The use of C8-octyl columns for the analysis of catecholamines by ion-pair reversed-phase liquid chromatography with amperometric detection

The chromatographic behavior of norepinephrine (NE), epinephrine, dopamine, 3,4-dihydroxyphenylalanine, and 3,4-dihydroxyphenylacetic acid on octylsilane (C₈) reversed-phase high-performance liquid chromatography columns was observed under various mobile phase conditions including manipulations of pH, pairing ion and methanol concentrations. The optimum isocratic conditions permitting quantitative resolution of these substances in minimum time and with maximum detector response were determined. Employing a pH 3.0–3.2 mobile phase comprising an aqueous buffer solution containing 0.1 M NaH₂PO₄, 0.1 mM EDTA, and 0.2 mM 1-octanesulfonate, admixed with a volume of methanol equal to 4% of the aqueous volume, the performance of the C₈ columns compares favorably to that of the more widely used C₁₈ columns. The column eluates were monitored with an amperometric detector utilizing a glassy-carbon flow-cell electrode. The detector response for NE was 1.5–2.0 nA/ng and the baseline noise was as little as 0.002 nA thereby permitting quantitation of 5-pg levels or more in the injected samples. By coupling the liquid chromatographic system to a procedure which eliminates non-catechol contaminants from the neuronal and body fluid specimens by alumina adsorption of the catechols, a sensitive and dependable method was developed and employed for the determination of catechol levels in discrete regions of rat brain, cat spinal cord, and in human plasma.     

Splicing Pattern of Gsα mRNA in Human and Rat Brain

The enzyme tyrosine hydroxylase catalyzes the first step in the biosynthesis of dopamine, norepinephrine, and epinephrine. Tyrosine hydroxylase is a substrate for cyclic AMP-dependent protein kinase as well as other protein kinases. We determined the Km and Vmax of rat pheochromocytoma tyrosine hydroxylase for cyclic AMP-dependent protein kinase and obtained values of 136 microM and 7.1 mumol/min/mg of catalytic subunit, respectively. These values were not appreciably affected by the substrates for tyrosine hydroxylase (tyrosine and tetrahydrobiopterin) or by feedback inhibitors (dopamine and norepinephrine). The high Km of tyrosine hydroxylase correlates with the high content of tyrosine hydroxylase in catecholaminergic cells. We also determined the kinetic constants for peptides modeled after actual or potential tyrosine hydroxylase phosphorylation sites. We found that the best substrates for cyclic AMP-dependent protein kinase were those peptides corresponding to serine 40. Tyrosine hydroxylase (36-46), for example, exhibited a Km of 108 microM and a Vmax of 6.93 mumol/min/mg of catalytic subunit. The next best substrate was the peptide corresponding to serine 153. The peptide containing the sequence conforming to serine 19 was a very poor substrate, and that conforming to serine 172 was not phosphorylated to any significant extent. The primary structure of the actual or potential phosphorylation sites is sufficient to explain the substrate behavior of the native enzyme.     

Medial Forebrain Bundle Stimulation or D-2 Dopamine Receptor Activation Increases Preproenkephalin mRNA in Rat Striatum

Electrical stimulation of the medial forebrain bundle, in a manner that augmented the release of dopamine in the forebrain, rapidly increased the striatal content of preproenkephalin (but not preprotachykinin) mRNA. This effect was mimicked by administration of either the indirect (dopamine-releasing) agonist methamphetamine or by the D-2 dopamine receptor agonist quinpirole, but not by the D-1 agonist SKF 38393. These data suggest that D-2 receptors, which mediate a stimulatory effect on enkephalin gene expression, may be subsaturated under basal conditions and, therefore, responsive to increases in synaptic dopamine.     

Choline acetyltransferase activity of spinal cord cell cultures increased by co-culture with muscle and by muscle-conditioned medium

Activity of the enzyme choline acetyltransferase (CAT), which mediates the synthesis of the neurotransmitter, acetylcholine, was increased up to 20- fold in spinal cord (SC) cells grown in culture with muscle cells for 2 wk. This increase was directly related to the duration of co-culture as well as to the cell density of both the SC and muscle involved and was not affected by the presence of the acetylcholine receptor blocking agent, α-bungarotoxin. Glutamic acid decarboxylase (GAD) activity was often markedly decreased in SC-muscle cultures while the activities of acetylcholinesterase and several other enzymes were little changed. Increased CAT activity was also observed when SC cultures were maintained in medium which had been conditioned by muscle cells or by undifferentiated cells from embryonic muscle. Muscle-conditioned medium (CM) did not affect the activities of SC cell GAD or acetylcholinesterase. Dilution or concentration of the CM directly affected its ability to increase SC CAT activity , as did the duration and timing of exposure of the SC cells to the CM. The medium could be conditioned by muscle cells in the presence or absence of serum, and remained effective after dialysis or heating to 58 degrees C. Membrane filtration data were consistent with the conclusion that the active material(s) in CM had a molecular weight in excess of 50,000 daltons. We conclude that large molecular weight material that is released by muscle cells is capable of producing a specific increase in CAT activity of SC cells.     

Concentrations of iron correlate with the extent of protein, but not lipid, oxidation in advanced human atherosclerotic lesions

Previous studies have provided compelling evidence for the presence of oxidized proteins and lipids in advanced human atherosclerotic lesions. The catalyst responsible for such oxidation is unknown and controversial. We have previously provided evidence for elevated levels of iron in lesions. In this study we hypothesized that if iron ions catalyzed protein and lipid oxidation in the artery wall, then there should be a positive correlation between these parameters. Iron concentrations in ex vivo healthy human arteries and advanced carotid lesions were quantified by electron paramagnetic resonance spectroscopy. Four specific side-chain oxidation products of proteins, and the lipid oxidation products 7-ketocholesterol and cholesterol ester alcohols and hydroperoxides, were quantified by HPLC in the same samples used for the iron measurements. Parent amino acids, cholesterol, and cholesterol esters were also quantified. Statistically elevated levels of iron, cholesterol, cholesterol esters, 7-ketocholesterol, and cholesterol ester alcohols and hydroperoxides were detected in advanced lesions compared with healthy control tissue. Iron levels correlated positively and strongly with all four markers of protein oxidation, but not with either marker of lipid oxidation. These data support the hypothesis that elevated levels of iron contribute to the extent of protein, but not lipid, oxidation in advanced human lesions.     

Pavement Ant Workers (<Emphasis Type="Italic">Tetramorium caespitum</Emphasis>) Assess Cues Coded in Cuticular Hydrocarbons to Recognize Conspecific and Heterospecific Non-Nestmate Ants

Most ants live in closed societies from which non-members are excluded through fighting or ritualized displays to protect colony resources. Nestmate recognition is the process by which ants discriminate nestmate from non-nestmate ants. Ants use cues coded in mixtures of long-chain hydrocarbon compounds on the cuticle as nestmate recognition cues. Pavement ants (Tetramorium caespitum) form conspicuous wars between neighboring colonies that are organized after workers meet and make the decision to fight after assessing nestmate recognition cues. These wars involve thousands of individuals. Fighting is ritualized and few ants die in the process. We identified 24 cuticular hydrocarbon compounds, above 1% in relative abundance, in the profile of pavement ants with chain lengths ranging from 15 to 31 carbon atoms. Cuticular lipids contained, in order of abundance: mono-methyl alkanes (45–56%), n-alkanes (range: 16–40% relative abundance), and alkenes (10–20%), with small or trace amounts of di-methyl, tri-methyl alkanes and fatty acids. Results from behavioral tests show that pavement ants assess information in cuticular hydrocarbon profiles to recognize both conspecific and heterospecfic (Pogonomyrmex occidentalis and Camponotus modoc) non-nestmate ants and that the relative abundance of methyl-branched alkanes and alkenes codes for nestmate status, at least for conspecific interactions. Our data add to a growing body of knowledge about how ants use cuticular hydrocarbon based nestmate recognition cues to prevent the intrusion of non-nestmates in to colony space.     

A kinetic model of bone marrow neutrophil production that characterizes late phenotypic maturation

Acute inflammatory stimuli rapidly mobilize neutrophils from the bone marrow by shortening postmitotic maturation time and releasing younger neutrophils; however, the kinetics of this change in maturation time remains unknown. We propose a kinetic model that examines the rate of change in neutrophil average age at exit from the bone marrow during active mobilization to quantify this response and use this model to examine the temporal profile of late neutrophil phenotypic maturation. Total and CD10(-)/CD16(low) circulating neutrophils were quantified in cardiac surgery patients during extracorporeal circulation (ECC). Net growth in the circulating neutrophil pool occurred during the procedural (0.04 +/- 0.02 x 10(9) x l(-1) x min(-1)), warming (0.14 +/- 0.02 x 10(9) x l(-1) x min(-1)), and weaning (0.12 +/- 0.06 x 10(9) x l(-1) x min(-1)) phases of ECC. When applied to our differential equation mathematical model, these results predict that neutrophil average age at exit from the bone marrow decreased continually during ECC, resulting in average neutrophil release 8.44 +/- 2.20 h earlier during the weaning phase than at the beginning of ECC sampling. Modeling of concurrent changes in CD10(-)/CD16(low) neutrophil numbers indicates that CD10 expression is directly related to neutrophil mean age and predicts that the proportion of mobilizable postmitotic neutrophils that are CD10(+) increases from 64 to 81% during these sampled 8.4 h of maturation.