Regulation of Vesicle Traffic and Neurotransmitter Release in Isolated Nerve Terminals

In this overview current insights in the regulation of presynaptic transmitter release, mainly acquired in studies using isolated CNS nerve terminals are highlighted. The following aspects are described. (i) The usefulness of pinched-off nerve terminals, so-called synaptosomes, for biochemical and ultrastructural studies of presynaptic stimulus-secretion coupling. (ii) The regulation of neurotransmitter release by multiple Ca²⁺ channels, with special emphasis on the specificity of different classes of these channels with respect to the release of distinct types of neurotransmitters, that are often co-localized, such as amino acids and neuropeptides. (iii) Possible molecular mechanisms involved in targeting synaptic vesicle (SV) traffic toward the active zone. (iv) The role of presynaptic receptors in regulating transmitter release, with special emphasis on different glutamate subtype receptors. Isolated nerve terminals are of great value as model system in order to obtain a better understanding of the regulation of the release of distinct classes of neurotransmitters in tiny CNS nerve endings.     

Three-color flowcytometric analysis of mature and immature hematological malignancies. A guideline of the Dutch Foundation for Immunophenotyping of Hematological Malignancies (SIHON)

Multiparameter flowcytometry offers an insight into differentiation pathways, maturation stages and abnormal features of cell (sub)populations thus helping to establish and classify hematological malignancies. The Dutch Foundation for Immunophenotyping of Hematological Malignancies (SIHON) has formulated a guideline for a rapid screening followed by confirmation and classification in a standardized way. For this aim seven carefully composed monoclonal antibody combinations are elucidated for screening the test sample in a first phase. In this phase a relative frequency distribution of the cells will be established and a decision will be made about abnormal cells present, as well as their mature or immature state and the cell lineage they belong to. In a second phase, panels with cell lineage dependent monoclonal antibody combinations may be used to confirm and classify the abnormal cell population indicated in phase 1, as well as to establish the presence or absence of an abberant immunophenotype.     

Development of an inexpensive, low attenuation styrofoam primate chair for use in a PET scanner

Pharmacokinetic modelling of radiotracers for positron emission tomography (PET) imaging of neuroreceptors can be performed with time-activity data for brain and blood. We aimed to develop an alternative to withdrawal of arterial blood samples for acquisition of a blood curve. A supportive primate chair was constructed out of styrofoam and fixed to the head portion of the bed of a PET scanner. A lightly anaesthetised rhesus monkey was positioned in the chair in a sitting position and injected with the radiotracer. The styrofoam chair provided sufficient support for the monkey. The presence of the chair in the PET scanner caused negligible attenuation of radiation, allowing simultaneous acquisition of dynamic data from the subject's brain and heart. We conclude that a styrofoam primate chair is an ideal tool to measure blood and brain data from a rhesus monkey with PET. Invasiveness to the animal is reduced, as well as experimenter time.     

Inhibition of L- and P-selectin by a rationally synthesized novel core 2-like branched structure containing GalNAc-Lewisx and Neu5Acalpha2- 3Galbeta1-3GalNAc sequences

The selectins interact in important normal and pathological situations with certain sialylated, fucosylated glycoconjugate ligands containing sialyl Lewisx(Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcN Ac). Much effort has gone into the synthesis of sialylated and sulfated Lewisxanalogs as competitive ligands for the selectins. Since the natural selectin ligands GlyCAM-1 and PSGL-1 carry sialyl Lewisxas part of a branched Core 2 O-linked structure, we recently synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(SE-3Galbeta1++ +-3)GalNAc1alphaOMe and found it to be a moderately superior ligand for L and P-selectin (Koenig et al. , Glycobiology 7, 79-93, 1997). Other studies have shown that sulfate esters can replace sialic acid in some selectin ligands (Yeun et al. , Biochemistry, 31, 9126-9131, 1992; Imai et al. , Nature, 361, 555, 1993). Based upon these observations, we hypothesized that Neu5Acalpha2-3Galbeta1-3GalNAc might have the capability of interacting with L- and P-selectin. To examine this hypothesis, we synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Neu5Acalpha2++ +-3Galbeta1-3)- GalNAc alpha1-OB, which was found to be 2- to 3-fold better than sialyl Lexfor P and L selectin, respectively. We also report the synthesis of an unusual structure GalNAcbeta1-4(Fucalpha1- 3)GlcNAcbeta1-OMe (GalNAc- Lewisx-O-methyl glycoside), which also proved to be a better inhibitor of L- and P-selectin than sialyl Lewisx-OMe. Combining this with our knowledge of Core 2 branched structures, we have synthesized a molecule that is 5- to 6-fold better at inhibiting L- and P-selectin than sialyl Lewisx-OMe, By contrast to unbranched structures, substitution of a sulfate ester group for a sialic acid residue in such a molecule resulted in a considerable loss of inhibition ability. Thus, the combination of a sialic acid residue on the primary (beta1-3) arm, and a modified Lexunit on the branched (beta1-6) arm on an O-linked Core 2 structure generated a monovalent synthetic oliogosaccharide inhibitor superior to SLexfor both L- and P-selectin.      

Beneficial effect of fluorocarbon emulsion media on the function of neuromuscular preparations in vitro

The effects of liquid fluorocarbons as bathing media were determined by use of in vitro neuromuscular preparations. Rat hemidiaphragms were bathed in either oxygenated fluorocarbon (FC) emulsion or standard oxygenated Krebs solution. Contractile force in response to simple supramaximal nerve stimuli as well as to high frequency stimulation was greater, while twitch:tetanus ratio was smaller in FC emulsion. With such medium, post-tetanic potentiation of contraction was also more consistently observed. Indirectly stimulated diaphragms survived longer in FC emulsion. After cessation of oxygenation, oxygen tension (ρO(2)) of the medium declined more rapidly with Krebs than with FC emulsion; ρO(2) directly correlated with force of contraction. Similarly, in the chick biventer cervicis preparation, FC emulsion enhanced nerve-stimulated force of contraction; returning the preparation to standard Krebs solution reversed this phenomenon. Dose-resonse curves of muscle contraction in response to acetycholine and KCl administration were shifted upward during FC emulsion superfusion. Frequency of miniature endplate potentials was lower in FC emulsion than that observed in Krebs solution, measured from the same cell of the rat diaphragm. Resting membrane potentials were also greater in muscle cells sampled from FC emulsion-bathed preparations. These data suggest that FC emulsion is superior to standard Krebs solution as a bathing medium for in vitro neuromuscular preparations by virtue of the high solubility of oxygen in it.     

Kinetic Modeling of 52Fe/52mMn-Citrate at tthe Blood-Brain Barrier by Positron Emission Tomography

The kinetics of iron at the blood-brain barrier of the monkey were studied in vivo using positron emission tomography (PET) and the tracer 52Fe/52mMn-citrate. 52mMn is the beta(+)-emitting daughter nuclide of 52Fe and therefore contributes to the observed signal and background in the PET images and may influence the quantification of physiological relevant iron parameters. The kinetics of pure (52m)Mn-citrate at the blood-brain barrier of the monkey were studied experimentally, and the analysis of the data with a reasonable compartment model led to equal efflux and influx parameters for Mn (1.35 +/- 0.3 x 10(-2) min(-1)). By using complexes between Mn and diethylenetriaminepentaacetic acid, the validity of the proposed model could be confirmed. To describe the observed kinetics of 52Fe/(52m)Mn-citrate, the manganese model was coupled to an iron model, which finally allowed the quantification of two iron-specific parameters: an input rate into global brain tissue of 7.15 +/- 2.6 x 10(-4) min(-1) and a time delay of roughly 24 min to account for the observed activities. The simpler linearization procedure has been proposed and could be applied to all our data sets and is able to replace the complicated nonlinear iron/manganese tracer kinetic model neglecting any influence of manganese on the analysis.     

Ghrelin is suppressed by glucagon and does not mediate glucagon-related growth hormone release

BACKGROUND: Glucagon stimulation is routinely used as a provocative test to assess growth hormone (GH) sufficiency in pediatrics. Ghrelin also markedly stimulates GH secretion. Because glucagon stimulates the promoter of the ghrelin gene in vitro as well as ghrelin secretion by the perfused rat stomach, we sought to determine whether ghrelin mediates glucagon-induced GH secretion. METHODS: We compared ghrelin, GH, insulin and glucose responses following administration of 0.03 mg/kg intravenously (iv; max. 1 mg) and 0.1 mg/kg intramuscularly (im; max. 2 mg) of glucagon in two groups (n = 10-11/group) of GH-sufficient children. We also measured ghrelin before and 6 min after iv administration of 1 mg glucagon in 21 adult subjects. RESULTS: In children, glucagon caused a 26% decrease in ghrelin and a 72% increase in glucose concentrations that were independent of the dose or administration route of glucagon. In contrast, the insulin response was 2-3 times higher following administration of 0.1 mg/kg im compared to 0.03 mg/kg of glucagon iv. There was a significant correlation between the maximum decrease in ghrelin and increases in glucose (p = 0.03) but not in insulin. There was a significant correlation between ghrelin and GH area under the curve after controlling for the dose of glucagon (p = 0.03) but not for the maximum increase in glucose.In normal adults, glucagon administration caused a 7% decrease in ghrelin concentrations after 6 min (p = 0.0002). CONCLUSION: Ghrelin does not play a causal role in the GH response to pharmacological glucagon administration, which suppresses ghrelin levels starting a few minutes after injection.