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Biomedical subjects

M G Castro

Publications and source records attributed to M G Castro.

At least 109 records · Page 6Linked to original sources
PubMed1994

Use of recombinant vectors derived from herpes simplex virus 1 mutant tsK for short-term expression of transgenes encoding cytoplasmic and membrane anchored proteins in postmitotic polarized cortical neurons and glial cells in vitro.

We constructed three recombinant vectors derived from the herpes simplex virus type 1 mutant tsK, each of which contained a different transgene under the control of the herpes simplex virus type 1 immediate early 3 promoter inserted into the thymidine kinase locus: the prokaryotic enzymes beta-galactosidase and chloramphenicol acetyl transferase, and a fusion gene consisting of human tissue inhibitor of metalloproteinases linked to the last exon of Thy-1, which encodes for a glycosyl-phosphatidyl-inositol membrane anchor. Infection of postmitotic neocortical and hippocampal neurons in low-density primary cultures with these vectors, achieved reliable expression of all three foreign gene products in various neocortical cell types, e.g. pyramidal neurons, non-pyramidal neurons, and glial cells. The percentage of neurons expressing transgenes ranged from 1 to 46% depending on the multiplicity of infection (highest assayed = 5); the percentage of glial cells expressing transgenes ranged from 0.5 to 98% (highest multiplicity assayed = 3.4). Expression of transgenes could be detected for up to three days in approximately 20% of neurons infected at a multiplicity of infection of 1. Infection of neurons with tk K-derived recombinant vectors inhibited their protein synthesis by 40-50% at a multiplicity of infection of 10, but no effect was observed at a multiplicity of infection of 1. Infection of glial cells with the same vectors at a multiplicity of infection of 1 inhibited protein synthesis by more than 90%. Analysis of neuronal viability at different times post-infection indicated that more than 98% of neurons expressing transgenes 48 h post-infection were viable. Thus, low-density neuronal cultures can be used to assess the efficiency of herpes simplex virus type 1-derived gene transfer vectors and transgene expression in developing cortical postmitotic cells, before and after they establish polarity. In addition, we show that two cytoplasmic enzymes, beta-galactosidase and chloramphenicol acetyl transferase, are able to diffuse freely in the cytoplasm reaching even growth cones in young neurons, while the chimeric protein tissue inhibitor of metalloproteinases/Thy-1 is correctly targeted to the plasma membrane via a glycosyl-phosphatidylinositol anchor. This model system should be useful for investigation of cellular and molecular aspects of the development and establishment of neuronal polarity, as well as for analysis of signals involved in protein targeting in postmitotic neurons.

Amino Acid Sequence↗
PubMed1994

Neuropeptide gene transfer into neuronal and glial cell lines.

Procorticotrophin-releasing hormone (ProCRH) is the precursor to the hypothalamic neuropeptide CRH(1-41) which mediates the neuroendocrine response to stress. In neuroendocrine cells and neurones, peptide hormones and neuropeptides are targeted to the dense-core vesicles of the regulated secretory pathway. These vesicles are transported to the ends of the cellular processes where they are stored until they are released upon an external stimulus. In order to study the post-translational processing and intracellular trafficking of neuropeptides in neuronal cells we have established stably transfected Neuro2A cells and NG115-401L cells expressing proCRH. The expression vector used contains glutamine synthetase (GS) coding sequences which are used as a dominant selectable marker in cells already containing GS genes. The various clonal cell lines isolated express different levels of CRH as assessed using a specific radioimmunoassay (RIA) thus indicating that the level of expression of the exogenous gene must depend upon the site of chromosome integration. Using immunofluorescence labelling, we have demonstrated that in Neuro2A and NG115-401L cells proCRH is packaged in vesicles which accumulate at the tips of cellular processes.

Animals↗
PubMed1994

HSV1 vectors to study protein targeting in neurones: are glycosyl-phosphatidylinositol anchors polarized targeting signals in neurones?

In order to characterize protein targeting signals in polarized postmitotic cortical neurones in vitro, we have developed recombinant and amplicon type vectors derived from herpes simplex virus 1 (HSV1) to transfer genes into these cells. We examined the targeting of both bacterial proteins, which lack specific targeting signals, as well as recombinant proteins containing mammalian targeting sequences, i.e. the sequence encoding for the addition of a glycosyl-phosphatidyl inositol (GPI) membrane anchor. Utilizing both HSV1 recombinant and amplicon vectors we demonstrate that while a GPI anchor is able to direct a foreign protein to the plasma membrane, the recombinant protein is targeted mainly to the dendritic, but also to the axonal plasma membrane; i.e. its distribution is not completely polarized. Thus, GPI anchors do not function as dominant polarized targeting signals in neurones, as they do in polarized epithelial cell lines.

Cells, Cultured↗
PubMed1993

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.

Cells, Cultured↗
PubMed1993

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.

Adrenocorticotropic Hormone↗
PubMed1993

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.

Algorithms↗
PubMed1993

Thymus-pituitary interactions during ageing.

We had previously shown that homoeostatic thymus hormone (HTH), an H2A-H2B histone dimer, possesses an age-dependent adrenostimulatory activity in vivo. It was therefore of interest to investigate the adrenocorticotropin (ACTH)-releasing activity of histones and other related preparations on perifused pituitary cells from young (2-4 months) and mature (16-18 months) rats. Histones and protamines gave a positive interference with the immunoradiometric assays (IRMA) for ACTH and corticotropin releasing hormone (CRH) whereas the histone fragment MB35, as well as nucleohistone and nucleoprotamine complexes did not. The latter three substances induced a significant ACTH response in pituitary cells from young and, to a lesser extent, mature rats. Our results suggest that nucleoprotein complexes released into the extracellular milieu may act as hypophysiotropic signals. On the other hand, the responsiveness of pituitary cells to these signals seems to decrease with the age of the donor.

Adrenocorticotropic Hormone↗
PubMed1993

Thymosin peptides stimulate corticotropin release by a calcium-dependent mechanism.

Thymosin fraction five (TF5), a well-characterized immunoregulatory thymic preparation, has been reported to stimulate corticotropin (ACTH) release from rat pituitary cells. Since a previous study in our laboratory had shown that TF5 was able to stimulate ACTH release from corticotropin-releasing hormone (CRH)-insensitive corticotropic tumor cells, it was of interest to assess the role of calcium in the mechanism of action of TF5 on corticotropic cells. A CRH-insensitive variant, denoted AtT-20(CI), of the wild-type corticotropic tumor cell line AtT-20 was used. Synthetic h/rCRH within a dose range of 0.1-100 nM was completely ineffective to stimulate basal ACTH release from AtT-20(CI) cells, although the same batch of neuropeptide displayed the expected ACTH-releasing activity on dispersed rat pituitary cells (for instance, 0.1 nM CRH induced a 3.7-fold increase in ACTH release in this cell system). Median eminence extracts (1/10) induced only a 12% increase in ACTH release from AtT-20(CI) cells as compared to the 395% stimulation induced in normal pituitary cells. As expected, TF5 induced a dose-dependent increase in ACTH release from AtT-20(CI) cells. However, this ACTH-releasing activity of TF5 was completely abolished when cells were incubated in Ca-free medium or Ca-free medium containing 0.5 mM EGTA. On the other hand, the presence of the Ca ionophore A23187 (5 microM) in medium containing normal Ca levels (2.5 mM) did not affect the ACTH-releasing activity of TF5 on AtT-20(CI) cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗
PubMed1993

Corticotropin-releasing (CRH) activity of thymic peptides on CRH-insensitive corticotropic tumor cells.

A number of thymic preparations are known to stimulate corticotropin (ACTH) release from pituitary cells but it remains unclear whether this effect is mediated by the corticotropin-releasing hormone (CRH) receptor-associated pathway. We report here that thymosin fraction five (TF5), peptide MB-35 and possibly calf thymus histones can stimulate the release of ACTH from a CRH-insensitive variant of the mouse corticotropic cell line AtT20. The effective concentration range at which TF5 and MB-35 displayed their ACTH-releasing activity in a dose-dependent manner was 100 to 2,000 micrograms/ml and 10 to 100 ng/ml, respectively, whereas neither preparation induced a significant depletion of intracellular ACTH stores. Our data suggest that thymosin peptides can stimulate ACTH release from corticotrophs by a CRH receptor-independent mechanism.

Adrenocorticotropic Hormone↗
PubMed1991

Expression and partial purification of human prolactin in Escherichia coli.

1. Human prolactin has been expressed in Escherichia coli. A cDNA fragment coding for the signal sequence and the full length prolactin molecule was cloned into the expression vector pUR291 which directs the synthesis of a beta-galactosidase prolactin fusion protein when expressed in E. coli. 2. Cultures of E. coli harbouring the recombinant plasmid pJMBG62 produced a fusion protein of the appropriate molecular weight which was detected by Western blot analysis using a polyclonal antibody raised against pituitary-derived human prolactin. 3. The fusion protein was isolated from inclusion bodies in a partially pure form and it was used as immunogen to raise antibodies against human prolactin. 4. When this partially purified fusion protein was injected into rabbits it generated antisera with good prolactin titres in animals which were rested for one year following a disappointing primary immunization with purified human prolactin.

Blotting, Western↗