Biomedical subjects

R Gallego

Publications and source records attributed to R Gallego.

At least 37 records · Page 2Linked to original sources

PubMed2001

Cellular localization of orexin receptors in human pituitary.

Orexins-A and -B are hypothalamic peptides derived from a precursor called prepro-orexin and relationated with the stimulation of food intake. They act on G protein receptors named orexin receptor 1 (OX(1)R) and orexin receptor 2 (OX(2)R), respectively. In the present study, we used RT-PCR and immunohistochemical techniques to detect the presence of OX(1)R and OX(2)R in human pituitary. A band of the expected size for both OX(1)R and OX(2)R was shown in human pituitary by RT-PCR. The cellular localization of OX(1)R and OX(2)R was carried out using histological techniques. By consecutive sections we demonstrated that OX(1)R was present in acidophil, diffusely distributed cells, which represent the half of the total adenohypophysis cell population. As was expected, these cells were shown to coexpress GH. OX(2)R was found in the pars intermedia and in clusters of basophil cells of the anterior pituitary, which coexpress ACTH. These results were confirmed by double immunofluorescence techniques. We also found focal positivity in axon terminals of neurohypophysis, more intense for OX(2)R than for OX(1)R. In conclusion, these results demonstrated for the first time that OX(1)R and OX(2)R were expressed by somatotrope and corticotrope cells, respectively.

Aged↗

PubMed2001

Cellular localization of orexin receptors in human pituitary.

Aged↗

PubMed2000

Quantitative tumor cell content of bone marrow and blood as a predictor of outcome in stage IV neuroblastoma: a Children's Cancer Group Study.

PURPOSE: This study investigated the prognostic value of quantifying tumor cells in bone marrow and blood by immunocytology in children with high-risk, metastatic neuroblastoma. PATIENTS AND METHODS: Patients with stage IV neuroblastoma (N = 466) registered on Children's Cancer Group study 3891 received five cycles of induction chemotherapy and were randomized either to myeloablative chemoradiotherapy with autologous purged bone marrow rescue or to nonmyeloablative chemotherapy. Subsequently, they were randomized to 13-cis-retinoic acid or no further treatment. Immunocytologic analyses of bone marrow and blood were performed at diagnosis, week 4, week 12, bone marrow collection, and end induction and were correlated with tumor biology, clinical variables, treatment regimen, and event-free survival (EFS). RESULTS: Immunocytology identified neuroblastoma cells in bone marrow of 81% at diagnosis, 55% at 4 weeks, 27% at 12 weeks, 19% at bone marrow collection, and 14% at end induction. Tumor cells were detected in blood of 58% at diagnosis and 5% at collection. There was an adverse effect on EFS of increasing tumor cell concentration in bone marrow at diagnosis (P =.04), at 12 weeks (P =.006), at bone marrow collection (P <.001), and at end induction (P =.07). Positive blood immunocytology at diagnosis was associated with decreased EFS (P: =.003). The prognostic impact of immunocytology was independent of morphologically detected bone marrow disease, MYCN status, and serum ferritin level in bivariate Cox analyses. CONCLUSION: Immunocytologic quantification of neuroblastoma cells in bone marrow and blood at diagnosis and in bone marrow during induction chemotherapy provides prognostic information that can identify patients with very high-risk disease who should be considered for experimental therapy that might improve outcome.

Adolescent↗

PubMed2000

Distinct mechanisms for activation of Cl- and K+ currents by Ca2+ from different sources in mouse sympathetic neurones.

We have investigated the roles of different voltage-dependent Ca2+ channels in the activation of the Cl- and K+ channels responsible for the afterdepolarization (ADP) and slow afterhyperpolarization (AHP) in sympathetic neurones of the isolated mouse superior cervical ganglion in vitro. The ADP and its associated Ca2+-activated Cl- current were markedly decreased by omega-agatoxin IVA (40-200 nM) and nifedipine (1-10 microM), but not by omega-conotoxin GVIA (300 nM). In contrast, the AHP and the apamin-sensitive Ca2+-activated K+ current that underlies this potential were blocked by omega-conotoxin GVIA, but were not affected by omega-agatoxin IVA and were only slightly reduced by nifedipine. Ryanodine (20 microM) reduced the Ca2+-activated Cl- current following an action potential by 75% but on average did not affect the Ca2+-activated K+ current. Evidence that R-type channels provide a proportion of the Ca2+ activating both types of Ca2+-dependent channel was obtained. We conclude that Ca2+ entering through L- and P-type Ca2+ channels preferentially activates the Cl- current responsible for the ADP in mouse sympathetic neurones, predominantly via Ca2+-induced Ca2+ release, whereas the Ca2+ that activates the K+ channels responsible for the AHP enters predominantly through N-type channels. The data can be explained by the selective association of each type of Ca2+ channel with particular intracellular mechanisms for activating other membrane channels, one indirect and the other direct, probably located at discrete sites on the soma and dendrites.

Animals↗

PubMed2000

Expression of growth hormone receptor in the human brain.

This study was designed to investigate the presence of growth hormone receptor (GHR) expression in the human brain tissue, both normal and tumoral, as well as in the human glioblastoma cell line U87MG. Reverse transcription-polymerase chain reaction revealed the presence of GHR mRNA in all brain samples investigated and in U87MG cells. GHR immunoreactivity was also detected in this cell line using both immunocytochemistry and western blotting. All together, our data demonstrate the existence of GHR expression within the central nervous system (CNS), thus supporting a possible role for GH in the CNS physiology.

Blotting, Western↗

PubMed2000

Role of growth hormone receptor in HL-60 cell survival.

Although it is presently well established that locally produced growth hormone (GH) plays a major role in the regulation of survival mechanisms in hemopoietic cells, the responsible mechanisms are poorly understood, and the involvement of the GH receptor (GHR) has not even been demonstrated to date. In this work we investigated the presence of GHR in the human promyelocytic leukemia cell line HL-60, as well as the ability of GH treatment to stimulate both GHR and survival signaling pathways downstream GHR. Our results demonstrate that (1) both GHR mRNA and GHR immunoreactivity are present in HL-60 cells; (2) GH treatment results in an increase in the phosphorylation of the GHR-associated Jak2 and Stat3 proteins, indicating the ability of the hormone to induce receptor activation; and (3) activation of GHR increases the activity of Akt, a serine/threonine kinase that plays a prominent role in the regulation of cell survival. Taken together, these results demonstrate that GHR activation promotes survival of HL-60 cells, thus suggesting that GH plays a major role in the regulation of cell survival in the hemopoietic system, via an autocrine/paracrine mechanism.

Animals↗

PubMed2000

Increased expression of growth hormone and prolactin receptors in hepatocellular carcinomas.

The liver is an essential target tissue for growth hormone (GH) and prolactin (PRL). The aim of this study was to determine the in situ expression of growth hormone receptor (GHR) and prolactin receptor (PRLR) in hepatocellular carcinomas and to compare the results with normal liver. For this purpose, in situ hybridization (ISH) and immunohistochemical techniques were performed and several tests were conducted to validate the results. By radioactive ISH, all the hepatocellular carcinomas studied showed labeling for GHR and PRLR mRNAs. Relative expression levels, determined by computer-assisted microdensity, were higher in hepatocellular carcinomas than in normal liver. Immunohistochemistry led us to confirm the constant expression of both receptor proteins in hepatocellular carcinomas and normal liver and to demonstrate their localization not only in the cytoplasm but also in the nucleus. These results confirm that the liver is a major GH and PRL target tissue and suggest that in hepatocellular carcinomas the proliferative effects of these hormones may be increased by a higher expression of their receptors.

Adult↗

PubMed2000

A computerised guideline for the management of diabetes.

This paper describes an experience of computerising a clinical guideline for the management of Diabetes. This guideline is being used in a National Programme for Diabetes supported by the Health Ministry in Portugal, who is interested in supporting its use by General Practitioners. The prototype system was developed according to the Prestige Protocol Model that is briefly outlined. The main conclusions regarding practical design decisions are then reported, partly based on a preliminary evaluation of the system by GPs.

Diabetes Mellitus, Type 2↗

PubMed2000

[Epidemiologic study of infection by hepatitis C virus in a hemodialysis unit].

OBJECTIVE: To analyze the prevalence of infection, the frequency of HCV genotypes and the epidemiology characteristics among the patients in hemodialysis treatment in one 25 years old hospital hemodialysis center and one 15 years old secondary unit by a transversal cross-section study in 1998. PATIENTS AND METHODS: 171 hemodialyzed-patients were studied. Patients sera were analyzed by the presence of HCV antibodies anti-VHC by a enzymoimmunoassay (Abbott Cientifica) and the presence of antibodies was confirmed by a line immunoassay (Inno-LIA HCV AbIII) and by the presence of VHC-RNA by reverse transcriptase PCR (Cobas Amplicor HCV). Genotypes were determinate by reverse hybridization (Inno-LIA HCV III). RESULTS: Fifty (29.2%) of the patients were HCV antibody positive. Forty-five (26.3%) were HCV-RNA positive, all of them with antibodies positive. The distribution of genotypes was: 1b, 34 (75.5%); 4f, 4 (8.9%); 1a, 3 (6.7%); 1, 3 (6.7%) and 1 case could not be typed (2.2%). In 14 patients (28.0%), seroconversions were documented Twenty-one patients (42.0%) were diagnosed when the routine tests were available and 15 patients (30.0%) were diagnosed pre-dialysis. The multivariate analysis showed that the risk of HCV infection was greater for patients who had been more 8 years on dialysis (OR: 6.22; 95% CI: 1.24-31.07). CONCLUSIONS: Data presented indicate that the prevalence of HCV infections in our hemodialysis units and the number of seroconversions were high and the HCV subtype 1b was more frequent; because of this, the screening by both serological and molecular methods is necessary, at least twice a year, to identify all the infected patients. Besides, we think that is necessary to increase the control of the completion of the Universal Precautions.

Cross-Sectional Studies↗

PubMed1999

Complex regulation of prothymosin alpha in mammary tumors arising arising in transgenic mice.

Expression of prothymosin alpha (PTA) has been related to cell proliferation, both normal and pathological. PTA has also been proposed to be a target of the c-myc protooncogene. To study PTA mRNA levels during pathological cell growth, and especially the effect of the activation of specific oncogenes on PTA expression, we have studied its expression in tumors that arise in transgenic mice. We found high PTA levels in mammary tumors arising in c-myc, c-neu, and v-ras transgenic mice. Levels of this protein were variable between different tumors, and there is a differential regulation of PTA respect to other putative c-myc target genes, such as Ornithine Decarboxylase (ODC). Furthermore, expression of PTA is not absolutely dependent of c-myc expression, as shown by MYC depletion experiments performed with antisense oligonucleotides. We conclude that regulation of PTA in these tumors is complex and depends on more than a single activated oncogene.

Animals↗

PubMed1999

Restricted presence of the growth hormone-releasing hormone receptor to somatotropes in rat and human pituitaries.

Specific binding of growth hormone-releasing hormone (GHRH) to its plasma membrane receptor represents the first step of cellular signals leading to exocytotic GH secretion in the anterior pituitary. The GHRH receptor (GHRH-R) has been cloned and belongs to the secretin/glucagon/vasoactive intestinal peptide subfamilly of G-protein-coupled receptors. To study its characteristics in rat and human pituitaries and examine its cellular and subcellular localization, a site-directed polyclonal antibody recognizing the C-terminal portion 392-404 of the rat and human GHRH-R was used. Immunohistochemistry was performed on paraffin-embedded pituitary sections while ultrastructural immunocytology was done on frozen and Lowicryl-resin-embedded ultrathin sections. GHRH-R-like immunoreactivity was restricted to somatotropes and colocalized with GH in both rat and human tissues. No signal was detected in gonadotropes, lactotropes, corticotropes and thyrotropes. At the subcellular level, gold particles were associated with the plasma membrane (observed on ultrathin frozen sections), secretory granule membrane, cytoplasmic matrix, nuclear membrane and nuclear matrix. In the nucleus, gold particles were mainly observed at the junction between eu- and heterochromatin. The highest density of labeling was observed in the cytoplasm (55 vs. 45% in the nucleus), mainly in secretory granules (59% of cytoplasmic labeling) and the plasma membrane. These results support the hypothesis that GHRH-mediated actions in the pituitary are specific to somatotropes and that GHRH-R isoforms and/or ligand-receptor complexes are involved in intracellular trafficking, recycling processes and nuclear functions.

Adolescent↗

PubMed1999

Orexin receptors are expressed in the adrenal medulla of the rat.

Two recently discovered hypothalamic peptides, orexin-A and orexin-B, play a role as mediators in the central mechanisms that regulate feeding behavior and sleep control. These peptides bind and activate two orexins receptors that belong to the G-protein coupled receptor superfamily. Morphological studies have detected mRNA expression of orexin receptors exclusively in the rat central nervous system. In this paper we demonstrate a strong level of expression of orexin receptor 1 and 2 in the adrenal medulla of the rat by RT-PCR and immunohistochemistry. The results of the present study provide the first evidence showing that the adrenal medulla expresses orexin receptors, and thus appears to be a target tissue for orexins. This could open a new loop in which the central and autonomous nervous system may be involved in body weight homeostasis and sleep control.

Adrenal Medulla↗

PubMed1998

Correlation of Pit-1 gene expression and Pit-1 content with proliferation and differentiation in human myeloid leukemic cells.

The transcription factor pituitary-1 (Pit-1) is a homeodomain-containing protein that is expressed mainly in the pituitary, where it drives the expression of growth hormone, prolactin, and thyroid-stimulating hormone beta chain genes. In addition, Pit-1 is required for adequate pituitary cell growth and may be involved in the pathogenesis of pituitary adenomas. Pit-1 expression has been also reported in nonpituitary tissues, where it might be involved in the control of cell proliferation. In order to elucidate such a possibility, we have investigated the changes in both Pit-1 mRNA and Pit-1 immunoreactivity in HL-60 cells following the addition of several differentiating agents. Our results show that while high Pit-1 levels are found in exponentially growing HL-60 cells, a significant decrease occurs after induction of cells to differentiate along the macrophage lineage with 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast no changes were observed when cells were treated with interferon-alpha, which also induces differentiation of HL-60 cells that, at odds with TPA, is not accompanied with growth arrest. In all, these findings suggest that Pit-1 expression is specifically associated with proliferation in HL-60 cells, thus supporting the idea that one of the functions of nonpituitary Pit-1 may be the control of cell proliferation.

Cell Differentiation↗

PubMed1998

Gender differences in both spontaneous and stimulated leptin secretion by human omental adipose tissue in vitro: dexamethasone and estradiol stimulate leptin release in women, but not in men.

Leptin is a hormone secreted by the adipocytes to serve as a signal to the central nervous system to regulate energy homeostasis. Circulating leptin mainly reflects both total fat mass and the size of constituent adipocytes, although other ancillary hormonal factors may contribute to its blood concentration. Relevant gender differences in leptin concentrations have been reported, but it is not clear whether the elevated leptin levels in women are an intrinsic property of their adipocytes or merely reflect a greater amount of fat reserves. To clarify these points, a systematic study with organ culture from human omental adipose tissue either stimulated or not with steroid hormones was undertaken in samples obtained at surgery from 67 nonobese donors (33 women and 34 men). The assay was standardized in periods of 24 h ending at 96 h, with no apparent tissue damage. Each adipose tissue sample from a single donor was incubated in triplicate, and leptin results are expressed as the mean +/- SEM of the integrated secretion to the medium (area under the curve; nanograms of leptin per g tissue/48 h). Control nonstimulated samples showed a steady leptin secretion along the 96 h studied, with the peak of secretory activity reached at 48 h; afterward, the in vitro secretion reached a plateau state. Spontaneous leptin secretion in samples from 33 women (3904 +/- 347) was significantly higher (P < 0.05) than that in samples from 34 men (2940 +/- 323). Coincubation of adipose tissue with 1 mumol/L dexamethasone induced a clear-cut leptin increase (P < 0.05) in samples from women (5848 +/- 624; n = 12), but did not change the spontaneous release of leptin in samples from men (3353 +/- 741; n = 6). Similarly, coincubation of adipose tissue with 1 mumol/L estradiol induced a notable leptin increase (P < 0.05) in samples from women (5698 +/- 688; n = 9), whereas it did not alter the secretion in the male samples (3373 +/- 444; n = 6). In samples from both sexes, coincubation with 1 mumol/L estrone or progesterone had no effect, whereas 1 mumol/L forskolin significantly (P < 0.05) reduced leptin release. In conclusion, leptin secretion from omental adipose tissue in vitro 1) is significantly higher in samples from women than in samples from men, 2) is stimulated by dexamethasone and estradiol in women but not in men, 3) is not modified by progesterone or estrone in both sexes, and 4) is inhibited by forskolin in both genders. This different response to the stimulation of adipose tissue may be the biological basis for the gender differences observed in circulating levels of human leptin.

Adipose Tissue↗

PubMed1998

Splenic peliosis with spontaneous splenic rupture in a patient with immune thrombocytopenia treated with danazol.

We present a 79-year-old man diagnosed with immune thrombocytopenia (ITP), treated with danazol, who died as a result of a spontaneously ruptured spleen. The histopathological diagnosis was splenic peliosis. This patient presents a chronological association between the treatment with danazol and the development of peliosis, which suggests a clear cause-effect relationship. Facing an individual patient with ITP, clinicians should weigh the potential benefits of danazol with the possible development of serious complications, such as hepatic failure or splenic rupture due to peliosis.

Aged↗

PubMed1997

Calcium-activated chloride current in normal mouse sympathetic ganglion cells.

1. In rat sympathetic ganglion cells, axotomy induces the appearance of a depolarizing after-potential (ADP) produced by a calcium-activated chloride current. Here we report that this current is also present in normal sympathetic neurones from the mouse. 2. In an in vitro preparation of the superior cervical ganglion, an ADP was observed after spike firing in 50% of the cells studied with single-electrode current- and voltage-clamp techniques. 3. When the cells were voltage clamped at -50 mV in the presence of tetrodotoxin (TTX) and tetraethylammonium chloride (TEA), depolarizing jumps evoked inward calcium currents which were contaminated by outward chloride currents, followed by slowly decaying inward chloride tail currents. 4. The ADP and the inward tail currents disappeared when calcium was removed from the extracellular solution or when cadmium was added. 5. The reversal potential for the inward tail current was approximately -24 mV and was displaced in agreement with the Nernst equation for chloride when the extracellular NaCl was replaced by sucrose or sodium isethionate. The chloride channel blocker anthracene-9-carboxylic acid (9AC) inhibited both the ADP and the tail current. 6. Using intracellular injection of neurobiotin, we found that cells with shorter dendrites had larger ADPs. In axotomized ganglia practically all cells showed very pronounced ADPs. 7. We conclude that normal mouse sympathetic ganglion cells have a calcium-activated chloride current that generates an ADP. The channels responsible for this current are probably located in the dendrites.

Action Potentials↗

PubMed1997

Cellular and subcellular distribution of 7B2 in porcine Merkel cells.

BACKGROUND: Merkel cells are neuroendocrine cells located in the skin and oral mucosa of various mammalian species. These cells express multiple peptides as well as serotonin. Although the precise function of Merkel cells is still unknown, different studies support its role as mechano-electric transducer. 7B2 granin (secretogranin V) is a polypeptide isolated from the pituitary gland and present in the dense-cored granules of neuronal and paraneuronal cells. METHODS: The expression of the 7B2 in Merkel cells of pig snout skin was analysed by immunohistochemical techniques. The streptavidin-biotin peroxidase complex procedure was employed for light microscopy. A postembedding method using immunoglobulin-colloidal gold complexes was employed for the ultrastructural studies. RESULTS: Immunoreactivity for 7B2 was observed in virtually all Merkel cells, both in epidermis and vibrissae. The immunostaining was shown in the basal side of cytoplasms where neuroendocrine granules were accumulated. Immunoelectron microscopy allowed us to demonstrate that 7B2 labelling was located on the electrondense granules. Nuclei and epidermal nerve terminals associated with merkel cells did not show immunoreactivity. CONCLUSIONS: The polypeptide 7B2 is present in the dense-cored granules of Merkel cells. This result is consistent with the possible role for 7B2 in secretory granules' processing. To our knowledge this is the first evidence of 7B2 protein in Merkel cells.

Animals↗

PubMed1997

Synthesis of leptin in human placenta.

UNLABELLED: Gender-based differences in serum leptin levels have been reported in umbilical cord blood, and leptin has been detectedin human amniotic fluid. In order to understand if leptin may be directly synthesized by human placentae an analysis made up of several steps was performed. First at all RT-PCR analysis from placenta-derived RNA was used to detect human leptin mRNA. The leptin-like immunoradioactivity detected in placentae extracts was identical to human leptin according to the criteria of charge, immunorecognition, SDS-PAGE analysis and blotting, indicating that intact leptin was found and no variants in size, charge or immunoactivity were present in the placentae. Finally an immunohistochemical analysis showed the presence of leptin in the cytoplasm of syncytiotrophoblast cells but not in the core of villi. IN CONCLUSION: leptin is synthesized as a single molecular variant identical to human recombinant leptin in human placentae at delivery.

Base Sequence↗