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  • 1
    ISSN: 1365-2826
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Corticotrophin-releasing hormone (CRH) is a 41 amino acid neuropeptide which plays a major role in regulating the endocrine response to stress. CRH acts by first binding to specific receptors on the plasma membrane of target cells. A CRH receptor from a human corticotroph adenoma and rat brain has recently been cloned (CRH-R1). In this paper, we have chosen three different peptide sequences within the CRH-R1 molecule which bear no similarity to other members of this receptor subfamily (or indeed any known protein) and which are likely to be exposed on the surface of the native protein, for antibody production. Some of these fragments produced anti-peptide antibodies of good titre which cross-reacted with the CRH-R1 receptor expressed in transiently transfected COS-7 cells and in tissue extracts from rat cerebellum, cortex, pituitary gland and human myometrium, both in Western blots and in liquid-phase radioimmunoassay. We used immunofluorescence techniques to localize the CRH receptor in transiently transfected COS-7 cells, primary cultures of rat anterior pituitary (AP) cells, the corticotroph-tumour cells AtT20 D16–16 and cortical neurons in primary culture. Our results indicate IR-CRH-R1 receptors have a punctate distribution on the plasma membrane of AP cells and AtT20 D16–16 cells. Whilst in AP cells their appearance is a fine punctate pattern, in AtT20 cells, they appear as large patches which could account for receptor clusters. Within primary cortical neurons, their distribution does not appear to be polarized. Our results suggest that distribution of CRH-R1 receptors within the different cell-types investigated depends not only on the amino acid sequence but also on cellular factors.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1365-2826
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Syrian hamsters exhibit a marked seasonal variation in prolactin secretion. The aim of this study was to analyse the nature of the photoperiodic regulation of prolactin gene expression, and to define the role of melatonin and the pars tuberalis of the anterior pituitary in this process. Pituitary prolactin gene expression, restricted to the pars distalis, was increased in hamsters maintained in long daylengths (16 h : 8 h, light : dark) compared to hamsters exposed to short daylengths (8 h : 16 h, light : dark) for 8–12 weeks. Analysis of single cells by in situ hybridization showed that photoperiod had no effect on the percentage of pars distalis cells expressing prolactin mRNA, but shifted the frequency distribution of prolactin mRNA expression per cell, such that in long photoperiods a greater proportion of cells were recruited to a higher expressing population. In vitro coculture of hamster pars tuberalis fragments increased prolactin promoter-driven luciferase activity in stably transfected GH3 cells in a dose- and duration-dependent manner. Conditioned medium from hamster and ovine pars tuberalis also activated the prolactin promoter. Furthermore, basal and forskolin-stimulated conditioned medium from hamster pars tuberalis increased prolactin mRNA expression in primary cultures of pars distalis cells. Melatonin attenuated the activity of pars tuberalis-conditioned medium but had no direct effect on either prolactin mRNA expression or secretion in pars distalis cell cultures. Finally, pars tuberalis fragments from long photoperiod hamsters stimulated prolactin gene promoter activity to a greater extent than those from short photoperiod hamsters. In conclusion, this study provides the first evidence in a seasonal mammal that the synthesis of prolactin depends on photoperiodic modulation of a pars tuberalis-derived factor. Our data support further the hypothesis that seasonal modulation of prolactin gene expression depends upon a melatonin-dependent paracrine action of the pars tuberalis on pars distalis lactotrophic cells.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Neurons are polarized secretory cells whose cytoplasm and plasma membrane are polarized to form two compartments: dendrites and axons. In mature, fully polarized neurons, the microtubule-associated protein Map2 is targeted to dendrites, while tau is mainly restricted to axons. However, the intraneuronal distribution of secretory pathway organelles, such as the endoplasmic reticulum and the Golgi complex, which give rise to all constitutive, regulated and lysosome vesicles, is poorly understood. Thus, to investigate the distribution of the trans-Golgi network during the development and maturation of rat neocortical neurons in vitro, we have utilized an antibody recognizing a 38 kDa trans-Golgi network-specific protein, TGN38, and immunofluorescence microscopy. Before neurons have established polarity, TGN38 immunoreactivity outlines several vesicles dispersed throughout the cell body cytoplasm; these converge close to a major Map2-immunopositive process during the establishment of neuronal polarity, and later merge into a single structure located at the base of a thick Map2-immunopositive process, ∼18 h after plating. At this stage TGN38 immunoreactivity is located within 45° of the major Map2-immunoreactive process in 54% of neurons, while in only 6% of cells it is located at the opposite pole. After 3 days in vitro, during the segregation of microtubule-associated proteins to either dendrites or axons, TGN38 immunoreactivity clusters continue to be located close to a major dendrite, and in some neurons these clusters begin to enter a major Map2-immunoreactive process. At 10 days in vitro TGN38 immunoreactivity extends into a major dendrite for 5–30 μm in many neurons. Thus, the distribution of TGN38 immunoreactivity becomes polarized, being localized within a single, usually the major, neocortical dendrite. Our results also show that the morphological appearance of TGN38-immunoreactive structures is microtubule-dependent, since nocodazole treatment of polarized neurons induces scattering of TGN38-immunoreactive vesicles throughout the cell body's cytoplasm. Treatment with brefeldin A induces scattering of small TGN38-immunoreactive vesicles throughout the neuronal cytoplasm and processes, a different response to that observed in non-neuronat cells.
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  • 4
    ISSN: 1432-0878
    Keywords: Key words: Autoimmune antibodies ; Corticotrophin-releasing hormone ; Double stranded DNA ; Nucleus ; Cell culture ; CHO-K1 cells (Chinese Hamster Ovary)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Human autoantibodies and corticotrophin-releasing hormone (CRH)-specific antibodies have been used in a double-labelling immunofluorescence technique to demonstrate that immunoreactive CRH structures are co-localised with immunostaining produced by double stranded DNA-specific human autoantibodies within the nucleus of cultured ovarian cells of Chinese hamsters (CHO-K1). This co-localisation was confirmed using confocal microscopy. A metabolic labelling technique was used to investigate the role of the cytoskeleton in mediating nuclear translocation of proCRH within stably transfected CHO-K1 cells and showed that microtubule and actin disrupting agents had no effect upon the nuclear translocation of proCRH. These results, therefore, suggest that nuclear translocation of proCRH is not affected by drugs which disrupt the cytoskeleton and, consequently, modify the diameter of the nuclear pores.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1432-0878
    Keywords: Autoimmune antibodies ; Corticotrophin-releasing hormone ; Double stranded DNA ; Nucleus ; Cell culture ; CHO-K1 cells (Chinese Hamster Ovary)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Human autoantibodies and corticotrophin-releasing hormone (CRH)-specific antibodies have been used in a double-labelling immunofluorescence technique to demonstrate that immunoreactive CRH structures are co-localised with immunostaining produced by double stranded DNA-specific human autoantibodies within the nucleus of cultured ovarian cells of Chinese hamsters (CHO-K1). This co-localisation was confirmed using confocal microscopy. A metabolic labelling technique was used to investigate the role of the cytoskeleton in mediating nuclear translocation of proCRH within stably transfected CHO-K1 cells and showed that microtubule and actin disrupting agents had no effect upon the nuclear translocation of proCRH. These results, therefore, suggest that nuclear translocation of proCRH is not affected by drugs which disrupt the cytoskeleton and, consequently, modify the diameter of the nuclear pores.
    Type of Medium: Electronic Resource
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