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Biomedical subjects

M G Castro

Publications and source records attributed to M G Castro.

At least 73 records · Page 4Linked to original sources
PubMed1999

Recent developments in gene therapy: applications for the treatment of pituitary tumours.

Pituitary tumours are normally benign, highly differentiated and slow growing neoplasms. Nevertheless, as many as half of them will show evidence of local invasion into the surrounding structures. Despite their benign growth characteristics and slow clinical progression, pituitary tumours commonly cause serious morbidity. The mass effects of large tumours, including headache and visual failure from optic chiasm compression, may cause lifelong disability. Hormone hypersecretion or deficiency causes major clinical problems that often require expensive and long-term medical therapy. Major advances have been made in the therapy of pituitary tumours over the past 20-30 years, but despite this, their treatment often remains an unsatisfactory compromise in practice. There is, therefore, a place for improvements in therapy, and to this end, gene therapy may come to hold a significant place in the future treatment of human pituitary tumours. With the development of new gene delivery vehicles, this concept can now be explored with a view to treating specific types of pituitary tumours.

Animals↗
PubMed1999

Neuronal and glial cell type-specific promoters within adenovirus recombinants restrict the expression of the apoptosis-inducing molecule Fas ligand to predetermined brain cell types, and abolish peripheral liver toxicity.

Gene therapy using Fas ligand (FasL) for treatment of tumours and protection of transplant rejection is hampered because of the systemic toxicity of FasL. In the present study, recombinant replication-defective adenovirus vectors (RAds) encoding FasL under the control of either the neuronal-specific neuronal-specific enolase (NSE) promoter or the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter have been constructed. The cell type-specific expression of FasL in both neurons and glial cells in primary cultures, and in neuronal and glial cell lines is demonstrated. Furthermore, transgene expression driven by the neuronal and glial promoter was not detected in fibroblastic or epithelial cell lines. Expression of FasL driven by a major immediate early human cytomegalovirus promoter (MIEhCMV) was, however, achieved in all cells tested. As a final test of the stringency of transgene-specific expression, the RAds were injected directly into the bloodstream of mice. The RAds encoding FasL under the control of the non-cell type-specific MIEhCMV promoter induced acute generalized liver haemorrhage with hepatocyte apoptosis, while the RAds containing the NSE or GFAP promoter sequences were completely non-toxic. This demonstrates the specificity of transgene expression, enhanced safety during systemic administration, and tightly regulated control of transgene expression of highly cytotoxic gene products, encoded within transcriptionally targeted RAds.

Adenoviridae↗
PubMed1999

Generation of a recombinant herpes simplex virus type 1 expressing the rat corticotropin- releasing hormone precursor: endoproteolytic processing, intracellular targeting and biological activity.

We describe the generation of a recombinant herpes simplex virus type 1 (HSV1) vector, tsK/CRH10, derived from the temperature-sensitive mutant tsK, expressing rat pre-procorticotropin-releasing hormone (ppCRH). In hypothalamic neurons, within the paraventricular and supraoptic nuclei, this neuropeptide precursor is processed to mature CRH (1-41), the key modulator of the hypothalamic-pituitary-adrenal stress response. We used the recombinant HSV1 tsK/CRH10 to study posttranslational processing, intracellular localization and biological activity of proCRH (pCRH) within neuronal, glial and epithelial cell lines. We showed that CRH-like immunoreactivity expressed in neuronal, glial and epithelial cells infected with tsK/CRH10 was biologically active, could be detected intracellularly and was also secreted. Our data also show that within Neuro2a and NG115 cells, the CRH precursor is cleaved to yield a CRH-like immunoreactive fragment of approximately 4.75 kD which could account for mature CRH (1-41). No endoproteolytic processing of the precursor takes place within the astrocytic 1321 NI cell line. Using immunocytochemistry techniques we detected CRH-like immunoreactivity within the endoplasmic reticulum-Golgi region in all cells and within secretory vesicles of Neuro2a and NG115 cells, suggesting correct targeting to the regulated secretory pathway within these cells. Our results demonstrate that the HSV1 recombinant vector expressing the full-length CRH precursor molecule constitutes an excellent delivery system for both cell lines and postmitotic neurons in vitro, which has enabled the study of targeting, endoproteolytic processing and biological activity of this neuropeptide precursor. Furthermore, it can also be used to generate transient transgenesis of the CRH precursor in vivo, to study neuroendocrine-immune interactions within the mammalian central nervous system.

Animals↗
PubMed1999

Wyeomyia (Prosopolepis) confusa (Lutz): subgeneric validation, species description, and recognition of Wyeomyia flui (Bonne-Wepster and Bonne) as the senior synonym of Wyeomyia kerri del Ponte and Cerqueira.

Prosopolepis Lutz is validated as a monotypic subgenus of Wyeomyia Theobald and the type species, Weomyia confusa (Lutz), is redescribed. The description includes illustrations of the male and female genitalia, the 4th-stage larva, and the pupa. Prosopolepis flui Bonne-Wepster and Bonne is resurrected from synonymy with Wy. confusa and recognized as the senior synonym of Wyeomyia kerri del Ponte and Cerqueira. Wyeomyia flui does not belong in the subgenus Prosopolepis and remains in the genus Wyeomyia without subgeneric placement. Trichoprosopon pusillum Lutz and Nuñez-Tovar is not synonymous with Wy. confusa and is provisionally regarded as a nomen dubium within Wyeomyia. The identity of Wy. confusa is fixed by neotype selection.

Animals↗
PubMed1998

Direct regulation of GnRH transcription by CRF-like peptides in an immortalized neuronal cell line.

The existence of a CRF-dependent inhibition of GnRH transcription was investigated using a neuronal GnRH-expressing cell line (Gn11) stably transfected with mouse (-611 bp) or chicken (-3000 bp) GnRH promoter/luciferase reporter constructs. The presence of the CRF-R1 receptor was established using a specific CRF-R1 antiserum. After 7 h of incubation, urotensin-I and sauvagine increased the mouse GnRH-reporter bioluminescence by 1.3- and 1.2-fold, respectively, compared with control cells. Subsequently, CRF, urotensin-I and sauvagine decreased luciferase reporter activity to about 60% of the control values after 14 h. Similar trends occurred with the chicken GnRH promoter with UI increasing reporter gene activity 2.4-fold over the controls after 14 h incubation. These data provide additional evidence for the direct regulation of GnRH transcription by CRF-like peptides.

Amphibian Proteins↗
PubMed1998

Procorticotrophin-releasing hormone: endoproteolytic processing and differential release of its derived peptides within AtT20 cells.

Procorticotrophin-releasing hormone (proCRH) is expressed mainly in the hypothalamus and in the placenta, where it undergoes tissue-specific endoproteolysis. Our results show that within stably transfected AtT20/D16V cells proCRH is cleaved to generate two fragments of approximately 8 and 3 kDa which could account for proCRH(125-194) and proCRH(125-151), respectively, and a 4.5 kDa product which could account for mature IR-CRH(1-41). The immunofluorescence staining patterns for IR-CRH and IR-ACTH and their response of secretagogues indicate targeting of proCRH and POMC to the secretory pathway in transfected AtT20 cells. In this work, we have used a unique set of specific RIAs and IRMAs to the full length POMC and proCRH molecules and several products of endoproteolytic processing to assess if they could be released differentially in response to stimulation. Although the release of both IR-ACTH and IR-CRH peptides from transfected AtT20 cells is stimulated in response to exposure to high potassium stimulation (51 mM KCl/SmM CaCl2), the sorting index (SI) suggests that mature ACTH is sorted to the regulated secretory pathway 2.1-fold more efficiently than mature CRH(1-41). Mature ACTH is also sorted to the regulated secretory pathway 9-fold more efficiently than IR-proCRH(125-151). Also, mature CRH(1-41) is sorted to the regulated secretory pathway 3-fold more efficiently than IR-proCRH(125-151). These results therefore indicate that the intracellular mechanisms for the storage and release of POMC, proCRH and their endoproteolytic products differ and would sustain the hypothesis that within mammalian peptidergic cells, different biologically active peptides originating from the same or different precursor molecules, could be differentially released in response to specific stimuli. This would give these cells the capacity to finely regulate neurotransmitter release in response to environmental and physiological demands.

Adrenocorticotropic Hormone↗
PubMed1998

Electrophysiological evidence against a neurotransmitter role of corticotropin-releasing hormone (CRH) in primary somatosensory cortex.

The possible neurotransmitter role of corticotropin-releasing hormone (CRH) was studied in the primary somatosensory cortex of the rat. Electrical activity of single neurones was recorded in layers II-VI of cortex, and in the region of the locus coeruleus. Iontophoresis and pressure ejection were employed to locally apply CRH, and changes in spontaneous, synaptically driven and iontophoretically driven firing were examined. In the cortex, of 62 neurones recorded most (51) were completely unaffected by high and prolonged current/pressure ejections of CRH. Depression of firing was occasionally seen (8 of 62), while a very few (3) were weakly excited. Of 25 cells studied with vibrissal stimulation to evoke excitatory synaptic responses, responses in two cells were depressed and in two they were enhanced. Activity that was evoked by iontophoretic ejection of excitatory amino acids, such as glutamate, was depressed in 6 of 40 cells (none were enhanced). Such effects as were seen were weak and often difficult to reproduce. The effect of CRH on depressions produced by GABA was also tested in four experiments. No effects on the amplitude or duration of the depressions were observed. In contrast recordings made in the midbrain, in the region of the locus coeruleus, resulted in over half the neurones (11 of 20) showing clear reproducible excitatory responses to CRH applications. Solutions used in the experiments were analysed using chromatography, radioimmunoassay and bioassay, and no significant degradation of the peptide was found compared with the synthetic standard (CRH (1-41)). The data provide evidence against CRH acting as a neurotransmitter or modulator in primary cortex, suggesting that the CRH which is localized in certain types of cortical cells is involved in other processes.

Animals↗
PubMed1998

Use of recombinant herpes simplex virus type 1 vectors for gene transfer into tumour and normal anterior pituitary cells.

In this paper we demonstrate the use of recombinant viral vectors derived from herpes simplex virus type 1 (HSV1) to transfer reporter genes in vitro into rat anterior pituitary cells grown in primary cultures and the anterior pituitary tumour cell lines GH3 and AtT20. The three vectors used were, tsK/beta-galactosidase (beta-gal), tsK/CRH and tsK/TIMP, the corresponding transgene products respectively being E. coli beta-gal, pre-procorticotropin releasing hormone (ppCRH), and the chimeric protein TIMP/Thy1 (tissue inhibitor of metalloproteinases (TIMP)/linked to the carboxy terminus of Thy1 which confers the addition of a glycolipid glycosyl-phosphatidylinositol anchor in the ER). Double labelling immunofluorescence experiments to detect reporter proteins and transduced cell types indicated that the three vectors could transfer and express the reporter genes in normal and tumour anterior pituitary cells. Virus infection of pituitary cells was characterised, and it was shown that infection with tsK/beta-gal at multiplicities of infection (MOI)=10, 100% of tumour and non-endocrine anterior pituitary cells expressed beta-gal, whereas 75% endocrine anterior pituitary cells expressed the transgene. Long-term expression studies after infection with tsK/beta-gal indicated that anterior pituitary cells in primary cultures expressed the transgene for significant longer periods than tumour anterior pituitary cells. Growth arrest by serum starvation markedly decreased the frequency of transgene expression in anterior pituitary cells following infection with tsK/beta-gal. Transgenic products expressed from tsK were targeted to their correct intracellular domain in both anterior pituitary cells in primary cultures and in pituitary tumour cell lines. We conclude that transgenes can be delivered into anterior pituitary cells in primary culture and pituitary tumour cell lines using tsK derived HSV1 vectors. The prospect of employing viral vectors to transfer genes into endocrine cells opens up the potential exploration of various molecular aspects of pituitary cell function both in vitro and in vivo, as well as the use of gene transfer into the pituitary for potentially therapeutic applications, such as the treatment of pituitary tumours.

Animals↗
PubMed1998

FasL induces Fas/Apo1-mediated apoptosis in human embryonic kidney 293 cells routinely used to generate E1-deleted adenoviral vectors.

Human embryonic kidney 293 cells contain the E1 region of adenovirus type 5, and thus sustain, through transcomplementation, the production of recombinant E1-deleted adenovirus vectors. During attempts to produce recombinant adenovirus expressing the apoptosis-inducing molecule Fas ligand (FasL) under the control of a very strong truncated major immediate-early human cytomegalovirus (MIEhCMV) promoter, we discovered that 293 cells were not surviving the initial cotransfection with a shuttle plasmid encoding the mouse FasL; and pJM17, a plasmid containing the genome of adenovirus type 5 with deletions in the E1-E3 regions, in an unpackagable form. Investigation of the reason for massive cell death after cotransfection led us to determine that 293 cells express the FasL receptor. Fas-Apo1 (CD95), and respond with apoptosis to the cross-linking of Fas-Apo1 with either IgM monoclonal antibodies or FasL. Therefore, we decided to generate adenoviral vectors expressing FasL, under the control of tissue-specific and/or-inducible promoter elements. Our findings can explain difficulties several groups have had in generating recombinant adenoviral vectors expressing FasL using 293 cells, as well as the lower titres reported.

Adenoviridae↗
PubMed1998

Intracellular retention of the corticotrophin-releasing hormone (CRH) precursor within COS-7 cells.

We investigated the intracellular localization of CRH in transiently transfected COS-7 cells expressing the full-length rat corticotropin-releasing hormone (CRH) precursor cDNA. CRH synthesized by transfected COS-7 cells is mainly stored intracellularly. In contrast, CHO-K1 cells expressing the same CRH precursor stored and released equal amounts of immunoreactive (IR)-CRH. Ultrastructural analysis revealed that CRH is stored in electron-dense aggregates in the RER of transiently transfected COS-7 cells and does not migrate into the Golgi apparatus. On the basis of the different intracellular localization, storage, and release of CRH in COS-7 and CHO-K1 cells, we hypothesize that the intracellular trafficking of CRH within the constitutive secretory pathway for protein secretion not only depends on its primary amino acid sequence but might also be influenced by intracellular conditions or factors.

Animals↗
PubMed1997

Evidence for sexual differences in the preoptic area regulation of blood glucose in rats.

The effect of noradrenaline (NA) injection (20 or 40 nmol) into the preoptic area (POA) on plasma glucose and insulin was studied in male and female rats. The rats were implanted with chronic jugular catheters for blood sampling and unilateral intracerebral cannulas placed just above the POA. Blood samples were taken before and at 5, 10, 15, 30 and 60 min after NA injection. As early as 5 min after NA injection, plasma glucose levels rose rapidly in both male and female rats, reaching a peak at 15 min poststimulus. NA injection into the POA caused a dose-dependent hyperglycemic response in both male and female rats, although the response was more intense and longer lasting in females than in males. However, NA injection into the POA induced an increase in plasma insulin concentration in male but not in female rats. In addition, the increase in plasma glucose induced by 40 nmol NA injection in males preceded that of insulin. Plasma levels of glucose after POA injection of NA were already significantly elevated (p < 0.01) within the first experimental interval (5 min), whereas a plasma insulin increase were first detected 15 min post injection. We conclude that, when administered locally into the POA, NA can activate the sympathetic outflow expressed by a neurally mediated hyperglycemia which is more intense in females than in males. These data demonstrate that the POA has a sexually differentiated function in the regulation of glycemia.

Animals↗
PubMed1997

Effect of the corticotrophin releasing hormone precursor on interleukin-6 release by human mononuclear cells.

A growing body of evidence suggests that corticotrophin releasing hormone (CRH) may exert direct modulatory effects on immune cells. In the present study we assessed the effects of its precursor molecule, proCRH, on interleukin-6 (IL-6) release by human peripheral blood mononuclear cells (MNC). Human MNC were incubated with the corresponding stimuli for 24 hr. The supernatants were collected and IL-6 measured by ELISA. Conditioned medium from CHO-K1 cells stably transfected with the recombinant plasmid pEE14/rat pre-proCRH cDNA was used as the source of proCRH. Western blot analysis of this medium, using an antibody specific for the intact precursor, showed that no proCRH degradation products were present. The proCRH had an inhibitory effect on basal and LPS-stimulated release of IL-6. These results suggest that the full length CRH precursor may possess immunomodulatory properties.

Adjuvants, Immunologic↗