Complex genetic regulation of bone mineral density and insulin-like growth factor-I in C57BL/6J-Chr #A/J/NaJ chromosome substitution strains.

Low bone mineral density (BMD) is a phenotype associated with osteoporosis and increased risk of fracture. Since 60-80% of variation in BMD is associated with genetic factors, we used the novel approach of chromosome substitution strains (CSS) to identify chromosomes that harbor genes that regulate BMD. Specifically, we evaluated 24 wk old C57BL/6J-Chr #(A/J)/NaJ CSS (n = 7 to 18) in which each chromosome in the host C57BL/6J strain is replaced by a corresponding chromosome from the donor A/J strain (19 autosomes, X, Y). We determined several A/J chromosomes contribute to body weight (BW), percent body fat (BF), whole body areal BMD (aBMD), and serum insulin-like growth factor (IGF)-I in both a positive and negative manner when compared with C57BL/6J. Specifically, C57BL/6J-Chr 5(A/J)/NaJ (B.A-5) (males) and B.A-13 (females) contributed to increased BW, whereas B.A-3, 4, 8, 9, 12, and 18 (males) and B.A-3, 4, and 11 (females) contributed to reduced BW. B.A-5 (males) and B.A-13 (females) contributed to increased BF, whereas B.A-12 (males) and B.A-3, 4, 10, and 11 (females) contributed to reduced BF. B.A-14 (females) contributed to increased aBMD and B.A-1, 2, 6, 9, 10, and 18 (males) and B.A-8, 9, and 10 (females) contributed to reduced aBMD. To determine if similar chromosomes regulate aBMD and IGF-I, we determined serum concentrations of IGF-I. B.A-14 and Y (males) and B.A-6 (females) contributed to increased IGF-I and male B.A-3 and female B.A-8 contributed to reduced IGF-I. Overall, we identified several sex-dependent and -independent chromosomes that regulate aBMD and IGF-I in adult mice.

Enhanced retroviral transduction of 5-fluorouracil-resistant human bone marrow (stem) cells using a genetically modified packaging cell line.

Pluripotent hematopoietic stem cells (PHSC) are rare cells capable of multilineage differentiation, long-term reconstituting activity and extensive self-renewal. Such cells are the logical targets for many forms of corrective gene therapy, but are poor targets for retroviral mediated gene transfer owing to their quiescence, as retroviral transduction requires that the target cells be cycling. To try and surmount this problem we have constructed a retroviral producer line that expresses the membrane-bound form of human stem cell factor (SCF) on its cell surface. These cells are capable, therefore, of delivering a growth signal concomitant with recombinant retroviral vector particles. In this report we describe the use of this cell line to transduce a highly quiescent population of cells isolated from adult human bone marrow using the 5-fluorouracil (FU) resistance technique of Berardi et al. Quiescent cells selected using this technique were transduced by cocultivation with retroviral producers expressing surface bound SCF or with the parent cell line that does not. Following coculture, the cells were plated in long-term bone marrow culture for a further 5 weeks, before plating the nonadherent cells in semisolid media. Colonies forming in the semisolid media over the next 14 days were analyzed by polymerase chain reaction for the presence of the retroviral vector genome. Over six experiments, the transduction frequency of the quiescent 5-FU resistant cells using the SCF-expressing producer line averaged about 20%, whereas those transduced using the parent producer line showed evidence of reduced levels or no transduction.

Analysis of the neuronal promoter of the rat aromatic L-amino acid decarboxylase gene.

The rat aromatic L-amino acid decarboxylase (AADC) gene contains alternative promoters directing expression of neuronal and nonneuronal mRNAs that differ only in their 5' untranslated regions (UTRs). We have analyzed the expression of the neuronal promoter of the AADC gene in cells synthesizing catecholamines and serotonin, as well as in non-AADC-expressing cells. We demonstrate the use of the neuronal-specific UTR in individual dopamine-, norepinephrine-, and serotonin-containing neurons. Transfection analyses show that the rat AADC neuronal promoter, containing 2,400 bp upstream of the transcription start site and including the 68-bp untranslated exon 2, can activate transcription from a reporter gene in both catecholaminergic and serotonergic cell lines. These analyses identified several positive and negative cis-active elements within this region. Unexpectedly, we observed that this promoter, when removed from its native context within the AADC gene, can also direct expression of a reporter gene in cells that do not normally express AADC mRNA. These results suggest that tissue-specific expression of the neuronal promoter may not be controlled by cis-active elements within the first 2,400 bp of the promoter. Additional information may be required to restrict neuronal promoter expression to appropriate cell types. This regulatory information could reside elsewhere within the promoter, within introns, or may be provided by interactions between the two AADC promoters.

Distribution of messenger RNAs encoding enkephalin, substance P, somatostatin, galanin, vasoactive intestinal polypeptide, neuropeptide Y, and calcitonin gene-related peptide in the midbrain periaqueductal grey in the rat.

The midbrain periaqueductal grey matter (PAG) has numerous functional roles that include mediating nociceptive inhibition and integrating behavioural and physiological responses to potentially threatening or stressful stimuli. Underlying these behaviours is the diverse interconnectivity of this region, and it is possible that neurochemical subdivisions within the PAG reflect the functional properties of the different PAG regions. In this study, using in situ hybridization, we have investigated the distribution in the rat PAG of the messenger ribonucleic acids (mRNAs) encoding seven neuropeptides: enkephalin (ENK), substance P (SP), somatostatin (SST), galanin (GAL), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), and calcitonin gene-related peptide (CGRP). Each peptide mRNA had a distinct topographical distribution in the PAG. Preproenkephalin A (ENK) mRNA-expressing cells were found at all levels of the PAG in three distinct longitudinal columns. Preprotachykinin A (SP)-expressing cells were found at all levels of the PAG, principally in the Edinger-Westphal nucleus and the lateral and dorsal PAG. There was a column of neurons producing mRNA-encoding somatostatin that extended along the rostrocaudal extent of the ventrolateral PAG; there were also labelled cells in the dorsal and dorsolateral subdivisions at some levels of the PAG. Galanin mRNA-producing neurones were limited to the dorsal raphe nucleus and to a second population in the ventral border of the aqueduct. VIP mRNA-producing neurones were found in very localized regions of the PAG, including the cell-sparse region immediately ventral to the aqueduct and the ventral part of the dorsal raphe nucleus. NPY mRNA-producing neurones were localized mainly in some cells of the Edinger-Westphal nucleus and dorsal raphe nucleus. CGRP mRNA-expressing neurons were limited to the oculomotor and trochlear nucleus. The results showed a topographical distribution of neuropeptides over the rostrocaudal extent of the PAG that is compatible with the emerging theory that the anatomical and functional specificity of the PAG is expressed in the form of longitudinally arranged neuronal columns that extend for varying distances along the rostrocaudal axis of the midbrain PAG.

Chronic D-fenfluramine decreases serotonin transporter messenger RNA expression in dorsal raphe nucleus.

In situ hybridization was used to measure the effects of chronic fenfluramine administration on serotonin transporter messenger RNA expression in cells of the dorsal raphe nucleus complex. Fenfluramine produced a significant, but transient, down-regulation of serotonin transporter mRNA in cells which lie in the ventral portion of the dorsal raphe nucleus, but not in the dorsal part of the dorsal raphe nucleus. Our findings suggest that cells which lie in the ventral part of the dorsal raphe nucleus are more sensitive to the effects of chronic fenfluramine administration, but that fenfluramine does not cause long-term changes in gene expression in serotonin cell bodies.

Effect of cortical kindling on [3H]D-aspartate uptake and glutamate metabolism in rats.

The effects of cortical kindling in rats on [3H]D-aspartate uptake and on glutaminase and glutamine synthetase activities has been studied. The high affinity uptake of [3H]D-aspartate in control cortical tissue (Km approximately 2 microM) was undetectable in the kindled tissue, whilst the enzyme activities were unchanged. A loss of the high-affinity uptake sites for excitatory amino acids may be a contributing factor to the kindling phenomenon.

Pharmacological studies on lamotrigine, a novel potential antiepileptic drug: II. Neurochemical studies on the mechanism of action.

Lamotrigine (LTG) [3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine] is a novel anticonvulsant chemically unrelated to current antiepileptic drugs and with a pharmacological profile similar to that of phenytoin. The effect of LTG has been compared with that of phenytoin, on the release of endogenous amino acids and radiolabelled acetylcholine evoked by veratrine or potassium, from slices of rat cerebral cortex in vitro. Both veratrine and potassium evoked a marked release of glutamate and gamma-aminobutyric acid (GABA), with a more moderate release of aspartate. LTG inhibited veratrine-evoked release of glutamate and aspartate, with ED50 values of 21 microM for both amino acids, but LTG was less potent in the inhibition of GABA release (ED50 = 44 microM). At concentrations up to 300 microM, LTG had no effect on potassium-evoked amino acid release or on spontaneous release. Also, LTG was some five times less potent in the inhibition of veratrine-evoked [3H]acetylcholine release (ED50 = 100 microM) than in glutamate or aspartate release. The total lack of effect of LTG on potassium-evoked release and the potent effect on veratrine-evoked release (at concentrations found in rat brain after anticonvulsant doses) strongly suggest that LTG acts at voltage-sensitive sodium channels to stabilise neuronal membranes and inhibit transmitter release, principally glutamate. The role of glutamate in the aetiology of epilepsy is discussed.

(+/-)-cis-2,3-Piperidine dicarboxylic acid is a partial N-methyl-D-aspartate agonist in the in vitro rat cerebellar cGMP model.

The effect of (+/-)-cis-2,3-piperidine dicarboxylic acid [+/-)-cis-2,3-PDA) on formation of cyclic GMP by immature (7-8 day) rat cerebellar slices has been studied. Using magnesium free medium containing the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX), (+/-)-cis-2,3-PDA behaves as an NMDA partial agonist. Thus in this medium, (+/-)-cis-2,3-PDA stimulates cyclic GMP formation, an effect completely blocked by the potent, specific NMDA antagonist (+/-)-2-amino-7-phosphonoheptanoic acid [+/-)-APH) with a Ki = 17.1 microM. The production of cyclic GMP by the full agonist (+/-)-trans-2,3-PDA, was also blocked by (+/-)-APH, suggesting that in this preparation it activates NMDA receptors. (+/-)-trans-2,3-PDA was approximately half as potent as NMDA. By constructing dose response curves to NMDA in the presence of increasing concentrations of (+/-)-APH or (+/-)-APV, these compounds were shown to be competitive NMDA antagonists using Schild analysis.

Changes in cortical amino acids during electrical kindling in rats.

The effect of kindling rats by electrical stimulation of the frontal cortex for approx. 3 months on the concentrations of amino acids (taurine, aspartate, glutamate, glutamine and GABA) in the cerebral cortex has been studied, as well as the release of endogenous amino acids from kindled slices of brain in vitro. In these kindled rats, killed 1 week after the last shock, there were no changes in any concentrations of amino acids. However, when cortical slices, prepared from either the control or kindled rats, were stimulated in vitro by exposure to veratrine, more endogenous glutamate and aspartate were released from the kindled tissue than from the control. The neurotransmitter amino acids, glutamate and aspartate, may be involved in the permanent effects of electrical kindling.