N1-Methyl-2-125I-Lysergic Acid Diethylamide, a Preferred Ligand for In Vitro and In Vivo Characterization of Serotonin Receptors

Methylation of 2-125I-lysergic acid diethylamide (125I-LSD) at the N1 position produces a new derivative, N1-methyl-2-125I-lysergic acid diethylamide (125I-MIL), with improved selectivity and higher affinity for serotonin 5-HT2 receptors. In rat frontal cortex homogenates, specific binding of 125I-MIL represents 80-90% of total binding, and the apparent dissociation constant (KD) for serotonin 5-HT2 receptors is 0.14 nM (using 2 mg of tissue/ml). 125I-MIL also displays a high affinity for serotonin 5-HT1C receptors, with an apparent dissociation constant of 0.41 nM at this site. 125I-MIL exhibits at least 60-fold higher affinity for serotonin 5-HT2 receptors than for other classes of neurotransmitter receptors, with the dopamine D2 receptor as its most potent secondary binding site. Studies of the association and dissociation kinetics of 125I-MIL reveal a strong temperature dependence, with very slow association and dissociation rates at 0 degree C. Autoradiographic experiments confirm the improved specificity of 125I-MIL. Selective labeling of serotonin receptors was observed in all brain areas examined. In vivo binding studies in mice indicate that 125I-MIL is the best serotonin receptor label yet described, with the highest frontal cortex to cerebellum ratio of any serotonergic radioligand. 125I-MIL is a promising ligand for both in vitro and in vivo labeling of serotonin receptors in the mammalian brain.     

Measurement of Acetylcholine Release in Freely Moving Rats by Means of Automated Intracerebral Dialysis

The present study demonstrates the feasibility of measuring acetylcholine in perfusion samples collected by means of in vivo brain dialysis in the striata of freely moving rats. The output of the dialysis device was directly connected to an automated sample valve of a HPLC-assay system that comprises a cation exchanger, a post-column enzyme reactor, and an electrochemical detector. The presence of an acetylcholinesterase inhibitor (neostigmine) in the perfusion fluid was required for the detection of acetylcholine in the perfusate. Increasing concentrations of neostigmine induced increasing amounts of acetylcholine. Continuous perfusion with a fixed concentration (2 microM) of neostigmine resulted in gradually increasing amounts of collected acetylcholine over time although a considerable variation between successive samples exists. The brain dialysis technique was further validated by studying the effect of various drugs. Systemically administered atropine increased the output of acetylcholine, whereas the addition of tetrodotoxin to the perfusion fluid resulted in a complete disappearance of the neurotransmitter.     

Host immunoglobulin G titre and antibody activity in haemolymph of the tick, Ornithodoros moubata

Immunoglobulin G (IgG) in tick haemolymph was analysed immunochemically and biochemically for its antigenicity, antibody activity and relative concentration in a soft tick, Ornithodoros moubata (Murray) sensu Walton 1962 (Acari: Argasidae). Ouchterlony immunodiffusion tests showed that haemolymph from a tick engorged on rabbit IgG (or human IgG) through an artificial membrane, reacted with anti-rabbit IgG (anti-human IgG) but not with anti-human IgG (anti-rabbit IgG). This indicates that haemolymph of the fed tick contains IgG with a similar antigen specificity to host blood IgG. IgG from tick haemolymph was demonstrated by enzyme immunoassay to have the same antibody activity as ingested IgG. The IgG concentration in tick haemolymph was measured by a quantitative single immunodiffusion test. Changes of IgG titre after a bloodmeal were correlated with IgG activity, which was low for 5 days after a bloodmeal and then suddenly increased. The IgG titre reached a maximum 7 days post-engorgement, and remained high for over 4 months during and after oviposition. 125I-labelled IgG was injected into the tick haemocoel to determine the persistence of IgG in the haemolymph. Recovery of labelled IgG was low at 1 and 3 days, and high at 5, 8 and 16 days after engorgement. The data suggest that IgG in haemolymph disappears quickly soon after engorgement possibly by degradation and/or absorption (adhesion to tissues).     

Detection and characterization of acidic compartments (vacuoles) in Chlorella vulgaris 11h cells by 31P-in vivo NMR spectroscopy and cytochemical techniques

Acidic inorganic phosphate (Pi) pool (pH around 6) was detected besides the cytoplasmic pool in intact cells of Chlorella vulgaris 11h by ³¹P-in vivo nuclear magnetic resonance (NMR) spectroscopy. It was characterized as acidic compartments (vacuoles) in combination with the cytochemical technique; staining the cells with neutral red and chloroquine which are known as basic reagents specifically accumulated in acidic compartments. Under various conditions, the results obtained with the cytochemical methods were well correlated with those obtained from in vivo NMR spectra; the vacuoles were well developed in the cells at the stationary growth phase where the acidic Pi signal was detected. In contrast, cells at the logarithmic phase in which no acidic Pi signal was detected contained only smaller vesicles that accumulated these basic reagents. No acidic compartment was detected by both cytochemical technique and ³¹P-NMR spectroscopy when the cells were treated with NH₄OH. The vacuolar pH was lowered by the anaerobic treatment of the cells in the presence of glucose, while it was not affected by the external pH during the preincubation ranging from 3 to 10. Possible vacuolar functions in unicellular algae especially with respect to intracellular pH regulation are discussed.Non-standard abbreviations EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MDP methylene diphosphonic acid - NMR nuelear magnetic resonance - PCA perchloric acid - PCV packed cell volume - Pi inorganic phosphate - Pic sytoplasmic inorganic phosphate - Piv vacuolar inorganic phosphate - ppm parts per million - SP sugar phosphates - TCA trichloroacetic acid     

Mutagenicity,sister chromatid exchange inducibility andin vitro cell transforming ability of particulates from Athens air

Airborne particulates were collected over a period of twelve months by the use of Hi-Vol samplers in the basin of Athens, Greece. N-Hexane extracts were tested in a battery ofin vitro tests for their ability to induce mutation in bacteria as well as mutation, sister chromatid exchange and morphological transformation in cultured mammalian cells. Positive results were found for mutagenicity withSalmonella strain TA98 in the Ames assay, for sister chromatid exchange induction in CHO cells and for transformation in BALB/c 3T3 cells in culture. They also showed weak non-doserelated induction of ouabain resistance in BALB/c 3T3 cells. The contribution of oxidizing and nitrating agents found in the Athens atmosphere, together with sunlight UV irradiation in the formation of direct acting mutagens and potential carcinogens from ambient polycyclic aromatic hydrocarbons, is suggested.Abbreviations FCS fetal calf serum - FPG fluorescent-plus-Giemsa technique - oua^R ouabain resistant - PAH polycyclic aromatic hydrocarbon - SCE sister chromatid exchange - TSP total suspended particulate     

Effects of etretinate on the distribution of elements in rats

We studied in the rat the effects of the drug etretinate (Tigason), given at three doses 3, 10, and 30 mg/kg body wt for 1 mo, on the concentrations of Na, K, Ca, Mg, Fe, S, P, Cu, and Zn in the plasma, brain, thymus, heart, liver, lung, kidney, testicle, muscle, and bone. The elements were simultaneously determined in tissues after nitric acid dissolution by inductively coupled plasma emission spectrometry using a JY 48 instrument. At the dose of 3 mg/kg, etretinate did not induce any statistically significant modifications of the element distribution. At the dose of 10 mg/kg, the main observed modifications were in plasma an increase of copper (+38%) and a decrease of zinc (-25%). At the highest dose of 30 mg/kg, some variations of the concentrations of elements in tissues were observed. But, on no account did retinoids induce an alteration of the mineral composition of bone, despite obvious macroscopic bone alterations.     

Multielement analysis of biological samples by inductively coupled plasma-mass spectrometry

Interest in the biological behavior of a growing number of elements, along with increasing recognition of the importance of interactions among them, demands a versatile and reliable technique for multielement analysis of biological samples. Significant improvements over the sensitivity achieved with conventional inductively coupled plasma (ICP) optical emission spectrometries have been realized with the introduction of quadrupole mass spectrometry (MS) for detection of ions in the plasma. The hybrid technique of ICP-MS promises to be a method of rapid multielement analysis, at detection limits that approach or surpass those of other technologies. However, the application of ICP-MS to analyses of biological interest is truly in its infancy. Here we report the use of ICP-MS for the determination of more than 30 elements of biological interest in a tissue and a biological fluid (rat liver and serum, respectively). Experimental values of the elements serve as a basis for discussion of analytical protocols, performance criteria, and certain problems peculiar to ICP-MS.     

In vitro development of angiosperm floral buds and organs

In the last twenty-five years, young inflorescences, floral buds and individual floral organs of a number of species have been cultured in vitro. There is considerable variability in the requirement of plant growth regulators and nutritional factors for flower development of different species. This variability is compounded by the fact that the hormonal and nutritional requirements are different at various stages of organ and floral development. Experimental studies on normal and mutant flowers in vitro have provided insights into some of the regulatory processes in floral organogenesis. The potential use of the in vitro technique in elucidating the various mechanisms in flower development is stressed.     

In vitro regeneration of Gaillardia pulchella Foug.

Young leaf and internodal stem segments of Gaillardia pulchella, collected from wild species re-established in the greenhouse, were used to initiate callus on Murashige & Skoog medium supplemented with NAA (2.0 mgl⁻¹) and BA (0.4 mgl⁻¹). Callus formed after 10 to 14 days in the dark. Cultures were transferred to fresh medium and placed under lighted conditions where shoot formation occurred approximately 14 to 30 days after initiation. Callus sub-cultured at 14 to 21-day intervals continued to produce primordia for several weeks. Flowers were produced by regenerated shoots maintained on MS medium, but roots did not develop until the plantlets were transferred to soil conditions.