Biomedical subjects

D Ruano

Publications and source records attributed to D Ruano.

At least 37 records · Page 2Linked to original sources

PubMed1996

Molecular and pharmacological characterization of native cortical gamma-aminobutyric acidA receptors containing both alpha1 and alpha3 subunits.

We have investigated the existence, molecular composition, and benzodiazepine binding properties of native cortical alpha1-alpha3 gamma-aminobutyric acidA (GABAA) receptors using subunit-specific antibodies. The co-existence of alpha1 and alpha3 subunits in native GABAA receptors was demonstrated by immunoblot analysis of the anti-alpha1- or anti-alpha3-immunopurified receptors and by immunoprecipitation experiments of the [3H]zolpidem binding activity. Furthermore, immunodepletion experiments indicated that the alpha1-alpha3 GABAA receptors represented 54.7 +/- 5.0 and 23.6 +/- 3.3% of the alpha3 and alpha1 populations, respectively. Therefore, alpha1 and alpha3 subunits are associated in the same native GABAA receptor complex, but, on the other hand, these alpha1-alpha3 GABAA receptors from the cortex constitute a large proportion of the total alpha3 population and a relatively minor component of the alpha1 population. The pharmacological analysis of the alpha1- or alpha3-immunopurified receptors demonstrated the presence of two different benzodiazepine binding sites in each receptor population with high (type I binding sites) and low (type II binding sites) affinities for zolpidem and Cl 218,872. These results indicate the existence of native GABAA receptors possessing both alpha1 and alpha3 subunits, with alpha1 and alpha3 subunits expressing their characteristic benzodiazepine pharmacology. The molecular characterization of the anti-alpha1-anti-alpha3 double-immunopurified receptors demonstrated the presence of stoichiometric amounts of alpha1 and alpha3 subunits, associated with beta2/3, and gamma2 subunits. The pharmacological analysis of alpha1-alpha3 GABAA receptors demonstrated that, despite the fact that each alpha subunit retained its benzodiazepine binding properties, the relative proportion between type I and II binding sites or between 51- and 59-61-kDa [3H]Ro15-4513-photolabeled peptides was 70:30. Therefore, the alpha1 subunit is pharmacologically predominant over the alpha3 subunit. These results indicate the existence of active and nonactive alpha subunits in the native alpha1-alpha3 GABAA receptors from rat cortex.

Amino Acid Sequence↗

PubMed1996

Age-related modifications on the GABAA receptor binding properties from Wistar rat prefrontal cortex.

In the present communication we have investigated the pharmacological properties of the GABAA receptor from adult (3 months old) and aged (24 months old) Wistar rat prefrontal cortex. The prefrontal cortex is implicated in cognitive functions and stress and both processes seem to be altered during aging. These changes could be mediated by modifications in the GABAA receptor properties. Our results indicated the absence of generalized age-related modifications on the pharmacological properties of the GABAA receptor from prefrontal cortical membranes. Saturation experiments using the non-selective benzodiazepine [3H]flunitrazepam revealed that neither the Kd values or the Bmax were modified during aging. Moreover, Cl 218 872 displacement of [3H]flunitrazepam showed no age-related modifications on either the Kis or the relative proportion between the Type I and Type II benzodiazepine binding sites. Therefore, the benzodiazepine binding sites are well preserved in aged prefrontal cortex. On the other hand, saturation experiments using the GABA agonist [3H]muscimol demonstrated in the Bmax of the low affinity [3H]muscimol binding sites in aged rats (4.3 +/- 0.8 pmol/mg protein vs. 2.3 +/- 0.2 pmol/mg protein in adult and aged rats, respectively). However, no age-dependent modifications were observed in the allosteric interaction between GABA and benzodiazepine binding sites. These results demonstrate that the benzodiazepine binding sites and the GABA binding sites of the GABAA receptor complex from rat prefrontal cortical membranes are differentially affected by the aging process.

Aging↗

PubMed1996

Aging-related subunit expression changes of the GABAA receptor in the rat hippocampus.

Aging-related changes in the subunit expression of some hippocampal GABAA receptors have been found. Quantitative in situ hybridization has revealed that alpha 1, subunit messenger RNA expression was significantly increased in the hippocampus (34%) of old rats. The largest increases were observed in the dentate gyrus (76%) and in the CA1 field (30%). Quantitative immunocytochemistry also showed increased protein expression of the alpha 1 subunit in the dentate gyrus (19%) and CA1 (14%) of old rats. The increased alpha 1 messenger RNA and protein expression led to increased proportions of assembled GABAA receptors that contained alpha 1 subunits, as revealed by quantitative immunoprecipitation of (3H)flunitrazepam and (3H)muscimol binding. In contrast, there were no significant changes in the expression of beta 2, beta 3 and total gamma 2 (gamma 2S + gamma 2L) subunits, although a slightly increased expression of gamma 2L peptide was detected in the hippocampus proper (7%), but not in the dentate gyrus. The results are consistent with the notion that in the rat hippocampus there is an aging-related change in the subunit composition of some GABAA receptors.

Aging↗

PubMed1996

Scanning electron microscopy of the human zonule of the lens (Zonula ciliaris).

In the present study eight human eyeballs were specifically prepared for scanning-electron-microscopic observation of the zonule. The zonule consisted of two main layers of radial fibres, an anterior and a posterior one, that inserted on the anterior and the posterior lens capsules, respectively. Some fibres inserted on the equator of the lens. Posterior zonular fibres originated at the pars plana, entered the dorsal part of the ciliary valleys and then changed their direction towards the posterior face of the lens. Posterior fibres inserted on the posterior capsule of the lens by branched endings 1 mm behind the equator of the lens. Anterior zonular fibres originated mainly at the pars plana and occasionally at the ciliary valleys. After running completely through the ciliary valleys in close contact with the lateral walls of the ciliary processes, they changed their direction at the anterior endings of the pars plicata and reached the anterior lens capsule. Anterior zonular insertions were achieved by webbed endings that diffused into the anterior capsule 2 mm in front of the lens equator. The extraordinary distension capacity of the zonular fibres was demonstrated by pulling the anterior lens capsule after hydrodissection. As a consequence, the anterior fibres were stretched up to four times their original length without breaking or disinserting.

Adult↗

PubMed1996

Non-tunneled catheters in children undergoing bone marrow transplantation.

We analyzed the use of non-tunneled (polyurethane double lumen) central venous catheters (CVCs) in 62 children undergoing bone marrow transplantation. The catheters were inserted in the Critical Care Unit without surgery or general anesthesia. The complications were pneumothorax in two patients and hemopneumothorax in two other patients (6.06%), entry site infection in six patients (9.6%), catheter-related infection in eight patients (12.9%) and catheter-related sepsis in nine patients (14.5%). The catheters were removed upon completion of therapy in 46 patients (74.1%), death in seven patients (11.3%) and in nine cases (14.5%) for infection. Despite the complications specific to non-tunneled catheter insertion, we believe this is indicated for patients during conditioning, transplantation and immediate post-transplantation periods.

Adolescent↗

PubMed1996

Pediatric experience with autologous peripheral blood progenitor cell transplantation: influence of CD34+ cell dose in engraftment kinetics.

The aim of this study was to analyze factors affecting mobilization and engraftment in 40 children undergoing autologous peripheral blood progenitor cell transplantation for different malignancies: 19 patients with haematological malignancies and 21 patients with solid tumors. Patients received 4-5 days of rhG-CSF (12 micrograms/kg/day) subcutaneously. Apheresis was performed by continuous flow blood cell separation beginning on the fifth day of rhG-CSF. For patients weighing < or = 25 kg, the extracorporeal line was primed with irradiated red blood cells. After myeloablative conditioning regimens, patients were grafted with 7.21 +/- 7.8 x 10(6)/kg CD34+ cells. Days to achieve an absolute neutrophil count > 0.5 x 10(9)/1 and a platelet count > 20 x 10(9)/1 without platelet support were 9.50 +/- 1.2 (range 7-13) and 18.1 +/- 8.3 (range 9-37), respectively. The number of CD34+ cells infused was highly correlated with engraftment kinetics (P = 0.0001). The patient's body weight and the number of previous chemotherapy courses had a negative influence on CD34+ cells collected.

Adolescent↗

PubMed1995

Kainate receptor subunits expressed in single cultured hippocampal neurons: molecular and functional variants by RNA editing.

To determine the kainate receptor subunits that are found in native kainate receptors, we have applied a multiplex PCR of cDNAs reverse transcribed from mRNA harvested from single cultured hippocampal neurons after electrophysiological recording. We found that all the cells showing rapidly desensitizing currents in response to kainate express the GluR6 subunit mRNA, and that some of them also express the GluR5 subunit mRNA. No GluR7, KA-1, or KA-2 subunit mRNAs were detected. Analysis of the editing sites of the GluR6 mRNA demonstrated that the three editing sites present in these subunits are edited to a different extent. Predominant expression of the unedited variant (Q) was observed, but edited and unedited variants may coexist in the same cell. In addition, we show that the Q/R site from the GluR6 subunit controls functional properties of native kainate receptors.

Animals↗

PubMed1995

Wrist denervation. Anatomical considerations.

In 1966, Wilhelm published a technique of denervation of the wrist joint based on his own anatomical studies of 1958. Other authors have published their experience in wrist denervations with similar results, but there are some discrepancies in descriptions of the innervation of the wrist. We have studied the innervation of the wrist by dissection under magnification on 20 cadaver limbs and by histological examination of five human foetal wrists. Based in our anatomical findings, we have demonstrated that Wilhelm's technique does not achieve total denervation of the wrist.

Cadaver↗

PubMed1995

Wrist denervation. Surgical considerations.

We have analyzed the results of 22 total wrist denervations following Wilhelm's technique and a series of 30 patients treated with denervation of the posterior interosseous nerve alone. Results of denervation have been classified depending on the pain (visual analogue scale) as other parameters (mobility and strength) have not been found to have any statistical significance. Results after partial denervation are worse than results achieved by total denervation.

Adult↗

PubMed1995

Aging-associated changes in the pharmacological properties of the benzodiazepine (omega) receptor isotypes in the rat hippocampus.

The aging-associated changes in hippocampal benzodiazepine (omega) receptor isotypes have been investigated in rats of the Wistar and Fischer 344 strains. Displacement experiments of [3H]flunitrazepam binding by zolpidem demonstrated that in hippocampal membranes from adult (3-month-old) Wistar strain rats, high (type I; omega 1)-, intermediate (type IIM; omega 2)-, and low (type IIL; omega 5)-affinity sites for this imidazopyridine account for 27.1 +/- 7.5, 44.2 +/- 7.5, and 28.8 +/- 5.1%, respectively. In hippocampal membranes from aged (24-month-old) rats of the same strain, the relative abundance of these sites was 42.8 +/- 9.3, 26.3 +/- 4, and 36.0 +/- 5.9%, respectively. In contrast, no significant difference was observed in the whole benzodiazepine (omega) binding site density between adult and aged rats. The increase in type I (omega 1) binding site density in the hippocampus of aged rats was also demonstrated in saturation experiments with [3H]zolpidem. This aging-induced increase in [3H]zolpidem binding was also observed in hippocampal membranes from Fischer 344 rats. Moreover, in both rat strains, GABA induced a greater enhancement of [3H]zolpidem (5 nM) binding to type I (omega 1) sites (GABA shift) in aged than in adult hippocampal membranes. Quantitative autoradiographic analysis of [3H]zolpidem binding to coronal brain sections from adult and aged Fischer 344 rats demonstrated that the aging-associated increases in the density of type I (omega 1) binding sites were restricted to the hippocampus. Moreover, increases in binding density were larger in the dentate gyrus and in the CA2 field than in the CA1 and CA3 fields.

Aging↗

PubMed1994

Molecular heterogeneity of the type I GABAA/benzodiazepine receptor complex.

We have investigated the presence of alpha 1, gamma 2 and beta 2-3 subunits as part of the type I (high affinity for [3H]zolpidem) GABAA/benzodiazepine receptor from rat cerebral cortex. Three subunit specific antibodies have been used (anti-alpha 1, to the C-terminal of rat alpha 1 subunit; anti-gamma 2, to the large intracellular loop of the gamma 2 subunit short form and the monoclonal 62-3G1, specific to beta 2-3 subunits) in immunoprecipitation experiments of the [3H]zolpidem binding activity or the 51,000 Da [3H]Ro 15-4513 photoaffinity labeled peptide (P51). The results indicated that alpha 1 subunit was present in the whole population (90%) of the GABAA/benzodiazepine receptors with high affinity for [3H]zolpidem. We cannot exclude the presence of other alpha subunits co-localized with alpha 1. On the one hand, 70-75% of type I GABAA/benzodiazepine receptor from rat cortex have co-existing alpha 1, beta 2-3 and gamma 2 subunits. We call this type Ia. On the other hand, 20-25% of the type I GABAA/benzodiazepine receptor complex should be constructed by the association of alpha 1 with subunits other than beta 2-3 and gamma 2. We call this type Ib. The identity of these subunits is currently unknown.

Animals↗

PubMed1994

Growth control of embryonic stem cells injected into mouse uterus on fifth day of pregnancy.

Previous studies have shown that embryonic stem cells (ES) may participate in normal embryonic development when they are injected into the blastocyst. In contrast, ES cells develop into tumors if injected in ectopic sites in adult mice. In this study we injected ES-D3 cells, with the LacZ gene incorporated, into 5-day pregnant mouse uteri, into pregnant unilaterally salpingectomized uteri, into pseudopregnant uteri and into non-pregnant uteri. X-gal staining enabled us to identify injected ES cells on the 7th, 9th, 10th, 12th and 15th days post-injection. In pregnant decidua, the ES cells were located initially in the mesometrial decidua and later distributed in the basal and capsular decidua and in the endodermic layer of the visceral yolk sac. In pregnant, unilaterally salpingectomized mouse uteri, ES cells were mainly located in the uterine lumen and tumors were not observed in either case. In contrast, ES cells injected into pseudopregnant uteri often developed into tumors and those injected into non-pregnant uteri always developed into teratocarcinomas. We conclude that the pregnant-uterine microenvironment may participate in the control of ES cell growth.

Animals↗

PubMed1994

Molecular characterization of type I GABAA receptor complex from rat cerebral cortex and hippocampus.

The molecular composition of the native gamma-aminobutyric acidA (GABAA) receptor complex is actually unknown. In the present communication we report a novel approach to characterize the minimal molecular conformation of the native GABAA receptor complex. This novel approach is based on the combination of subunit specific antibodies and specific 3H-labeled ligands in immunoprecipitation experiments. We have determined the presence of beta 2/3 and gamma 2 subunits in the Type I GABAA receptor complex, from rat cerebral cortex and hippocampus, by using two antibodies, the monoclonal 62-3G1 (specific for beta 2/3) and the polyclonal anti-gamma 2 (to the large intracellular loop of the gamma 2 short form) together with the Type I-specific ligand [3H]zolpidem. The association of gamma 2 and beta 2/3 subunits with the GABAA receptor complex was also tested using [3H]flumazenil. The results indicated that both gamma 2 and beta 2/3 were the most abundant subunits associated to either Type I or total benzodiazepine receptors from both cortex and hippocampus. Between 70-80% of Type I or total benzodiazepine binding activity was immunoprecipitated by either antibody. In addition, we have also investigated the coexistence of both subunits as part of the same population of Type I GABAA receptor complex by cross-immunoprecipitation experiments with 62-3G1 and anti-gamma 2. The results indicated that, in cerebral cortex, both gamma 2 and beta 2/3 subunits were part of the same population of Type I receptors. In hippocampus, an additional 20% of Type I receptors displayed either gamma 2 or beta 2/3 but not both subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Absence of modifications of the pharmacological properties of the GABAA receptor complex during aging, as assessed in 3- and 24-month-old rat cerebral cortex.

We have investigated the possible modifications of the pharmacological properties of Type I and Type II benzodiazepine binding sites of the gamma-aminobutyric acidA (GABAA) receptor complex in cortical membranes from 3- and 24-month-old Wistar or Fischer rats. No major changes were found in the binding parameters of [3H]zolpidem (a Type I-specific ligand) or [3H]flunitrazepam (a non-selective benzodiazepine). Neither the Kd values nor the Bmax for either ligand were modified during aging in cortical membranes from Wistar or Fischer rats. Consequently, the proportion of Type I binding sites was also unmodified in aged cortical membranes. The absence of modifications of Type I and Type II binding sites was also confirmed by Cl 218,872 displacement of [3H]flunitrazepam binding in aged cortical membranes from Wistar rats. Furthermore, the [3H]muscimol binding and the allosteric interactions between Type I or total benzodiazepine binding sites and GABA binding sites also remained unaltered with aging in cortical membranes from Wistar rats. Moreover, the pattern and proportion of the [3H]flunitrazepam photoaffinity labeled peptides were also unmodified by aging. These results demonstrate the absence of modifications in Type I or total benzodiazepine binding sites of the GABAA receptor complex from adult and aged cortical membranes in Fischer or Wistar rats.

Affinity Labels↗

PubMed1993

Comparative autoradiographic distribution of central omega (benzodiazepine) modulatory site subtypes with high, intermediate and low affinity for zolpidem and alpidem.

Pharmacological characterization of [3H]benzodiazepine binding to membrane preparations of adult rat hippocampus and neonatal rat brain have demonstrated, in addition to the omega 1 and omega 2 populations of central omega benzodiazepine binding sites associated with GABAA receptors, the existence of binding sites with microM affinity for the imidazopyridines zolpidem and alpidem. In the present study we have investigated their comparative autoradiographic distribution using [3H]flumazenil as a ligand. In the neonatal rat CNS, the imidazopyridine derivatives zolpidem and alpidem were found to discriminate two [3H]flumazenil binding site populations with an IC50 value ratio of more than 200-fold. In the different regions investigated (spinal cord, striatum, neocortex and inferior colliculus) the low affinity component had IC50 values of 20-40 microM (zolpidem) and 5-15 microM (alpidem) and accounted for ca. 50% of the total binding site population. In the adult rat, these imidazopyridine derivatives displayed a greater displacing potency in the cerebellum (IC50 = 6 and 36 nM, respectively) than in the hippocampus (IC50 = 37 and 403 nM, respectively). In the cerebellum, [3H]flumazenil binding was fully displaced by 1 microM of either compound and Hill coefficients of displacement curves were close to unity. In the hippocampus, 25% of [3H]flumazenil binding were resistant to 3 microM zolpidem or 1 microM alpidem, but were displaced by 100 microM of either compound. CL 218,872 also displayed a greater displacing potency in the cerebellum (IC50 = 83 nM) than in the hippocampus (IC50 = 711 nM) but [3H]flumazenil binding in the hippocampus was fully displaced by 10 microM of this compound. In adult rat hippocampus, zolpidem and alpidem were found to discriminate between three central omega site subtypes which display high (IC50 = 31 and 6.1 nM, for these imidazopyridine derivatives. In contrast, CL 218,872 discriminated between omega 1 and omega 2 sites but not between two omega 2 receptor subpopulations. omega 1 sites were mainly localized in layer IV of the sensorimotor cortex, cerebellum, substantia nigra, olfactory bulb and inferior colliculus. omega 2I sites were present in the cortical mantle (with higher levels in the cingulate and olfactory than in the sensorimotor cortex) and in subcortical (hippocampus, hypothalamus and nucleus accumbens) limbic structures. In the hippocampus, hypothalamus, spinal cord and nucleus accumbens, omega 2L sites accounted for more than 25% of the specific [3H]flumazenil binding; the density of these sites was minor in the cortex and in most pyramidal and extrapyramidal system structures.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Regional differences in the enhancement by GABA of [3H]zolpidem binding to omega 1 sites in rat brain membranes and sections.

The potential heterogeneity in the allosteric coupling between GABA and omega 1 binding sites within the native GABAA receptor complex has been evaluated in the rat by measuring the enhancement by GABA of [3H]zolpidem binding to omega 1 site in membranes from three rat brain structures (neocortex, cerebellum and hippocampus) and brain sections. The maximal stimulatory effect of GABA was significantly higher (265 +/- 47%) in cortical membranes than in cerebellar (165 +/- 48%) or in hippocampal (118 +/- 17%) membranes. These differences are not due either to the presence of different amounts of residual GABA in the membrane preparations or to the labeling, in presence of GABA, of binding sites other than omega 1 since: (1) the pharmacological properties of the [3H]zolpidem binding sites were similar in the three regions; (2) the degree of allosteric enhancement was unrelated to the relative proportion of omega 1 sites in each structure; and (3) GABA did not increase the Bmax for [3H]zolpidem. Regional differences in the effect of 100 microM GABA on [3H]zolpidem binding were also observed by quantitative autoradiography. Regions where the strongest (3-4-fold) effects of GABA in [3H]zolpidem binding were observed included the substantia nigra, lateral geniculate body, olfactory tubercule and red nucleus. A large increase in [3H]zolpidem binding was also demonstrated in the cingulate and frontoparietal cortices with higher effects in deep (4.2-fold) rather than in superficial layers (3.3-fold). Heterogeneous subregional increases in [3H]zolpidem binding in the presence of GABA were quantified within the cerebellum, hippocampus and superior colliculus.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗

PubMed1992

Heterogeneity in the allosteric interaction between the gamma-aminobutyric acid (GABA) binding site and three different benzodiazepine binding sites of the GABAA/benzodiazepine receptor complex in the rat nervous system.

In the present communication we have investigated the allosteric coupling between the gamma-aminobutyric acidA (GABAA) receptor and the pharmacologically different benzodiazepine (BZD) receptor subtypes in membranes from various rat nervous system regions. Two types of BZD receptors (type I and type II) have been classically defined using CL 218.872. However, using zolpidem, three different BZD receptors have been identified by binding displacement experiments in membranes. These BZD receptor subtypes displayed high, low, and very low affinity for zolpidem. The distribution of the high- and low-affinity binding sites for zolpidem was similar to that of type I and type II subtypes in cerebellum, prefrontal cortex, and adult cerebral cortex. On the other hand, the very-low-affinity binding site was localized in relative high proportion in spinal cord, hippocampus, and newborn cerebral cortex and, to a minor extent, in superior colliculus. The allosteric coupling between the GABAA receptor and the BZD receptor subtypes was different. The high- and low-affinity binding sites for zolpidem seemed to have a similar high degree of coupling, except in spinal cord. On the other hand, the very-low-affinity binding site for zolpidem displayed a low degree of coupling with the GABAA receptor. These results seem to indicate that the different efficacy of GABA in enhancing the [3H]flunitrazepam binding could be due to the different BZD receptor subtypes present in the GABAA/BZD receptor complex and, moreover, led us to speculate that the low GABA efficacy found in membranes from spinal cord, hippocampus, and newborn cerebral cortex might be due to the presence in relatively high proportion of the very-low-affinity binding site for zolpidem.

Allosteric Site↗

PubMed1991

Laminin and fibronectin expression during in vivo growth of embryoid bodies derived from teratocarcinoma.

Embryoid bodies (EB) derived from teratocarcinoma OTT6050 are a useful model for the study of early embryogenesis and tumorigenesis and are found in two forms: cystic and simple. After injection of cystic EB intraperitoneally into isogenic 129/Sv mice, their growth and the expression of laminin (LAM) and fibronectin (FN) were studied at 5, 7 and 9 days post-injection. LAM was highly expressed on the internal face of endodermal and on some internal cells in cystic type EB and in both endodermal cell faces in simple EB. Only slight FN expression was observed in cystic and simple EB. Extracellular matrix proteins can be studied in this model.

Animals↗