The effect of oral administration of various doses of ascorbic acid, 0 (control), 1.0, 1.5, 2.0, 3.0, and 5.0 g, on its concentration in aqueous humour, obtained from volunteer cataract patients was studied. RESULTS: The concentration of ascorbic acid in aqueous humour of control group (without administration of vitamin-C tablet or drug containing ascorbic acid was (254 +/- 119) mg.L-1. This study revealed that the administration of 2.0 g of ascorbic acid saturate the aqueous humour and further increase in the dose (3.0 g and 5.0 g) did not increase its concentration in aqueous humour, although its concentration was increased in plasma. CONCLUSION: Oral administration of 2.0 g of Vc is sufficient to saturate the aqueous humour where it may be helpful in controlling the intra-ocular pressure.

Analysis of a residual diamine in a pharmaceutical polymer using solid phase extraction with analysis by gas chromatography mass spectrometry.

A method was developed for the analysis of 4,4'-methylenebiscyclohexylamine (DMDA) and 4,4'-methylenedicyclohexylisocyanate (DMDI) in a pharmaceutical polymer. The DMDA was extracted from the polymer with either buffer (0.1 M potassium phosphate pH 3.1) and the extract was passed through a SCX solid phase extraction cartridge. It was eluted from the cartridge with methanolic ammonia and then converted to its heptafluorobutyramide (HFB) derivative prior to analysis by gas chromatography-negative chemical ionisation mass spectrometry (GC-MS) in the negative ion chemical ionisation (NICI) mode. It was not possible to directly measure DMDI and it was thus analysed by selecting extraction conditions such that it would decompose to DMDA. The quantification of the residues in the polymer was based on the method of standard additions since this gave a better indication of the recovery from the complex matrix.

Investigation of the chemical equivalence of the trypanocidal products, Samorin and Veridium.

A procedure for the evaluation of chemical equivalence of proprietary formulations of isometamidium is described. The method combines the analysis of the principal component (isometamidium), HPLC profiling of related substances and determination of the inorganic impurity, ammonium chloride, using a modification of the Berthelot (Indophenol) reaction. Application of these procedures to analyses of commercially available sachets from four different batches of Samorin and four different batches of Veridium has demonstrated that there are marked qualitative and quantitative differences between batches from these two sources. Whilst Samorin samples showed inter-batch consistency of composition, there was considerable inter-batch variation between the samples of Veridium.

HPTLC and HPLC determination of isometamidium in the presence of its manufacturing and degradation impurities.

The determination of the phenanthridine trypanocide, isometamidium chloride hydrochloride (ISM), in the presence of four process-related and degradation impurities, by RP-HPLC using a Licrospher-60 RP-select B column with a mobile phase composition of acetonitrile/KH2PO4 (PH 3.0, 20 mM) (25:75 v/v) with UV detection at 320 nm, is described. The method is selective, reproducible and precise with a limit of detection of 45 ng ml-1 for ISM. A HPTLC system (Kieselgel 60 F254, pyridine/acetonitrile/butanol/formic acid, 6:6:4:1, v/v), with UV densitometric evaluation at 320 nm, suitable for the separation of ISM and the related substances is reported.

The expression of a cloned Drosophila octopamine/tyramine receptor in Xenopus oocytes.

The expression of a cloned Drosophila octopamine/tyramine receptor (OctyR99AB) is described in Xenopus oocytes. Agonist stimulation of OctyR99AB receptors increased intracellular Ca2+ levels monitored as changes in the endogenous inward Ca2+-dependent chloride current. The receptor is preferentially sensitive to biogenic amines with a single hydroxyl on the aromatic ring. The G-protein, Galphai, appears to be involved in the coupling of the receptor to the production of intracellular calcium signals, since the effect is pertussis-toxin sensitive and is blocked or substantially reduced in antisense knockout experiments using oligonucleotides directed against Galphai but not by those directed against Galphao, Galphaq and Galpha11. The increase in intracellular calcium levels induced by activation of the OctyR99AB receptor can potentiate the ability of activation of a co-expressed beta2-adrenergic receptor to increase oocyte cyclic AMP levels. A comparison of the pharmacological coupling of OctyR99AB to different second messenger systems when expressed in Xenopus oocytes with previous studies on the expression of the receptor in a Chinese hamster ovary cell line suggests that the property of agonist-specific coupling of the receptor to different second messenger systems may be cell-specific, depending upon the G-protein environment of any particular cell type.

Determination of molecular weight of hyaluronic acid in bovine vitreous humour and Healon by high performance gel permeation chromatography and its depolymerization with ascorbic acid.

A method has been developed for the determination of the molecular weight of hyaluronic acid by high performance gel permeation chromatography. The molecular weight distribution was determined using three polymeric columns. Columns were calibrated using Pullulan Polymer molecular weight standards. The average molecular weight (+/- SD) of the hyaluronic acid in bovine vitreous humour and Healon were 1.20 x 10(6) Da (+/- 0.49 x 10(6) and 3.01 x 10(6) Da (+/- 0.14 x 10(6), respectively. Furthermore, the depolymerization of the hyaluronic acid in the presence of ascorbic acid (the concentration found in human eye) was studied. Samples incubated in the presence of atmospheric air showed more depolymerization than those where atmospheric air had been flushed-out with nitrogen.

Selective inhibition of adenylyl cyclase by octopamine via a human cloned alpha 2A-adrenoceptor.

1. In this study we have compared the abilities of the enantiomers of the structural isomers of the phenolamines, octopamine and synephrine, and the catecholamines, noradrenaline and adrenaline, to couple selectively a human cloned alpha 2A-adrenoceptor, stably expressed in a Chinese hamster ovary (CHO) cell line, to G-protein linked second messenger pathways mediating an increase and a decrease in cyclic AMP production. 2. The catecholamines couple the alpha 2A-adrenoceptor to both an increase and a decrease in the rate of cyclic AMP production. In the absence of pertussis toxin pretreatment both catecholamines tested showed a dose-dependent decrease with a maximum at 100 nM. After pertussis toxin pretreatment they both produced a dose-dependent increase in cyclic AMP production with a maximum at 10 microM. 3. The phenolamines, octopamine and synephrine were only able to couple the alpha 2A-adrenoceptor to a dose-dependent decrease in cyclic AMP production at concentrations up to 1 mM, with the synephrine isomers being more potent than the corresponding octopamine isomers. The meta-isomers of both phenolamines were more potent than the corresponding para-isomers and the (-)-enantiomers were more potent than the (+)-enantiomers. Thus, (-)-meta-synephrine [(-)-phenylephrine] was the most effective isomer tested with an observable decrease occurring between 100 nM and 1 microM. 4. The effects of octopamine and the catecholamines on the decrease in cyclic AMP production were additive at submaximal concentrations, whilst octopamine reduced the stimulant effect of submaximal concentrations of noradrenaline on cyclic AMP production after pertussis toxin pretreatment. 5. The time courses of the inhibitory effects of both meta-octopamine and noradrenaline were parallel and peaked after a 1 min exposure to the agonist. In contrast, the stimulant effects of noradrenaline after pertussis toxin pretreatment were of a much slower time course with a maximum effect occurring after a 5 min incubation period. 6. Since octopamine and synephrine occur naturally in, and are co-released with catecholamines from, mammalian tissues, the results of the present study suggest that the human cloned alpha 2A-adrenoceptor can be coupled selectively by different endogenous agonists to G-protein pathways mediating the regulation of adenylyl cyclase activity.

Determination of oestrone, 17alpha- and 17beta-oestradiol in bovine aqueous humor using gas chromatography-negative ion chemical ionization mass spectrometry.

Perfluorotolyl (PFT)-ether and perfluorotoly-trimethylsilyl (PFT-TMS) ether derivatives of oestrone, 17alpha- and 17beta-oestradiol were prepared under phase transfer conditions. The former derivatives under negative ion chemical ionization conditions gave significant ions in the mass spectrometer but 17alpha- and 17alpha-oestradiol gave poor resolution. However, the PFT-TMS derivatives of 17alpha- and 17beta-oestradiol showed good resolution. These derivatives were used for the analysis of oestrogens in bovine aqueous humour, vitreous humour and retina. The mean (+/-SEM) concentrations of oestrone in bovine aqueous humour (n=18), vitreous humour (n=18) and bovine retina (n=4) were 0.47+/-0.11, 0.46+/-0.14 and 1.10+/-0.24 ng.ml(-1), respectively. 17alpha-Oestradiol was detected in 16 out of 18 samples of bovine aqueous humour and vitreous humour and the mean (+/-SEM) concentrations were 0.30+/-0.10 and 0.08+/-0.02 ng.ml(-1), respectively. The mean (+/-SEM) concentration of 17beta-oestradiol in aqueous humour (n=7) and vitreous homour (n=11) 0.83+/-0.26 ng ml(-1) and 0.39+/-0.09 ng ml(-1), respectively. In retina the concentrations of both steroids were below the detection limit.

Neuromuscular blockade: offset anomalies. Are they simply potency-related receptor bonding effects?

Rapid making and breaking of bonds between quaternary ammonium compounds and cholinergic receptors is typical of ion-pair bonding, which is weak, and ion-pair reactions, which are extremely fast. These properties explain the observed rapid association and dissociation of turbocurarine at receptors. The time course receptor offset is determined by two factors, buffered diffusion due to repetitive bonding, and a potency-related offset-retarding effect. The strength of the latter is a function of chemical structure, which determines the microscopic molecular rate of drug-receptor association and dissociation. Together, buffered diffusion and the potency-related offset-retarding effect provide a complete rational physico-chemical explanation for the marked, yet variable, differences between onset and offset times of non-depolarizing neuromuscular blocking agents. The influence of a potency-related offset-retarding effect together with differing structural requirements for neuromuscular blocking potency and plasma carboxyesterase hydrolysis, suggests that a high potency ultrashort duration block is unlikely to be achieved in a non-depolarizing compound metabolized by plasma esterases alone.

Synthesis and characterisation of acyl glycines. Their measurement in single blood spots by gas chromatography-mass spectrometry to diagnose inborn errors of metabolism.

Acyl glycines are normally minor metabolites of fatty acids; however, the excretion of certain acyl glycines is increased in several inborn errors of metabolism. Therefore measurement of the metabolites in body fluids can be used to diagnose these metabolic disorders. The chemical synthesis of a range of acyl glycines is described, together with that of their 13C2-isotopomers for use as internal standards. An analytical method for the measurement of hexanoyl, octanoyl, 3-phenylpropionyl and suberyl glycines in urine, employing gas chromatography-mass spectrometry with negative-ion chemical ionisation was adapted to measure a larger range of acyl glycines in a single blood spot on a standard Guthrie card. Diagnoses of a case of medium-chain acyl-CoA dehydrogenase deficiency and a case of isovaleric acidaemia were confirmed using a single blood spot from each patient.

Approaches to short-acting neuromuscular blocking agents: nonsymmetrical bis-tetrahydroisoquinolinium mono- and diesters.

Nonsymmetrical bisquaternary mono- and diesters combining the potency-enhancing properties of the (1R)-laudanosinium group with a second unhindered quaternary ammonium moiety have been studied as a means of promoting short action with high-potency neuromuscular block. Atracurium-related nonsymmetrical diesters showed high potency, freedom from vagal blockade at neuromuscular blocking doses, and short action. Nonsymmetrical monoesters were short acting but showed varying degrees of vagal block.

Analysis of unconjugated morphine, codeine, normorphine and morphine as glucuronides in small volumes of plasma from children.

A sensitive method for the analysis of unconjugated morphine, codeine, normorphine and total morphine after hydrolysis of glucuronide conjugates is described. The method was applicable to 50-microliters volumes of plasma. The analytes were converted to heptafluorobutyryl (HFB) derivatives before analysis by gas chromatography-negative ion chemical ionization mass spectrometry. Morphine and codeine were quantified against their [2H3]-isotopomers. Linearity, precision and accuracy were quite acceptable (in the 10(-10)-10(-9) g range), and the absolute limits of detection were < 1 pg.