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  • 1
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Concentrations of vasopressin (VP) precursor and oxytocin (OT) precursor mRNA were measured in magno-cellular cell groups of the rat hypothalamus by newly developed solution hybridization assays. The assays employed single-stranded 35S-labeled VP-specific and OT-specific DNA probes that were prepared by primer extension on re-combinant M13 DNA templates. Solution hybridization assays were standardized by known amounts of cloned DNA. The detection limit was 〈1 pg DNA equivalent of the respective mRNA. In total RNA preparations of microdis-sected supraoptic nucleus (SON) mean (SEM) basal levels of 1.370.18pgVP mRNA and 1.950.I4pgOT mRNA were measured. RNA of the microdissected paraventricular nucleus (PVN) contained 0.35 ± 0.02 pg VP mRNA and 1.77 ± 0.15 pg OT mRNA. Elevation of plasma osmolality induced by drinking of 2% saline for 25 days resulted in a 1.85-fold increase in VP mRNA levels of the SON and a 1.6-fold increase in VP mRNA levels of the PVN. The solution hybridization assays are suitable tools to study the regulation of VP and OT mRNAs in magnocellular neurons of the brain.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Incubation of β-endorphin with cytosolic and particulate fractions of rat brain resulted in the formation of several peptides, including γ-endorphin [β-endorphin-(1–17)] and β-endorphin-(18–31), indicating the presence of enzyme activity cleaving the Leu17-Phe18 bond of β-endorphin. An assay for this Leu-Phe cleaving activity, based on the cleavage of the 14C-labeled substrate acetyl-Val-Thr-Leu-Phe-[ε ([14C]CH3)2]Lys-NHCH3, was used to examine the properties of this enzyme activity. β-Endorphin-(1–31) competitively inhibited the Leu-Phe-cleaving enzyme activity on the pentapeptide substrate. Over 90% of activity was recovered in the cytosolic fraction. Leu-Phe-cleaving activity behaved like a thiol endopeptidase because it was inhibited by low concentrations of N-ethylmaleimide, p-chloromercuribenzoate, p-chloromercuribenzoyl sulfate, and low concentrations of Hg2+. Low concentrations of sulfhydryl compounds stimulated Leu-Phe-cleaving activity. The activity was optimal between pH 8.5 and 9.0. The Km of Leu-Phe-cleaving activity in the cytosolic fraction was 35 μM and in the particulate fraction 88 μM with Vmax values of 193 and 15 nmol mg protein−1 h−1, respectively. The apparent molecular mass of the Leu-Phe-cleaving enzyme was estimated by gel filtration to be ∼200 kilodaltons. These properties of Leu-Phe-cleaving activity indicate that the Leu-Phe-cleaving enzyme is distinct from any known brain endopeptidase.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Three different transcripts of the homeodomain gene termed pituitary homeobox (Ptx) 2 (Pitx2/Brx/Rieg/Solurshin/Arp) were cloned from different species encoding proteins belonging to the paired-like family of homeodomain proteins. Ptx2a (324 amino acids), Ptx2b (271 amino acids), and Ptx2c (318 amino acids) share the C terminus, including the homeodomain, and have different N termini. Here we report the comparative analysis of all three different Ptx2 splice variants for their transcriptional activity and their expression pattern in the adult rat brain. Ptx2 is able to trans-activate via different model promoters in different cell lines. A mild difference in trans-activating potential is observed among the splice variants, but the underlying mechanism is at present unknown. It is surprising that all Ptx2 transcripts displayed an identical expression pattern in the brain. This markedly restricted pattern is limited to the following brain areas: the anterior and intermediate lobes of the pituitary gland, the subthalamic nucleus, the posterior hypothalamic nucleus, the mammillary bodies, the red nucleus, and the deep gray layer of the superior colliculus. The data presented suggest that all variants of Ptx2 are involved in the development and regulation of distinct neuronal cell groups and the pituitary gland.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Oxford UK : Blackwell Science Ltd.
    Journal of neurochemistry 74 (2000), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of catecholamines, which takes place in different types of neuronal systems and nonneuronal tissues. The transcriptional regulation of the TH gene, which is complex and highly variable among different tissues, reflects this heterogeneity. We recently isolated a homeodomain transcription factor, named Ptx3, that is uniquely expressed in the dopaminergic neurons of the substantia nigra pars compacta and ventral tegmental area, which together form the mesencephalic dopaminergic system. This strict localization and its coinciding induction of expression with the TH gene during development suggested a possible role for this transcription factor in the control of the TH gene. We report here the presence of a responsive element for Ptx3 located at position -50 to -45 of the rat TH promoter. Transient transfections using TH promoter constructs and electrophoretic mobility shift assays using Ptx3-containing nuclear extracts demonstrated that this region binds Ptx3 protein and confers a transcriptional effect on the TH gene. Depending on the cell type, the effect of Ptx3 was an eight- to 12-fold enhancement of TH promoter activity in Neuro2A neuroblastoma cells, or a 60-80% repression in nonneuronal human embryonic kidney 293 cells. Despite the close association of the Ptx3-binding site and the major cyclic AMP-response element in the TH gene, no interplay was found between Ptx3 and cyclic AMP-modulating agents. In combination with the orphan nuclear receptor Nurr1, which is required for the induction of the TH gene in mesencephalic dopaminergic neurons, the TH promoter activity to Ptx3 was enhanced in Neuro2A cells. Nurr1 alone displayed only very weak activity on the TH promoter in this cell type. The results demonstrate that the homeodomain protein Ptx3 has the potential to act on the promoter of the TH gene in a markedly cell type-dependent fashion. This suggests that Ptx3 contributes to the regulation of TH expression in mesencephalic dopaminergic neurons.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Annals of the New York Academy of Sciences 684 (1993), S. 0 
    ISSN: 1749-6632
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Natural Sciences in General
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Annals of the New York Academy of Sciences 652 (1992), S. 0 
    ISSN: 1749-6632
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Natural Sciences in General
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neuroendocrinology 3 (1991), S. 0 
    ISSN: 1365-2826
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neuroendocrinology 1 (1989), S. 0 
    ISSN: 1365-2826
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Post-translational modifications of vasopressin and oxytocin in pituitary and brain were investigated in view of recent evidence that oxytocin is partly Nα-acetyfated in the bovine pineal gland. Two peptides were isolated from the neurointermediate lobe of the rat pituitary gland and characterized as Nα-acetyl-vasopressin and Nα-acetyl-oxytocin, based on chromatographic and immunological properties as well as the blocked N-terminus. In the neurointermediate pituitary the acetylated forms represented approximately 1% of the vasopressin and oxytocin contents. These two peptides were also detected in some, but not all, investigated brain areas. The highest degree of acetylation was found in the pineal gland. In all regions acetylation of oxytocin was more abundant than that of vasopressin. The data indicate that acetylation of vasopressin and oxytocin generally occurs as a post-translational modification. They support the concept that acetylation may represent a mechanism aimed to control bioactivity of the neurohypophyseal hormones.
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The hypothalamo-neurohypophysial system (HNS) is composed of hypothalamic magnocellular neurons and neural lobe pituicytes that accommodate the nerve terminals. Here we have investigated if the communication of the peptidergic neurons of the HNS with neighbouring cells plays a role in the development and assembly of the HNS. We employed munc18–1-deficient mice, which completely lack neurotransmitter secretion. Morphological and immunohistological analysis of the HNS in these mutant embryos during brain development showed that this peptidergic system was formed normally during early embryogenesis. However, the development arrested at embryonal day 14.5, the stage when terminal differentiation has to take place. The peptidergic neurons targeted axons in the correct direction, but few arrived at their final location and the neurons were not maintained in later stages. The pituicytes in the neural lobe of the pituitary were generated, but failed to organize normally. Our results indicate that peptide gene expression, axon outgrowth and migration are intrinsic developmental events in these peptidergic neurons, that are initiated in the munc18–1 null mutant. The further expansion and the integration of outgrowing axon terminals with neural lobe pituicytes requires munc18–1-dependent processes, probably exocytosis, at multiple levels. Firstly, to maintain and propagate neuronal outgrowth and guidance, and secondly, to control the cellular organization of the pituicytes. Thus, the communication between the outgrowing neurons and the pituicytes could serve to integrate these two cell types to constitute a functional peptidergic system.
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Chronic treatment with the opioid antagonist naltrexone induces functional supersensitivity to opioid agonists, which may be explained by receptor up-regulation induced by opioid receptor blockade. In the present study, the levels of opioid receptor subtypes through the brain of mice were determined after chronic naltrexone treatment using quantitative in vitro autoradiography. This is the first complete mapping study in mice for µ-, δ- and κ-opioid receptors after chronic naltrexone exposure. Treatment with naltrexone clearly induced up-regulation of µ- (mean 80%) and, to a lesser extent, δ-opioid receptors (mean 39%). The up-regulation of µ- and δ-opioid receptors was evident throughout the brain, although there was variation in the percentage change across brain regions. In contrast, consistent up-regulation of κ-opioid receptors was observed in cortical structures only and was not so marked as for µ- and δ-opioid receptors. In noncortical regions κ-opioid receptor expression was unchanged. Taken together, the present findings suggest opioid receptor subtype-selective regulation by chronic naltrexone treatment in mice.
    Type of Medium: Electronic Resource
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