Application of magnetic techniques in the field of drug discovery and biomedicine

Magnetic separation technology, using magnetic particles, is quick and easy method for sensitive and reliable capture of specific proteins, genetic material and other biomolecules. The technique offers an advantage in terms of subjecting the analyte to very little mechanical stress compared to other methods. Secondly, these methods are non-laborious, cheap and often highly scalable. Moreover, techniques employing magnetism are more amenable to automation and miniaturization. Now that the human genome is sequenced and about 30,000 genes are annotated, the next step is to identify the function of these individual genes, carrying out genotyping studies for allelic variation and SNP analysis, ultimately leading to identification of novel drug targets. In this post-genomic era, technologies based on magnetic separation are becoming an integral part of todays biology laboratory. This article briefly reviews the selected applications of magnetic separation techniques in the field of biotechnology, biomedicine and drug discovery.     

Metabolism of angiotensin II is required for its in vivo effect on dopamine release in the striatum of the rat

The effect of angiotensin (Ang) IV, an inhibitor of insulin-regulated aminopeptidase (IRAP), on extracellular dopamine levels in the striatum of freely moving rats was examined using in vivo microdialysis. The Ang IV was administered locally in the striatum through the microdialysis probe. A concentration-dependent (10-100 microm) increase in extracellular striatal dopamine was observed. The effect of Ang II (10-100 microm), which has only a weak affinity for IRAP, was similar to that observed for Ang IV. The effects of both peptides could not be blocked by the AT1 antagonist candesartan (10 nm and 1 microm) nor by the AT2 antagonist S-(+)-1-([4-(dimethylamino)-3-methylphenyl]methyl)-5-(diphenyl-acetyl)-4,5,6,7-tetrahydro-1H-amidazo(4,5-c) pyridine-6-carboxylic acid (1 microm), suggesting that the observed effects are both AT1 and AT2 independent. The effect of Ang II could be blocked by the aminopeptidase-A inhibitor (S)-3-amino-4-mercaptobutylsulphonic acid as well as the aminopeptidase-N inhibitor 2-amino-4-methylsulphonylbutane thiol, indicating that the effect of Ang II is mediated via metabolism into Ang IV. Other IRAP inhibitors, such as Divalinal-Ang IV and LVV-haemorphin-7, had similar effects on extracellular dopamine levels as compared with Ang IV. We propose a role for IRAP as mediator for the effects of Ang IV and related peptides on extracellular dopamine levels in the striatum of the rat.     

Ventricular but not atrial electro-mechanical delay of the embryonic heart is altered by anoxia-reoxygenation and improved by nitric oxide

BACKGROUND/AIM: Excitation-contraction coupling is modulated by nitric oxide (NO) which otherwise has either beneficial or detrimental effects on myocardial function during hypoxia-reoxygenation. This work aimed at characterizing the variations of electromechanical delay (EMD) induced by anoxia-reoxygenation within the developing heart and determining whether atrial and ventricular EMD are modulated by NO to the same extent. METHODS: Hearts of 4 or 4.5-day-old chick embryos were excised and submitted in vitro to normoxia (45 min), anoxia (30 min) and reoxygenation (60 min). Electrocardiogram and atrial and ventricular contractions were simultaneously recorded throughout experiment. Anoxia-reoxygenation-induced chrono-, dromo-and inotropic disturbances and changes in EMD in atrium (EMDa) and ventricle (EMDv) were investigated in control hearts and in hearts exposed to 0.1, 1, 10, 50 and 100 microM of DETA-NONOate (a NO donating agent) or to 50 microM of L-NAME (a NOS inhibitor). RESULTS: Under normoxia, heart rate, PR interval, ventricular shortening velocity, EMDa and EMDv were similar in control, L-NAME-treated and DETA-NONOate-treated hearts. Under anoxia, cardiac activity became markedly erratic within less than 10 min in all groups. At the onset of reoxygenation, EMDv was increased by about 300% with respect to the preanoxic value while EMDa did not vary significatively. Compared to control conditions, L-NAME or DETA-NONOate had no influence on the negative chrono-, dromo- and inotropic effects induced by anoxia-reoxygenation. However, L-NAME prolonged EMDv during anoxia and delayed EMDv recovery during reoxygenation while 100 microM DETA-NONOate had the opposite effects. EMDa was neither affected by NOS inhibitor nor NO donor. At the end of reoxygenation, all the investigated parameters returned to their basal values. CONCLUSION: This work provides evidence that a NO-dependent pathway is involved in regulation of the ventricular excitation-contraction coupling in the anoxic-reoxygenated developing heart.     

Acute dopamine/norepinephrine reuptake inhibition increases brain and core temperature in rats.

The purpose of the present study was to examine the effects of an acute dose of the dual dopamine (DA) and norepinephrine (NE) reuptake inhibitor bupropion (Bup) on brain (T(brain)), body core (T(core)), and tail skin (T(tail)) temperature in freely moving rats and to simultaneously monitor the extracellular neurotransmitter concentrations in the preoptic area and anterior hypothalamus (PO/AH). A microdialysis probe was inserted in the PO/AH, and samples for NE, DA, and serotonin (5-HT) were collected every 20 min before and after the injection of 17 mg/kg of Bup, for a total sampling time of 180 min. T(core) was monitored using a biotelemetry system. T(brain) and T(tail), an index of heat loss response, were also measured. Both NE and DA levels in the PO/AH significantly increased after Bup injection compared with the baseline levels, reaching approximately 450 and 230%, respectively, 40 min after injection. There was no effect on 5-HT release. The neurotransmitter changes were accompanied by a significant decrease in T(tail) and an increase in both T(brain) and T(core) compared with the baseline levels. The present results demonstrate that inhibition of NE and DA reuptake suppresses heat loss mechanisms and elevates T(brain) and T(core) in freely moving rats.     

Short chain oligogalacturonides induce ethylene production and expression of the gene encoding aminocyclopropane 1-carboxylic acid oxidase in tomato plants

Oligogalacturonic acids (OGAs), derived from plant cell wall pectin, have been implicated in a number of signal transduction pathways involved in growth, development and defense responses of higher plants. This study investigates the size range of OGAs capable of inducing ethylene synthesis in tomato plants, and demonstrates that in contrast with many other effects, only short chain OGAs are active. Oligomers across a range of DP from 2-15 were separated and purified to homogeneity by QAE-Sephadex anion exchange chromatography using a novel elution system. The OGAs were applied to tomato plants and assayed for their ability to induce ethylene gas release and changes in steady state levels of mRNA encoding the ethylene forming enzyme aminocyclopropane-1-carboxylic acid oxidase (ACO). The study demonstrated that only OGAs in the size range of DP4-6 were active both in eliciting ACO expression and in the production of ethylene.      

Studies of esterase 6 in Drosophila melanogaster. XVIII. Biochemical differences between the slow and fast allozymes

Most natural populations of Drosophila melanogaster are polymorphic for two major electrophoretic variants at the esterase-6 locus. The frequency of the EST 6F allozyme is greatest in populations in warmer latitudes, whereas the EST 6S allozyme is predominant in colder latitudes. Latitudinal clines in electromorph frequencies are found on three continents. Purified preparations of the allozymes have been characterized for their pH optimum, substrate specificity, organophosphate inhibition, alcohol activation, thermal stability, and kinetic parameters. These and previous analyses of the EST 6 allozymes reveal that the two variants have differences in their physical and kinetic properties that may provide a basis for the selective maintenance of the polymorphisms and an explanation of the clinal variation observed in natural populations.      

The phosphorylation of eukaryotic initiation factor 2: a principle of translational control in mammalian cells

In eukaryotic cells, protein biosynthesis is controlled at the level of polypeptide chain initiation. During the initiation process, eukaryotic initiation factor 2 (eIF-2) catalyzes the binding of Met-tRNAf and GTP to the 40S ribosomal subunit. In a later step, eIF-2 is released from the ribosomal initiation complex, most likely as an eIF-2.GDP complex, and another initiation factor termed eIF-2B is necessary to recycle eIF-2 by displacing GDP by GTP. In rabbit reticulocytes, inhibition of protein synthesis is accompanied by the phosphorylation of the alpha-subunit of eIF-2, a process that does not render eIF-2 inactive, but prevents it from being recycled by eIF-2B. First described in rabbit reticulocytes as inhibitors of translation, two distinct eIF-2 alpha kinases are known: the haemin-controlled kinase (termed HCI) and the double-stranded RNA-activated kinase (termed DAI). eIF-2 alpha phosphorylation appears to be a reversible control mechanism since corresponding phosphatases have been described. Recent reports indicate a correlation between eIF-2 alpha phosphorylation and the inhibition of protein synthesis in several mammalian cell types under a range of physiological conditions. In this review, the physical and functional features of the known eIF-2 alpha kinases are described with respect to their role in mammalian cells and the mode of activation by cellular signals. Furthermore, the possible impact of the eIF-2/eIF-2B ratio and of the subcellular compartmentation of these factors (and the eIF-2 alpha kinases) on mammalian protein synthesis is discussed.     

Presence of haemin-controlled eIF-2 alpha kinases in both undifferentiated and differentiating mouse erythroleukaemia cells.

In rabbit reticulocytes, globin synthesis is regulated by a haemin-controlled translational inhibitor (HCI) which acts by phosphorylating the alpha-subunit of eukaryotic initiation factor 2 (eIF-2). With purified eIF-2 as substrate, haemin-controlled eIF-2 alpha kinases could be partially purified from cultured mouse erythroleukaemia cells (MEL cells), which can be induced in vivo to erythroid differentiation. The eIF-2 alpha kinases from both uninduced and induced MEL cells are clearly distinct from the double-stranded-RNA-activated eIF-2 alpha kinase described for many mammalian cell types. A rough quantitative estimation indicates that, on a per-cell basis, induced MEL cells contain the same amount of haemin-controlled eIF-2 alpha kinase activity as rabbit reticulocytes, whereas uninduced MEL cells contain about one-tenth as much. As to their chromatographic behavior on CM-Sephadex and DEAE-cellulose and their sensitivity towards physiological concentrations of haemin (5-10 microM), the eIF-2 alpha kinases from MEL cells are indistinguishable from HCI. They differ from HCI with respect to their response towards activating stimuli such as prolonged incubation at 37 degrees C or brief exposure to the thiol reagent N-ethylmaleimide.     

Cycling exercise and the determination of electromechanical delay.

The main aim of the present paper was to address the validity of a methodology proposed in a previous paper [Li L, Baum BS. Electromechanical delay estimated by using electromyography during cycling at different pedaling frequencies. J Electromyogr Kinesiol 2004;14(6):647-52], aimed at determining the electromechanical delay from pedaling exercise performed at various cadences. Twelve trained subjects undertook pedaling bouts corresponding to combinations of cadences ranging from 50 to 100 RPM and power output from 37.5% to 75% of Pmax. As cadence increased, peak torque angle was found to shift forward in crank cycle (from 60-65 degrees at 50 RPM to 75-80 degrees at 100 RPM, depending on the power output level), while muscle bursts shifted backward in accordance with previous works. It is therefore suggested to take into account this peak torque angle lag to improve the methodology proposed by Li and Baum. The present results also evidenced that the central strategy, consisting in earlier muscle activation in crank cycle as cadence increases, is only partial. Neural strategy seems to be a trade-off between mechanical efficiency of muscular force output and coactivation.