Tyrosine Hydroxylase Activity in Rat Brain Synaptosomes: Direct Measurement Using High Performance Liquid Chromatography

Abstract: By use of high performance liquid chromatography with electrochemical detection to measure dopamine production, tyrosine hydroxylase (EC 1.14.16.2) activity has been measured in rat brain synaptosomes from striatum and forebrain. Normal specific activities three- to fivefold higher than previously reported in the literature for radiochemical methods of assay were found. It is suggested that synaptosomes contain a significant amount of endogenous substrate for tyrosine hydroxylase, which causes dilution of the added labelled tyrosine and hence underestimation of the activity of this enzyme when radiochemical methods are used.     

A Rapid and Simple Method for the Determination of Picogram Levels of 3-Methoxytyramine in Brain Tissue Using Liquid Chromatography with Electrochemical Detection

Abstract: A rapid and simple technique using solvent extraction, ion-pairing extraction, and high pressure liquid chromatography with electrochemical detection has been developed for the determination of 3-methoxytyramine in striata of rats killed by microwave irradiation. The method is specific and reproducible (coefficient of variation among replications, ±4%); recovery of authentic 3-methoxytyramine added to the samples is 45–50%. 3-Methoxytyramine levels found with this technique in rat striata were 15 ± 1.7 ng/g. The method has a sensitivity of about 0.2 pmol per brain sample. Monoamine oxidase inhibition with pargyline increased 3-methoxytyramine levels in rat striata, while catechol- O -methyltransferase inhibition with 3',4'-dihydroxy-2 methylpropiophenone completely depleted 3-methoxytyramine. The effects of nomifensine, quipazine, caroxazone, piribedil, and D-amphetamine were also examined. The 3-methoxytyramine concentrations in the brains of animals killed by decapitation or by microwave irradiation were compared.     

Luminometric measurement of population activity of genetically modified Pseudomonas fluorescens in the soil

Genetically modified cells of Pseudomonas fluorescens, chromosomally marked with genes for bioluminescence, were inoculated into sterile soil microcosms. During incubation for 90 days, viable cell concentration did not change significantly but light output, measured by luminometry, decreased, indicating reduced metabolic activity due to lack of substrates. Amendment with nutrients resulted in parallel increases in both luminescence and dehydrogenase activity. Luminometry therefore enables rapid monitoring of the activity of populations of luminescence-marked microbial inocula in the soil, with greater sensitivity and selectivity than traditional techniques.     

Off‐host aggregation in the non‐fed,female brown dog tick,Rhipicephalus sanguineus (Latreille), is induced by tick excreta and enhanced by low relative humidity

We report that Rhipicephalus sanguineus (Ixodida: Ixodidae) faeces and its main component, guanine, act as assembly pheromones in short‐range Petri plate bioassays. Arrestment activity in response to guanine was lower than that in response to natural excreta, indicating the presence of other active ingredients in natural excreta. The selective removal of appendages was used to establish the important roles played by the palps and the front pair of legs in the detection of the pheromone. Reaction to chemically pure guanine at varying concentrations occurred without a dose response; thus only the presence of guanine, not a critical amount, is required to induce assembly. Higher speed and intensity of clustering occurred at 33% relative humidity (RH). We conclude that female adults of R. sanguineus are more prone to assemble under dry conditions that match the arid microhabitats preferred by this species and that this tendency allows this tick to reside in human dwellings and dog kennels that maintain standards of comfort at 30–50% RH. Cleaning or removing tick excreta‐covered surfaces on which ticks aggregate from within and around human dwellings may prove useful as a means of interfering with the establishment of off‐host clusters of R. sanguineus.     

Commentary Salicylate Trapping of -OH as a Tool for Studying Post-Ischemic Oxidative Injury in the Isolated Rat Heart

The use of salicylate as a chemical trap for -OH represents a simple and convenient alternative to the use of spin trapping techniques to study oxidative injury in isolated perfused organs. In these systems, salicylate is included in the perfusion buffer at concentrations ranging from 0.1 to 2mM depending on the detection apparatus employed. In our studies, we have used a coulometric detector, which has a theoretical efficiency of 100% as compared to 1-5% for the standard glassy carbon electrode. We have been able to generate reproducible results by inclusion of only 100 μM salicylate, a concentration demonstrated not to affect pre- or post-ischemic cardiac function. In initial studies, we observed an increase in perfusate 2,5-dihydroxybenzoic acid consistent with an early post-ischemic burst of -OH, not unlike that reported using spin trapping techniques. Since then we and others have used this technique to examine possible relationships between -OH formation and treatments that alter post-ischemic cardiac functional recovery. For example, preischemic loading of hearts with copper results in increases in postischemic dysfunction and LDH release that were associated with an increase in 2,5-dihydroxybenzoate and by inference, -OH formation. Alternatively, we have reported that the nitroxide spin label, TEMPO, reputed to be a superoxide dismutase mimetic, decreased post-ischemic arrhythmias and 2,5-dihydroxybenzoate formation. Most recently, we have observed that preischemic loading of hearts with zinc-bis-histidinate results in improved post-ischemic cardiac function and decreased LDH release; changes that were associated with decreased 2,5-dihydroxybenzoate formation. These studies indicate that under certain conditions, salicylate is a valuable alternative to spin trapping techniques to probe the role of -OH in cardiac oxidative injury, particularly when applied to the isolated perfused heart preparation.