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  • 1
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Frozen and vibratome sections from the adrenal gland of the rat were hybridized in situ using a biotinylated oligonucleotide probe specific for tyrosine hydroxylase (TH) messenger ribonucleic acid (mRNA). Hybridization was detected using the streptavidin-peroxidase-diaminobenzidine (DAB) system in combination with silver-gold postintensification. The signal appeared as a black coloration and was localized to the cytoplasm of catecholamine-synthesizing chromaffin cells in the adrenal medulla. This coloration was due to the deposition of the silver-gold intensified DAB chromogen onto the probe hybridized to mRNA in carrier organelles. Compared with the conventional peroxidase-DAB labelling, the silver-gold amplified version was more sensitive in detecting TH mRNA. Using this modification, we were able to adapt the procedure to electron microscopy, thereby further localizing the hybridized signal to ribosomes. Because this hybridization detection system produces grains, not just color, this method has the potential for measurement of changes in mRNA levels at the ultrastructural level.
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  • 2
    ISSN: 1365-2826
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Beta-endorphin is thought be an important inhibitor of LHRH neuronal activity and also to play a role in conveying information about changes in steroid levels to LHRH neurons. We have previously shown that the mRNA encoding the precursor of ß-endorphin, proopiomelanocortin (POMC), fluctuates during the estrous cycle with the most dramatic changes occurring on proestrus. POMC mRNA levels decline before the onset of LH surge release but then dramatically rise and remain elevated during the surge. In the present studies we tested the hypothesis that the decline in POMC mRNA levels immediately before the proestrous LH surge is mediated by estrogen and the rise during the surge by progesterone. To test this hypothesis, we compared changes in POMC mRNA levels between ovariectomized (OVX) and OVX estrogen (E2)-treated rats and between OVX E2-treated rats with and without progesterone. Animals were examined at hourly intervals after the administration of progesterone, then at every 4h during the LH surge. Using in situ hybridization histochemistry, we found that E2 decreased POMC mRNA levels in OVX rats before the onset of the LH surge and further suppressed levels during the surge. Compared to animals treated with E2 alone, progesterone advanced the time at which both the LH surge began and the time at which POMC mRNA levels declined. After a transient decline, POMC mRNA levels rose in these progesterone-treated animals and remained elevated throughout the period of the LH surge. These results support the hypothesis that progesterone times the LH surge and limits its appearance to one day by exerting a biphasic effect on the activity of ß-endorphinergic neurons of the arcuate nucleus.
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  • 3
    ISSN: 1365-3083
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Allogeneic bone marrow transplantation (BMT) is a potentially curative therapy for patients with haematologic malignancies. Several lines of evidence demonstrate that donor T cells are involved in the antitumour effects observed after BMT. Thus, patients receiving T-cell-depleted BMT have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated BMT, and patients experiencing graft-versus-host disease (GVHD) have a lower risk of disease relapse than patients who do not experience GVHD. Although the importance of donor T cells for the curative action of BMT has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. In a recently initiated project, we have conducted a longitudinal study of T-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. Peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (MNCs) were isolated and cryopreserved. CD8+ T cells were isolated from the MNCs by use of immunomagnetic beads or FACS and analysed for the presence of clonally expanded cells by T-cell receptor clonotype mapping based on RT-PCR and denaturing gradient gel electrophoresis (DGGE). Using this gel-based methodology, clonally expanded T cells were monitored after transplant and compared to the clinical data of the patients. The preliminary results demonstrates the presence of clonally expanded CD8+ T cells at all time points analysed. Furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. The appearance of newly emerged clonotypes which coincided with clinical GVHD could indicate a role for these T cells in the pathogenesis of GVHD.
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  • 4
    ISSN: 1365-2826
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Oestradiol (E2) induces luteinizing hormone-releasing hormone (LHRH) hypersecretion, thereby triggering LH surge release in ovariectomized (OVX) rats. Neural signals responsible for the surge are marked by a morning increase in LHRH gene expression and an afternoon increase in LHRH release. Evidence suggests that subpopulations of noradrenergic neurones may be responsible for one or both of these signals. To further investigate this issue, we examined effects of E2 on the activity of A1 and A2 noradrenergic neurones, as reflected in changes in tyrosine hydroxylase (TH) mRNA expression, on the day of LH surge release. We then used dual-label in situ hybridization to determine whether E2-induced changes occurred primarily in A1 and A2 subdivisions wherein most noradrenergic neurones expressed oestrogen receptor (ER)α and/or ERβ mRNA. We found that in all subdivisions, levels of TH mRNA were higher in E2- than oil-treated rats at 12.00 h. These differences resulted from a decline in TH mRNA expression in oil-treated rats, as well as a rise in levels in E2-treated rats between 10.00 h and 12.00 h. During the afternoon, TH mRNA expression in most A1 and A2 subdivisions peaked at 14.00 h when LH surge release began. However, in all but the middle and caudal A2 subdivisons, levels were similar in E2-treated and control rats at this time. This was attributable to a widespread increase in TH mRNA expression between 12.00 h and 14.00 h in OVX rats. There was no evidence that E2 induced changes in TH mRNA expression preferentially in regions wherein most neurones contained ERα or ERβ mRNA. Our findings suggest that E2 activation of middle and caudal A2 neurones, in conjunction with the widespread E2-independent activation of noradrenergic neurones in other subdivisions, may play a role in the induction of LH surge release.
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  • 5
    ISSN: 1365-2826
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: GABA neurones in the preoptic area (POA) are critical for oestradiol (E2)-dependent surge release of luteinizing hormone (LH); however, it is not clear which population(s) of POA GABA neurones is involved. The goals of the present studies were: (i) to determine whether E2 regulates GABA neurones similarly in two subdivisions of the POA that play a role in LH surge release, the rostral POA region that contains the organum vasculosum of the lamina terminalis (rPOA/OVLT), and the region containing the anteroventral periventricular nucleus (AVPV) and medial preoptic nucleus (MPN) and (ii) to determine whether GABA neurones in either or both regions exhibit temporal changes consistent with a role in the regulation of LH surge release. To accomplish these goals, we measured glutamic acid decarboxylase (GAD) 65 and 67 mRNA levels at several time points in ovariectomized (OVX), E2-treated OVX rats exhibiting LH surge release, and in E2-treated OVX rats in which LH surge release was blocked by prior administration of progesterone (P4). Our findings demonstrate that, despite their close proximity, GABA neurones in the AVPV/MPN region are regulated differently from those in the rPOA/OVLT. Only neurones in the AVPV/MPN region show temporal changes in GAD 67 mRNA expression that appear to be linked to positive-feedback effects of E2 on luteinizing hormone-releasing hormone (LHRH) and LH release. Our findings also indicate that a morning rise and an afternoon fall in GAD 67 mRNA levels marks two E2-dependent signals required for LHRH and LH surge release. Finally, our results suggest that there are distinct E2-induced signals to the rPOA/OVLT and AVPV/MPN regions and that these signals differentially regulate GAD 65 and 67 gene expression.
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  • 6
    Electronic Resource
    Electronic Resource
    Oxford, UK; Malden, USA : Blackwell Science Inc
    Restoration ecology 12 (2004), S. 0 
    ISSN: 1526-100X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Establishment of native plant populations on disturbed roadsides was investigated at Bryce Canyon National Park (BCNP) in relation to several revegetation and seedbed preparation techniques. In 1994, the BCNP Rim Road (2,683–2,770 m elevation) was reconstructed resulting in a 23.8-ha roadside disturbance. Revegetation comparisons included the influence of fertilizer on plant establishment and development, the success of indigenous versus commercial seed, seedling response to microsites, methods of erosion control, and shrub transplant growth and survival. Plant density, cover, and biomass were measured 1, 2, and 4 years after revegetation implementation (1995–1998). Seeded native grass cover and density were the highest on plots fertilized with nitrogen and phosphorus, but by the fourth growing season, differences between fertilized and unfertilized plots were minimal. Fertilizers may facilitate more rapid establishment of seeded grasses following disturbance, increasing soil cover and soil stability on steep and unstable slopes. However the benefit of increased soil nutrients favored few of the desired species resulting in lower species richness over time compared to unfertilized sites. Elymus trachycaulus (slender wheatgrass) plants raised from indigenous seed had higher density and cover than those from a commercial seed source 2 and 4 years after sowing. Indigenous materials may exhibit slow establishment immediately following seeding, but they will likely persist during extreme climatic conditions such as cold temperatures and relatively short growing seasons. Seeded grasses established better near stones and logs than on adjacent open microsites, suggesting that a roughened seedbed created before seeding can significantly enhance plant establishment. After two growing seasons, total grass cover between various erosion-control treatments was similar indicating that a variety of erosion reduction techniques can be utilized to reduce erosion. Finally shrub transplants showed minimal differential response to fertilizers, water-absorbing gels, and soil type. Simply planting and watering transplants was sufficient for the greatest plant survival and growth.
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