Maria G. Castro

Biosynthesis of corticotropin-releasing hormone in human T-lymphocytes

Corticotropin-releasing hormone (CRH) is a 41-amino acid neuropeptide which increases the transcription of the proopiomelanocortin (POMC) gene, as well as the biosynthesis and secretion of POMC-derived peptides. Using a specific human CRH radioimmunoassay we have shown that human T-lymphocytes contain immunoreactive CRH. We studied the effects of phytohemagglutinin (PHA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the biosynthesis of CRH in human T-lymphocyte cell cultures. A significant increase in CRH mRNA levels was observed in human lymphocytes after 12 h of PHA/TPA treatment, while the levels decreased after 22 h. These findings could imply an immunomodulatory role for CRH that could be due to autocrine and/or paracrine interactions.

Polarized Distribution of the Trans‐Golgi Network Marker TGN38 During the In Vitro Development of Neocortical Neurons: Effects of Nocodazole and Brefeldin A

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 bodys 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.

Simultaneous detection of amplicon and HSV-1 helper encoded proteins reveals that neurons and astrocytoma cells do express amplicon-borne transgenes in the absence of synthesis of virus immediate early proteins

HSV-1 amplicon vectors were used to express either a cytoplasmic (beta-galactosidase) or a membrane targeted protein (TIMP-Thy1) in primary neuronal cultures, and a human astrocytoma cell line. Whereas some cells became infected by vector particles alone others were simultaneously infected by both vector and helper particles. Our results show that IEHCMV and HSV-1 IE3 promoters are able to direct transgene expression in these cells in the absence of synthesis of helper virus transacting proteins, and stress the need of monitoring expression from both partners of an amplicon population, in order to differentiate transgene expression in cells singly infected with amplicon particles, from those infected by both amplicon and helper particles.

Expression of biologically active procorticotrophin-releasing hormone (proCRH) in stably transfected CHO-K1 cells: characterization of nuclear proCRH.

Corticotrophin‐releasing hormone (CRH) is a 41 amino acid neuropeptide which is cleaved at a pair of dibasic amino acids from a larger precursor molecule (pre‐proCRH) by the action of endopeptidases. In cells possessing a regulated secretory pathway, sorting of proneuropeptides and prohormones occurs within the trans‐Golgi network, where they are finally packaged into secretory vesicles to be released in response to an external stimulus. Such cells also possess a constitutive secretory pathway, and neuropeptides are also translocated into this subcellular compartment. We have recently established stably transfected CHO‐K1 cells expressing the rat pre‐proCRH cDNA, and shown that proCRH was localized within the secretory pathway and the nucleus of transfected cells. Both the cytoplasmic and nuclear species of IR‐CRH displayed an apparent molecular weight of approximately 19 kDa, consistent with the size of the uncleaved CRH precursor molecule.

Synaptogenesis and distribution of presynaptic axonal varicosities in low density primary cultures of neocortex: an immunocytochemical study utilizing synaptic vesicle-specific antibodies, and an electrophysiological examination utilizing whole cell recording

SummaryLow-density primary cultures of neocortical neurons were utilized to examine: (i) early interactions of growing neurites with morphological characteristics of axons with other neuronal elements, and (ii) the distribution of presynaptic axonal varicosities closely apposed to MAP-2 immunoreactive, putatively postsynaptic, dendrites. At the light microscopical level axonal varicosites, presumably presynaptic terminals, were identified using immunocytochemistry incorporating antibodies specific for the synaptic vesicle antigens synaptophysin and synapsin. The presence of synaptophysin- and synapsin-immunoreactive swellings along axonal processes was first detected at 5 days post-plating and was also apparent in axons growing in isolation. At 5–7 daysin vitro, immunolabelled axonal varicosities in close apposition to putative postsynaptic dendrites (MAP-2 immunoreactive) dendrites were detected. Electrophysiologically active synaptic contacts can also readily be detected at this stage. After 3 weeksin vitro presynaptic contacts do appear to be distributed heterogeneously along postsynaptic dendrites of many neurons in culture. As the culture matures a higher number of presynaptic profiles can be seen along dendrites, with a centrifugal distribution, e.g. a higher density of presynaptic axonal terminals in close apposition to more distal regions of larger dendrites, putatively considered to be apical dendrites of pyramidal-like neurons. In our cultures, the overall increase in the density and the pattern of distribution of presynaptic axon terminals immunoreactive for synaptic vesicle antigens closely apposed to putative post-synaptic structures mimics the general postnatal increase of synaptic density in the neocortexin vivo. Thus, low density primary cultures of neocortical neurons offer a valuable system to explore and manipulate (i) the molecular and cellular basis of neocortical synaptogenesis, and (ii) the pharmacology of neocortical synaptic transmission.

Effects of corticotrophin-releasing factor and arginine-vasopressin on proopiomelanocortin (POMC) mRNA levels, release and storage of adrenocorticotrophin from mouse anterior pituitary cells

1. The present studies were undertaken to determine the effects of arginine vasopressin (AVP), corticotrophin-releasing factor (CRF) and AVP in combination with CRF on proopiomelanocortin (POMC) gene expression, and in the release and biosynthesis of POMC products (i.e. ACTH). 2. After a 3 hr treatment, AVP (10(-7) M), CRF (10(-7) M) and AVP (10(-7) M) in combination with CRF (10(-7) M) stimulated ACTH release by 291 +/- 19.2%, 377.4 +/- 25.6% and 462.1 +/- 38.4% (P < 0.01), respectively, with respect to basal secretion; while ACTH content diminished by 84.0 +/- 4.6%, 81.3 +/- 2.1% and 71.0 +/- 1.5% (P < 0.01), respectively, with respect to basal. Total POMC mRNA levels were not affected after a 3 hr treatment. 3. When cells were treated with a wide range of AVP concentrations (10(-11)-10(-7) M) to which we added different concentrations of CRF, modulation of AVP-induced ACTH release was most effective at CRF concentrations of < 10(-10) M. 4. Prolonged (3-6 hr) exposure to as low as 10(-10) M CRF or 10(-9) M AVP resulted in homologous desensitization of ACTH secretion. However, these pretreated cells were able to respond to a further challenge of CRF and AVP. This paper provides new information on the dose ranges over which desensitization and cross-sensitization between CRF and AVP secretory effects take place in mouse anterior pituitary cells.

Generation and characterization of an antiserum reactive with a proteolytic processing site within rat procorticotrophin-releasing hormone

In this paper we report the generation of an antibody specific for the cleavage site within procorticotrophin-releasing hormone (proCRH) at the N-terminus proCRH/CRH (1-41) junction. Using radioimmunoassay techniques were show that the antibody generated (781) cross-reacts specifically with the proCRH (137-150) Tyr fragment, corresponding to the cleavage site within the full length precursor molecule. The anti-cleavage site antibody does not crossreact with the endoproteolytic products originated from the CRH precursor molecule, i.e. CRH (1-41) or proCRH (125-151) or with any of the CRH-immunoreactive fragments tested i.e. CRH (36-41), CRH (1-20) and CRH (30-41). It also shows no cross-reactivity with CRH-related substances from other species, i.e. urotensin I (fish) and sauvagine (frog). The cleavage site antibody (781), recognizes the full length proCRH molecule in Western blotting and in liquid phase radioimmunoassay from transfected CHO-K1 cells expressing the full length pre-proCRH cDNA. Using immunofluorescence and immunoprecipitation techniques followed by SDS-PAGE and autoradiography, we confirm the presence of the intact CRH precursor molecule within the nucleus and the cytoplasm of stably transfected CHO-K1 cells expressing immunoreactive proCRH. The immunofluorescence studies using primary cultures of hypothalamic neurons, show that immunoreactive (IR) proCRH is localized within the perinuclear region and was also seen along the neuronal processes where it accumulates at their tips. Our results, therefore, show that this antibody will be an invaluable tool in the study of intracellular trafficking in relation to the endoproteolytic processing of the CRH precursor molecule.

Co-localisation of autoimmune antibodies specific for double stranded DNA with procorticotrophin-releasing hormone within the nucleus of stably transfected CHO-K1 cells

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.

Prediction of protein antigenic sites in human corticotrophin-releasing hormone precursor

1. The primary structure of human corticotrophin-releasing hormone precursor (h pre-proCRH) has been analysed using a number of computer algorithms to identify the areas of highest predicted antigenicity. 2. These results were correlated with crossreactivity data obtained from studies of antibodies produced in rabbits by immunizing with h pre-proCRH, and a number of related peptides. 3. Six areas of high predicted antigenicity were identified in h pre-proCRH by the prediction routines utilized. Two of these corresponded almost exactly to the two putative cleavage sites of the prohormone, and a third lay within the C-terminal region of one of the products of post-translational processing of the prohormone, i.e. CRH(1-41). 4. Experimental crossreactivity data also indicated that a number of structural factors (e.g. Omega loops, peptide conformation) may also be involved in recognition of peptide fragments by antibodies.