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G Alonso

Publications and source records attributed to G Alonso.

At least 37 records · Page 2Linked to original sources
PubMed2001

High voltage-activated Ca2+ currents in rat supraoptic neurones: biophysical properties and expression of the various channel alpha1 subunits.

The diversity of Ca2+ currents was studied in voltage-clamped acutely dissociated neurones from the rat supraoptic nucleus (SON), and the expression of the various corresponding pore-forming alpha1 subunits determined by immunohistochemistry. We observed the presence of all high voltage-activated L-, N-, P/Q- and R-type currents. We did not observe low-voltage-activated T-type current. The multimodal current/voltage relationships of L- and R-type currents indicated further heterogeneity within these current types, each exhibiting two components that differed by a high (-20 mV) and a lower (-40 mV) threshold potential of activation. L- and R-type currents were fast activating and showed time-dependent inactivation, conversely to N- and P/Q-type currents, which activated more slowly and did not inactivate. The immunocytochemical staining indicated that the soma and proximal dendrites of SON neurones were immunoreactive for Cav1.2, Cav1.3 (forming L-type channels), Cav2.1 (P/Q-type), Cav2.2 (N-type) and Cav2.3 subunits (R-type). Each subunit exhibited further specificity in its distribution throughout the nucleus, and we particularly observed strong immunostaining of Cav1.3 and Cav2.3 subunits within the dendritic zone of the SON. These data show a high heterogeneity of Ca2+ channels in SON. neurones, both in their functional properties and cellular distribution. The lower threshold and rapidly activating L- and R-type currents should underlie major Ca2+ entry during action potentials, while the slower and higher threshold N- and P/Q-type currents should be preferentially recruited during burst activity. It will be of key interest to determine their respective role in the numerous Ca2+-dependent events that control the activity and physiology of SON neurones

Animals↗
PubMed2000

Differential activation of p38 mitogen-activated protein kinase isoforms depending on signal strength.

We have investigated the ability of the mitogen-activated protein kinase (MAPK) kinase MKK6 to activate different members of the p38 subfamily of MAPKs and found that some MKK6 mutants can efficiently activate p38alpha but not p38gamma. In contrast, a constitutively active MKK6 mutant activated both p38 MAPK isoforms to similar extents. The same results were obtained upon co-expression in Xenopus oocytes and in vitro using either MKK6 immunoprecipitates from transfected cells or bacterially produced recombinant proteins. We also found that the preferential activation of p38alpha by MKK6 correlated with more efficient binding of MKK6 to p38alpha than to p38gamma. Furthermore, increasing concentrations of constitutively active MKK6 differentially activated either p38alpha alone (low MKK6 activity) or both p38alpha and p38gamma (high MKK6 activity), both in vitro and in injected oocytes. The determinants for selectivity are located at the carboxyl-terminal lobe of p38 MAPKs but do not correspond to the activation loop or common docking sequences. We also showed that different stimuli can induce different levels of endogenous MKK6 activity that correlate with differential activation of p38 MAPKs. Our results suggest that the level of MKK6 activity triggered by a given stimulus may determine the pattern of downstream p38 MAPK activation in the particular response.

Amino Acid Sequence↗
PubMed2000

On the mechanism of resistance to channel-forming colicins (PacB) and tellurite, encoded by plasmid Mip233 (IncHI3).

Plasmids of the H incompatibility complex confer protection against all known channel-forming colicins (PacB character) and resistance to potassium tellurite (Te(r)) to Escherichia coli strains. A DNA clone (2.2 kbp) from plasmid Mip233 (IncHI3) expressing PacB-Te(r) phenotypes was studied. DNA sequence analysis revealed a high degree of homology with the enzyme O-acetylserine sulfhydrylase. Size of the PacB-Te(r) transcript was estimated as 1200 bases. A single polypeptide was found on SDS-polyacrylamide gel with a molecular mass estimated of 34 kDa. The effect of channel-forming colicins and tellurite was analyzed at physiological and transcriptional levels. Results suggest that the pacB gene product could be a reductase-like enzyme. It is also suggested that presence of the PacB character among H plasmid confers selective advantage on cells sharing an ecological niche.

Amino Acid Sequence↗
PubMed2000

Human NT2 neurons express a large variety of neurotransmission phenotypes in vitro.

The NT2 cell line, which was derived from a human teratocarcinoma, exhibits properties that are characteristic of a committed neuronal precursor at an early stage of development. NT2 cells can be induced by retinoic acid to differentiate in vitro into postmitotic central nervous system (CNS) neurons (NT2-N cells). The commitment of NT2-N cells to a stable neuronal phenotype is irreversible. Because it may be possible to transplant these human neurons to compensate for neuronal loss after traumatic injuries or neurodegenerative diseases of the CNS, knowledge of their phenotype is essential. This study aimed to characterize in detail the neurotransmission phenotype of NT2-N cells by using immunocytochemical methods. Single peroxidase immunostaining demonstrated that NT2-N cells expressed the gamma-aminobutyric acidergic (GABAergic), catecholaminergic, and cholinergic phenotypes to a large extent and expressed the serotonergic phenotype to a minor extent. NT2-N cells also expressed different neuropeptides, such as neuropeptide Y, oxytocin, vasopressin, calcitonin gene-related peptide, and Met- and Leu-enkephalin. Double fluorescence immunostaining further indicated that a large number of NT2-N cells could express GABA and another neurotransmitter or neuropeptide at the same time. Finally, electron microscopy demonstrated that these NT2 neurons elaborate classical synaptic contacts. The multipotentiality of these neurons, combined with their apparent functionality, suggests that they may represent useful material for a variety of therapeutic approaches aimed at replacing dead neurons after neurodegenerative diseases or lesions of the CNS.

Cell Differentiation↗
PubMed2000

Prolonged corticosterone treatment of adult rats inhibits the proliferation of oligodendrocyte progenitors present throughout white and gray matter regions of the brain.

It is well established that glucocorticoids inhibit the proliferation of progenitor cells that occurs in the hippocampal dentate gyrus of adult mammals. Active cell proliferation also occurs in the subventricular zone (SVZ) of the lateral ventricle and, to a lesser extent, throughout white and gray matter regions of the adult brain. The aim of the present study was to determine whether extrahippocampal cell proliferation is also affected by glucocorticoids. The cell proliferation marker bromodeoxyuridine (BrdU) was administered to control rats, to adrenalectomized rats, and to rats treated with a daily injection of corticosterone (10 mg/kg) for a period of 15 days. In control and adrenalectomized rats, high to low numerical densities of BrdU-labeled nuclei were detected within the different forebrain regions examined. In rats treated with corticosterone, a dramatic decrease of cell proliferation was detected in the dentate gyrus, but also throughout all white and gray matter regions examined, except for the SVZ of the lateral ventricle. Double-labeling experiments indicated that throughout the different white and gray forebrain regions examined, except for the SVZ, BrdU-labeled nuclei were essentially associated with cells immunostained for the marker of oligodendrocyte progenitors NG2. These data indicate that glucocorticoids inhibit the proliferation of oligodendrocyte precursors located throughout the white and gray matter regions of the adult rat brain. Since the proliferation of oligodendrocyte precursors plays a major role in the processes of remyelination, these data raise the question of possible detrimental effects of therapeutic treatments of CNS trauma based on the administration of glucocorticoids.

Age Factors↗
PubMed2000

Tanycytes transplanted into the adult rat spinal cord support the regeneration of lesioned axons.

During past years a number of therapeutic strategies have been developed in order to stimulate axonal regeneration after traumatic injuries of the spinal cord. Recently, encouraging data have been obtained by grafting specific glial cells such as Schwann cells or olfactory ensheathing glial cells, known to support the regeneration of peripheral or central axons, respectively. In a recent series of studies, we have shown that tanycytes, a particular glial cell type present in the mediobasal hypothalamus, were able to support the regeneration of a variety of axons innervating this region. The aim of the present study was to determine whether tanycytes could also support the regeneration of lesioned spinal axons. Cultured hypothalamic tanycytes and cortical astrocytes were prelabeled with Fast blue (FB) and grafted into the thoracic spinal cord of adult rats. Three weeks after the transplantation, the animals were fixed and spinal cord sections treated for multiple fluorescence detection of the FB-labeled transplanted cells on the one hand and of various glial and neuronal markers on the other hand. We show here that in all the spinal cords examined, transplanted tanycytes or astrocytes formed large spherical clusters of about 0.5 mm in diameter, located in the mediolateral spinal cord layer. The immunodetection of glial markers showed that transplanted astrocytes exhibited intense immunostaining for both glial fibrillary acidic protein (GFAP) and vimentin (VIM), whereas transplanted tanycytes were intensely immunostained for VIM, but GFAP negative. The immunodetection of axonal markers showed that contrasting with astrocyte transplants, tanycyte transplants were invaded by numerous axonal fibers. These data indicate that tanycyte transplants may represent a useful therapeutic tool for the reparation of the lesioned spinal axons.

Age Factors↗
PubMed2000

Immunocytochemical localization of the sigma(1) receptor in the adult rat central nervous system.

In order to characterize the localization of the sigma(1) receptor in the adult rat central nervous system, a polyclonal antibody was raised against a 20 amino acid peptide, corresponding to the fragment 143-162 of the cloned sigma(1) receptor protein. Throughout the rostrocaudal regions of the central nervous system extending from the olfactory bulb to the spinal cord, intense to moderate immunostaining was found to be associated with: (i) ependymocytes bordering the entire ventricular system, and (ii) neuron-like structures located within the parenchyma. Double fluorescence studies confirmed that, throughout the parenchyma, sigma(1) receptor-immunostaining was essentially associated with neuronal structures immunostained for the neuronal marker betaIII-tubulin. In all rats examined, high levels of immunostaining were always associated with neurons located within specific regions including the granular layer of the olfactory bulb, various hypothalamic nuclei, the septum, the central gray, motor nuclei of the hindbrain and the dorsal horn of the spinal cord. In contrast, only faint immunostaining was associated with neurons located in the caudate-putamen and the cerebellum. Electron microscope studies indicated that sigma(1) receptor immunostaining was mostly associated with neuronal perikarya and dendrites, where it was localized to the limiting plasma membrane, the membrane of mitochondria and of some cisternae of the endoplasmic reticulum. At the level of synaptic contacts, intense immunostaining was associated with postsynaptic structures including the postsynaptic thickening and some polymorphous vesicles, whereas the presynaptic axons were devoid of immunostaining. These data indicate that the sigma(1) receptor antibody prepared here, represents a promising tool for further investigating the role of sigma(1) receptors.

Animals↗
PubMed2000

Growth-associated protein-43 messenger ribonucleic acid expression in gonadotropin-releasing hormone neurons during the rat estrous cycle.

We have shown previously at the ultrastructural level that morphological changes occur in the external zone of the median eminence allowing certain GnRH nerve terminals to contact the pericapillary space on the day of proestrus. The present study was designed to determine whether the intrinsic determinant of neuronal outgrowth, growth-associated protein-43 (GAP-43), was expressed in GnRH neurons of adult female rats, and whether its expression varied throughout the estrous cycle. To accomplish this, we perfusion-fixed groups of adult female rats at 0800 and 1600 h on diestrous day 2 (diestrous II), at 0800 h and 1600 h on proestrus, and at 0800 and 1600 h on estrus (n = 4 rats/group) and used double labeling in situ hybridization and quantification to compare the levels of GAP-43 messenger RNA (mRNA) in cells coexpressing GnRH mRNA. GnRH mRNA was detected with an antisense complementary RNA (cRNA) probe labeled with the hapten digoxigenin, whereas the GAP-43 cRNA probe was labeled with 35S and detected by autoradiography. In addition, GAP-43 protein was identified with immunohistochemistry in the median eminence. The results show that many GnRH neurons expressed GAP-43 mRNA and that GAP-43 protein was present in many GnRH axon terminals in the outer layer of the median eminence. The number of GnRH neurons expressing GAP-43 mRNA was significantly higher on proestrus (64 +/- 5%) than on diestrous II (40 +/- 2%; P < 0.001) or on estrus (45 +/- 8%; P < 0.05), and the GAP-43 mRNA levels in GnRH neurons also varied as a function of time of death during the estrous cycle. The GAP-43 mRNA levels in GnRH neurons were higher on proestrus and estrus than on diestrous II (P < 0.05). These data show that 1) GAP-43 is expressed in adult GnRH neurons; 2) GAP-43 mRNA expression in GnRH neurons fluctuates during the estrous cycle; and 3) GAP-43 mRNA content in GnRH neurons is highest on the day of proestrus, before and during the onset of the LH surge. These observations suggest that the increased GAP-43 mRNA expression in GnRH neurons on the day of proestrus could promote the outgrowth of GnRH axon terminals to establish direct neurovascular contacts in the external zone of the median eminence and thus facilitate GnRH release into the pituitary portal blood.

Animals↗
PubMed2000

Increased hip bone mineral density in a woman with gluteal silicon implant.

Conditions and artifacts such as aortic calcification, osteophytes, hip prostheses, and metallic objects can mislead the results of dual energy X-ray absorptiometry (DXA) scanning of the spine and hip. Gluteal silicon implants increasingly are being used for aesthetic purposes by women in Brazil, some of whom are at an age of high risk for the development of postmenopausal osteoporosis. We herein report a case of a woman whose hip bone mineral density by DXA clearly increased after the placement of bilateral gluteal implants of silicon. This case demonstrates the importance of inquiring about the presence of this artifact to avoid unnecessary evaluation of hip bone densitometry in these situations.

Absorptiometry, Photon↗
PubMed2000

Essential role of p38alpha MAP kinase in placental but not embryonic cardiovascular development.

p38alpha MAP kinase is activated in response to many cellular stresses and also regulates the differentiation and/or survival of various cell types in vitro, including skeletal muscle cells and cardiomyocytes. Here we show that targeted inactivation of the mouse p38alpha gene results in embryonic lethality at midgestation correlating with a massive reduction of the myocardium and malformation of blood vessels in the head region. However, this defect appears to be secondary to insufficient oxygen and nutrient transfer across the placenta. When the placental defect was rescued, p38alpha(-/-) embryos developed to term and were normal in appearance. Our results indicate that p38alpha is required for placental organogenesis but is not essential for other aspects of mammalian embryonic development.

Animals↗
PubMed2000

Construction of a cassette for cloning and analysis of replicons.

The aim of this work was the construction of a cassette, i.e., a non-replicative molecule formed by linkage of an antibiotic resistance gene and a multiple cloning site. This cassette would allow the cloning and analysis of a wide range of replicons. The aac(6')-lc amikacin gene was isolated and ligated to the multiple cloning site of the pUC18 vector. This construction was HindIII digested and cloned in the HindIII site of the vector. The resulting pHJ13 clone conferred to the recipient cells the ability to grow in presence of amikacin (cassette marker) and ampicillin (vector gene). By restriction analysis, the cassette orientation was established. Cassette versatility is provided by the presence of the unaltered multiple cloning site segment, and also because it allows sequencing of any replication origin inserted. Cassette functionality was demonstrated by ligation to a replicative region of H plasmid pHH1457. Presence of the ori region from pHH1457 and the aac(6')-lc gene was confirmed in E. coli transformed clones. The incompatibility properties of the pHH1457 and its capability to replicate in a Poll defective strain were preserved in the pHJII14 construct. Currently, the amikacin cassette is being used in the characterization of H Complex plasmids.

Amikacin↗
PubMed2000

How bacteria protect themselves against channel-forming colicins.

Here we review the mechanisms that bacterial cells use to protect themselves against channel-forming colicins. Four mechanisms are examined: immunity, resistance, tolerance and PacB character. Immunity confers protection to colicinogenic cells against the colicin they produce, since the colicinogenic plasmid bears the genetic determinant for such immunity protein. Resistance is provided by modifications on colicin receptors located on the outer membrane. It prevents colicin adsorption and protects against those colicins sharing a common receptor. Tolerance is achieved by changes in the translocation system. The adsorbed colicin is not translocated toward the periplasmic space. This impedes its insertion into the cell membrane as well as the formation of the transmembrane channel. Tolerance confers protection against colicins that share the same translocation system. Finally, we discuss the PacB character, that confers protection against all known channel-forming colicins. The latter property is encoded by non-colicinogenic plasmids in the H-incompatibility complex.

Bacterial Outer Membrane Proteins↗
PubMed2000

[Magnetic resonance imaging in central diabetes insipidus in children and adolescents. findings at diagnosis and during follow-up].

AIM: The absence of the hyperintense signal of the posterior pituitary in magnetic resonance imaging (MRI) is considered by some authors to be evidence of neurohypophyseal dysfunction. To evaluate the utility of MRI as a complementary diagnostic aid in patients with central diabetes insipidus (CDI), we studied the MR images of pediatric patients at diagnosis and during follow-up. METHODS: MR images from 14 patients (4 females, 10 males; mean age 8.5 years) who were referred for polyuria and polydipsia and whose diagnosis was central diabetes insipidus (CDI) were analyzed. Mean time of evolution from onset of polyuria until the first MRI was 1.5 years. In 11 patients more than one MR image was obtained during follow-up. Mean time of follow-up was 2.8 years. RESULTS: In 10 patients CDI was idiopathic, in 3 it was secondary to a hypothalamic tumor and in 1 it was secondary to histiocytosis. In one patient with idiopathic CDI, the hyperintense signal was present at diagnosis but disappeared during the following 15 months. Four of the patients with idiopathic CDI developed thickening of the pituitary stalk, some at their diagnosis and others during follow-up. Of the three patients in whom CDI was secondary to a germinoma, the hyperintense signal was absent in two of them, while in one the signal was ectopic and associated with a thickened pituitary stalk. In the patient with histiocytosis, the hyperintense signal was absent at diagnosis. CONCLUSIONS: 1. In most of the patients with CDI the hyperintense signal of the posterior pituitary was absent at diagnosis; however in one patient this signal disappeared during follow-up and consequently its presence does not rule out a diagnosis of CDI. 2. Although a thickened pituitary stalk could reflect only a non-specific, transient inflammatory process, its presence makes ruling out tumoral or infiltrative disease obligatory.

Adolescent↗
PubMed2000

Immuno and cytochemical localization of Trypanosoma cruzi nitric oxide synthase.

The localization and subcellular distribution of Trypanosoma cruzi nitric oxide synthase was investigated in epimastigote cells by immunocytochemistry at electron and light microscope level, using a polyclonal antibody to neuronal nitric oxide synthase, and also, at light microscope level, by the nicotinamide adenine dinucleotide phosphate-diaphorase histochemical reaction. The immunoreactivity was ultrastructurally localized by electron microscopy in the inner surface of cell membranes and in free cytosolic clusters in the body, flagellum and apical extreme. Light microscopy showed that immunoprecipitates, specific for the Trypanosoma cruzi nitric oxide synthase, co-localized with the formazan precipitates generated by the diaphorase reaction in the same areas identified by electron microscopy. These results, taken together with previous finding from our laboratory could help to explain the involvement of the nitric oxide transduction pathway in T. cruzi epimastigote motility.

Animals↗
PubMed1999

Neuronal progenitor-like cells expressing polysialylated neural cell adhesion molecule are present on the ventricular surface of the adult rat brain and spinal cord.

In the adult rodent brain, it is now well established that neurons are continuously generated from proliferating neuronal progenitor cells located in the subventricular zone of the lateral ventricle (SVZ) and the dentate gyrus of the hippocampus. Recently, it has been shown that neurons can also be generated in vitro from various regions of the adult brain and spinal cord ventricular neuroaxis. As the highly polysialylated neural cell adhesion molecule (PSA-NCAM) has been shown to be specifically expressed by neuronal progenitor cells of the SVZ and the hippocampus, the present study was designed to determine whether cells expressing this molecule could be detected in the vicinity of the ventricular system of the adult rat brain and spinal cord. After double or triple immunostaining for different neuronal and glial markers, confocal microscopy was used to examine the surface of the ventricular neuroaxis in either 40- to 50-microm-thick transverse vibratome sections cut through different brain regions, or in 200- to 300-microm-thick tissue slices including the intact surface of the brain ventricles or of the spinal cord central canal. In untreated rats, PSA-NCAM, microtubule associated protein 2 (MAP2) and class III-beta-tubulin were found to be associated with a number of neuron-like cells located on the surface of the third and fourth ventricles and of the spinal cord central canal. The proliferation of the PSA-NCAM-immunoreactive (IR) neuron-like cells detected on the surface of the third and fourth ventricles was not affected by injection of epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) into these ventricles, but was stimulated by the combined injection of EGF + bFGF. These data indicate that cells exhibiting features of neuronal progenitors are present on the ependymal surface of the adult rat brain and spinal cord ventricular axis.

Age Factors↗
PubMed1999

Cloning and characterization of a replicon region of the IncHII plasmid pHH1457.

A replicative region of the large conjugative plasmid pHH1457 (incompatibility group HII (IncHII)) was cloned. A 1.4-kbp region, in a stable pSBII14 clone, containing a PolI-independent replicon and determinants for the HII incompatibility phenotype, was selected and characterized. High incompatibility with IncHII plasmids was corroborated. Independent replication of the insert was demonstrated by ligation to an antibiotic resistance cassette. pSBII14 was used as a probe to identify IncHII plasmids from other members of the H complex: IncHI (IncHI1, IncHI2 and IncHI3 subgroups). Hybridization experiments revealed a high homology with the replication region of IncHII plasmids, but not with IncHI1 or IncHI3 plasmid prototypes. Homology with IncHI2 plasmids was observed, suggesting the presence of IncHII-like replicons among this subgroup of plasmids. This is the first report of the characterization of an IncHII plasmid maintenance region.

Cloning, Molecular↗