Astrocyte-specific inactivation of the neurofibromatosis 1 gene (NF1) is insufficient for astrocytoma formation

Individuals with the neurofibromatosis 1 (NF1) inherited tumor syndrome develop low-grade gliomas (astrocytomas) at an increased frequency, suggesting that the NF1 gene is a critical growth regulator for astrocytes. In an effort to determine the contribution of the NF1 gene product, neurofibromin, to astrocyte growth regulation and NF1-associated astrocytoma formation, we generated astrocyte-specific Nf1 conditional knockout mice (Nf1(GFAP)CKO) by using Cre/LoxP technology. Transgenic mice were developed in which Cre recombinase was specifically expressed in astrocytes by embryonic day 14.5. Successive intercrossing with mice bearing a conditional Nf1 allele (Nf1flox) resulted in GFAP-Cre Nf1flox/flox (Nf1(GFAP)CKO) animals. No astrocytoma formation or neurological impairment was observed in Nf1(GFAP)CKO mice after 20 months, but increased numbers of proliferating astrocytes were observed in several brain regions. To determine the consequence of Nf1 inactivation at different developmental times, the growth properties of embryonic day 12.5 and postnatal day 2 Nf1 null astrocytes were analyzed. Nf1 null astrocytes exhibited increased proliferation but lacked tumorigenic properties in vitro and did not form tumors when injected into immunocompromised mouse brains in vivo. Collectively, our results suggest that loss of neurofibromin is not sufficient for astrocytoma formation in mice and that other genetic or environmental factors might influence NF1-associated glioma tumorigenesis.     

A compartmental model of an external urethral sphincter motoneuron of Onuf's nucleus

This article discusses a model of the electrical behavior of an external urethral sphincter motoneuron, based on morphological parameters like soma size, dendritic diameters and spatial dendritic configuration, and several electrical parameters. Because experimental data about the exact ion conductance mix of external urethral sphincter neurons is scarce, the gaps in knowledge about external urethral sphincter motoneurons were filled in with known data of alpha-motoneurons. The constructed compartmental model of motoneurons of Onuf's nucleus contains six voltage-dependent ionic conductances: a fast sodium and potassium conductance and an anomalous rectifier in the soma; a fast delayed rectifier type potassium conductance and a fast sodium conductance in the initial axon segment; an L-type calcium channel in the dendritic compartments. This paper considers the simulation of external urethral sphincter motoneuron responses to current injections that evoke bistable behavior. Simulations show self-sustained discharge following a depolarizing pulse through the microelectrode; the firing was subsequently terminated by a short hyperpolarizing pulse. This behavior is highly functional for neurons that have to exhibit prolonged activation during sphincter closure. In addition to these 'on' and 'off ' responses, we also observed a particular firing behavior in response to long-lasting triangular current pulses. When the depolarizing current was slowly increased and then decreased (triangular pulse) the firing frequency was higher during the descending phase than during the initial ascending phase.     

Bioactivatable, membrane-permeant analogs of cyclic nucleotides as biological tools for growth control of C6 glioma cells

In the present study, the cAMP analogs 8-bromo-cAMP (8-Br-cAMP), N6-2'O-dibutyryl-cAMP (DBcAMP) and 8-para-chlorophenylthio-cAMP (8-CPT-cAMP), as well as the corresponding cAMP-acetoxymethyl (AM)-ester-prodrugs were tested in a HPLC study for their membrane permeability, intracellular accumulation and biotransformation. Antiproliferative activities of these compounds were studied in the rat C6 glioma cell line. Chromatographic analysis revealed that the AM-ester analogs of the cyclic nucleotides penetrate quantitatively into rat C6 glioma cells and generate high amounts of their parent cyclic nucleotides intracellularly within 60 min; however, long-term growth inhibition tested in C6 cells is only slightly enhanced with the AM-ester prodrugs of 8-Br-cAMP or DBcAMP.     

Interactions between Allosteric Modulators and 4-DAMP and Other Antagonists at Muscarinic Receptors: Potential Significance of the Distance between the N and Carboxyl C Atoms in the Molecules of Antagonists

Allosteric enhancement of the affinity of muscarinic receptors for their ligands offers a new way to influence cholinergic neurotransmission. The structure of the allosteric binding domain(s) and the features of agonists, antagonists and modulators which determine the occurrence of either positive or negative cooperativity require clarification. We tested interactions between allosteric modulators alcuronium, strychnine and brucine and eight antagonists at muscarinic receptors expressed in CHO cells. In experiments with unlabeled antagonists, all three modulators enhanced the affinity for 4-diphenylacetoxy-N-dimethylpiperidinium (4-DAMP) at the M₂ receptors, and strychnine did so also at the M₄ receptors. Positive interactions were also observed between alcuronium and L-hyoscyamine (M₂) and scopolamine (M₂), between strychnine and butylscopolamine (M₄), L-hyoscyamine (M₂ and M₄) and scopolamine (M₄), and between brucine and scopolamine (M₂). Positive effects of alcuronium, strychnine and brucine on the affinity of the M₂ receptors for 4-DAMP have been confirmed by direct measurements of the binding of [³H]-4-DAMP. A comparison of molecular models of several antagonists which are esters revealed that antagonists in which the distance between the N and the carboxyl C atoms corresponds to five chemical bonds are more likely to display positive cooperativity with alcuronium at the M₂ receptors than the antagonists in which the N-carboxyl C distance corresponds to four chemical bonds.     

Expression of cucumber lipid-body lipoxygenase in transgenic tobacco: lipid-body lipoxygenase is correctly targeted to seed lipid bodies

 A particular isoform of lipoxygenase (LOX, EC 1.13.11.12) localized on lipid bodies has been shown by earlier investigations to play a role during seed germination in initiating the mobilization of triacylglycerols. On lipid bodies of germinating cucumber (Cucumis sativus L.) seedlings, the modification of linoleoyl moieties by this LOX precedes the hydrolysis of the ester bonds. We analyzed the expression and intracellular location of this particular LOX form in leaves and seeds of tobacco (Nicotiana tabacum L.) transformed with one construct coding for cucumber lipid-body LOX and one construct coding for cucumber LOX fused with a hemagglutinin epitope. In both tissues, the amount of lipid-body LOX was clearly detectable. Biochemical analysis revealed that in mature seeds the foreign LOX was targeted to lipid bodies, and the preferred location of the LOX on lipid bodies was verified by immunofluorescence microscopy. Cells of the endosperm and of the embryo exhibited fluorescence based on the immunodecoration of LOX protein whereas very weak fluorescent label was visible in seeds of untransformed control plants. Further cytochemical analysis of transformed plants showed that the LOX protein accumulated in the cytoplasm when green leaves lacking lipid bodies were analyzed. Increased LOX activity was shown in young leaves of transformed plants by an increase in the amounts of endogenous (2E)-hexenal and jasmonic acid. Received: 9 August 1999 / Accepted: 28 September 1999     

Developmental expression of neuromodulators in the central complex of the grasshopper Schistocerca gregaria

The central complex is a major integrative region within the insect brain with demonstrated roles in spatial orientation, the regulation of locomotor behavior, and sound production. In the hemimetabolous grasshopper, the central complex comprises the protocerebral bridge, central body (CB), ellipsoid body, noduli, and accessory lobes, and this modular organization develops entirely during embryogenesis. From a biochemical perspective, a range of neuroactive substances has been demonstrated in these modules of the adult central complex, but little is known about their developmental expression. In this study, we use matrix‐assisted laser desorption/ionization‐imaging mass spectrometry on single brain slices to confirm the presence of several peptide families (tachykinin, allatostatin, periviscerokinin/pyrokinin, FLRFamide, and neuropeptide F) in the adult central complex and then use immunohistochemistry and histology to examine their developmental expression, together with that of the indolamin serotonin, and the endogenous messenger nitric oxide (NO; via its synthesizing enzyme). We find that each neuromodulator is expressed according to a unique, stereotypic, pattern within the various modules making up the central complex. Neuropeptides such as tachykinin (55%) and allatostatin (65%), and the NO‐synthesizing enzyme diaphorase (70%), are expressed earlier during embryonic development than the biogenic amine serotonin (80%), whereas periviscerokinin‐like peptides and FLRFamide‐like peptides begin to be expressed only postembryonically. Within the CB, these neuroactive substances are present in tangential projection neurons before they appear in columnar neurons. There is also no colocalization of serotonin‐positive and peptide‐positive projections up to the third larval instar during development, consistent with the clear dorsoventral layering of the neuropil we observe. Our results provide the first neurochemical fingerprint of the developing central complex in an hemimetabolous insect. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Timing of reproduction in a Darwin''s finch: temporal opportunism under spatial constraints

We investigated whether predation by the minor grison ( Galictis cuja , a small mustelid) played a key role in limiting a wild cavy population ( Cavia magna ), ultimately leading to its local extinction. Radio-telemetry and capture-mark-recapture techniques were used to estimate grison predation rates (kill rates), time-specific probabilities of apparent mortality (population loss rate), overall mortality and grison predation for the cavy population. Additionally, we present data on alternative prey species, grison diet and reproduction to show potential proximate mechanisms of grison predation on wild cavies. The predictions specified were mostly confirmed: (1) grison predation was responsible for almost 80% of the cavies killed by known predators; (2) grison predation probabilities paralleled those of overall mortality of cavies over time; and (3) also those of the apparent mortality of the population. Thus, the population dynamics and the local extinction of the cavy population were not due to emigration processes. (4) Grison predation rates were not density-dependent, but showed pronounced peaks during the austral summer. The grison mainly preyed on small mammals: two water-rat species and the wild cavies. When the availability of alternative prey decreased in summer, the grison appeared to specialise on cavies. The onset of grison reproduction was somewhat delayed in relation to the onset of cavy reproduction. The lack of alternative prey coincided with high grison food demands due to reproduction, leading to a very high predation pressure ultimately resulting in the local extinction of the cavy population. We conclude that grison predation was indeed the main factor driving changes of the cavy population studied and speculate why caviomorph rodents might be especially susceptible to local extinction processes.