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Biomedical subjects

A Alonso

Publications and source records attributed to A Alonso.

At least 109 records · Page 6Linked to original sources
PubMed2001

Effect of treatment with different doses of 17-beta-estradiol on insulin receptor substrate-1.

CONTEXT: Ovarian hormones modulate insulin sensitivity, but their exact role remains unclear. OBJECTIVE: We tried to determine whether different doses of 17-beta-estradiol cause changes in the regulation of insulin receptor substrate (IRS-1) levels, and if so, the possible implications in insulin sensitivity. DESIGN: Ovariectomized rats were treated with different doses of 17-beta-estradiol at 6, 11 and 16 days. MAIN OUTCOME MEASURES: Immunoprecipitation and Western blotting for IRS-1 were performed in different tissues. RESULTS: We found that estradiol treatment has an influence on the amount of IRS-1 but that it acts in different ways depending on the tissue studied, on the length of treatment, and on the doses employed. CONCLUSIONS: Our results suggest that low concentrations of 17-beta-estradiol could be responsible for the upregulation of insulin receptor substrate 1, increasing insulin sensitivity in muscle and adipose tissue. However, insulin receptor substrate 1 is downregulated with high concentrations of 17-beta-estradiol, thus these high hormone plasma levels could favour insulin resistance in peripheral tissues. The role of 17-beta-estradiol seems to modulate insulin receptor substrate 1 levels in insulin dependent tissues, but in a different manner in each tissue. These novel findings are important for improving knowledge about the possible risk for insulin resistance in women taking oral contraceptives or receiving hormone replacement therapy at menopause.

Adipose Tissue↗
PubMed2000

Discharge profiles of juxtacellularly labeled and immunohistochemically identified GABAergic basal forebrain neurons recorded in association with the electroencephalogram in anesthetized rats.

The basal forebrain ostensibly plays a dual role in the modulation of cortical activation and behavioral state. It is essential for stimulating cortical activation in association with waking (and paradoxical sleep), yet also important for attenuating cortical activation and promoting slow wave sleep. Using juxtacellular recording and labeling of neurons with Neurobiotin followed by immunohistochemical staining for glutamic acid decarboxylase (GAD), we studied the discharge properties of identified GABAergic basal forebrain neurons in relation to electroencephalographic (EEG) activity in urethane-anesthetized rats to determine the part or parts that they may play in this dual role. The GABAergic neurons displayed distinct discharge profiles in relation to somatosensory stimulation-evoked cortical activation. Whereas a significant minority increased its average discharge rate, the majority decreased its average discharge rate in association with cortical activation. Moreover, subgroups displayed distinct discharge patterns related to different cortical activities, including very regular high-frequency tonic spiking within a gamma EEG frequency range and rhythmic cluster spiking within a theta-like frequency range during cortical activation. During irregular slow EEG activity in absence of stimulation, one subgroup displayed spike bursts correlated with cortical slow oscillations. As relatively large in size and also antidromically activated from the cortex, many GABAergic neurons recorded were considered to be cortically projecting and thus capable of directly modulating cortical activity. Subgroups of GABAergic basal forebrain neurons would thus have the capacity to promote cortical activation by modulating gamma or theta activity and others to attenuate cortical activation by modulating irregular slow oscillations that normally occur during slow wave sleep.

Action Potentials↗
PubMed2000

Spectroscopic techniques in the study of membrane solubilization, reconstitution and permeabilization by detergents.

This review focuses on the use of spectroscopic techniques for the study of membrane solubilization, reconstitution, and permeabilization by detergents. Turbidity and light scattering, visible and infrared spectroscopic methods, fluorescence, nuclear magnetic resonance, electron spin resonance and X-ray diffraction are examined from the point of view of their applicability to the above detergent-mediated phenomena. A short introduction is provided about each of the techniques, and references are given for further study.

Detergents↗
PubMed2000

Lipids favoring inverted phase enhance the ability of aerolysin to permeabilize liposome bilayers.

Channel formation by the bacterial toxin aerolysin follows oligomerization of the protein to produce heptamers that are capable of inserting into lipid bilayers. How insertion occurs is not understood, not only for aerolysin but also for other proteins that can penetrate membranes. We have studied aerolysin channel formation by measuring dye leakage from large unilamellar egg phosphatidylcholine vesicles containing varying amounts of other lipids. The rate of leakage was enhanced in a dose-dependent manner by the presence of phosphatidylethanolamine, diacylglycerol, cholesterol, or hexadecane, all of which are known to favor a lamellar-to-inverted hexagonal (L-H) phase transition. Phosphatidylethanolamine molecular species with low L-H transition temperatures had the largest effects on aerolysin activity. In contrast, the presence in the egg phosphatidylcholine liposomes of lipids that are known to stabilize the lamellar phase, such as sphingomyelin and saturated phosphatidylcholines, reduced the rate of channel formation, as did the presence of lysophosphatidylcholine, which favors positive membrane curvature. When two different lipids that favor hexagonal phase were present with egg PC in the liposomes, their stimulatory effects were additive. Phosphatidylethanolamine and lysophosphatidylcholine canceled each other's effect on channel formation.

Animals↗
PubMed2000

Leaky vesicle fusion induced by phosphatidylinositol-specific phospholipase C: observation of mixing of vesicular inner monolayers.

Large unilamellar vesicles containing phosphatidylinositol (PI), neutral phospholipids, and cholesterol are induced to fuse by the catalytic activity of phosphatidylinositol-specific phospholipase C (PI-PLC). PI cleavage by PI-PLC is followed by vesicle aggregation, intervesicular lipid mixing, and mixing of vesicular aqueous contents. An average of 2-3 vesicles merge into a large one in the fusion process. Vesicle fusion is accompanied by leakage of vesicular contents. A novel method has been developed to monitor mixing of lipids located in the inner monolayers of the vesicles involved in fusion. Using this method, the mixing of inner monolayer lipids and that of vesicular aqueous contents are seen to occur simultaneously, thus giving rise to the fusion pore. Kinetic studies show, for fusing vesicles, second-order dependence of lipid mixing on diacylglycerol concentration in the bilayer. Varying proportions of PI in the liposomal formulation lead to different physical effects of PI-PLC. Specifically, 30-40 mol % PI lead to vesicle fusion, while with 5-10 mol % PI only hemifusion is detected, i.e., mixing of outer monolayer lipids without mixing of aqueous contents. However, when diacylglycerol is included in the bilayers containing 5 mol % PI, PI-PLC activity leads to complete fusion.

Cholesterol↗
PubMed2000

Inhibitory role for dual specificity phosphatase VHR in T cell antigen receptor and CD28-induced Erk and Jnk activation.

The 21-kDa dual specific protein phosphatase VH1-related (VHR) is one of the smallest known phosphatases, and its function has remained obscure. We report that this enzyme is expressed in lymphoid cells and is not induced by T cell antigen receptor like other dual specificity phosphatases. Introduction of exogenous VHR into Jurkat T cells caused a marked decrease in the transcriptional activation of a nuclear factor of activated T cells and an activator protein-1-driven reporter gene in response to ligation of T cell antigen receptors. The inhibition was dose-dependent and was similar at different doses of anti-receptor antibody. Catalytically inactive VHR mutants caused an increase in gene activation, suggesting a role for endogenous VHR in this response. In contrast, the activation of a nuclear factor kappaB-driven reporter was not affected. The inhibitory effects of VHR were also seen at the level of the mitogen-activated kinases Erk1, Erk2, Jnk1, Jnk2, and on reporter genes that directly depend on these kinases, namely Elk, c-Jun, and activator protein-1. In contrast, p38 kinase activation was not affected by VHR, and p38-assisted gene activation was less sensitive. Our results suggest that VHR is a negative regulator of the Erk and Jnk pathways in T cells and, therefore, may play a role in aspects of T lymphocyte physiology that depend on these kinases.

CD28 Antigens↗
PubMed2000

Neurotensin-induced bursting of cholinergic basal forebrain neurons promotes gamma and theta cortical activity together with waking and paradoxical sleep.

Cholinergic basal forebrain neurons have long been thought to play an important role in cortical activation and behavioral state, yet the precise way in which they influence these processes has yet to be fully understood. Here, we have examined the effects on the electroencephalogram (EEG) and sleep-wake state of basal forebrain administration of neurotensin (NT), a neuropeptide that has been shown in vitro to potently and selectively modulate the cholinergic cells. Microinjection of (0.1-3.0 mm) NT into the basal forebrain of freely moving, naturally waking-sleeping rats produced a dose-dependent decrease in delta ( approximately 1-4 Hz) and increase in both theta ( approximately 4-9 Hz) and high-frequency gamma activity (30-60 Hz) across cortical, areas with no increase in the electromyogram. These EEG changes were accompanied by concomitant decreases in slow wave sleep (SWS) and transitional SWS (tSWS), increases in wake, and most remarkably, increases in paradoxical sleep (PS) and transitional PS (tPS), despite the virtual absence of SWS. The effects were attributed to direct action on cholinergic neurons as evidenced by selective internalization of a fluorescent ligand, Fluo-NT, in choline acetyltransferase (ChAT)-immunoreactive cells and stimulation by NT of bursting discharge in juxtacellularly recorded, Neurobiotin-labeled, ChAT-immunoreactive neurons. We conclude that NT-induced rhythmic bursting of cholinergic basal forebrain neurons stimulates rhythmic theta oscillations and gamma across the cerebral cortex. With the selective action of NT on the cholinergic cells, their bursting discharge promotes theta and gamma independent of motor activity and thereby also stimulates and enhances PS.

Animals↗
PubMed2000

Mixed membranes of sphingolipids and glycerolipids as studied by spin-label ESR spectroscopy. A search for domain formation.

The temperature dependences of the ESR spectra from different positional isomers of sphingomyelin and of phosphatidylcholine spin-labeled in their acyl chain have been compared in mixed membranes composed of sphingolipids and glycerolipids. The purpose of the study was to identify the possible formation of sphingolipid-rich in-plane membrane domains. The principal mixtures that were studied contained sphingomyelin and the corresponding glycerolipid phosphatidylcholine, both from egg yolk. Other sphingolipids that were investigated were brain cerebrosides and brain gangliosides, in addition to sphingomyelins from brain and milk. The outer hyperfine splittings in the ESR spectra of sphingomyelin and of phosphatidylcholine spin-labeled on C-5 of the acyl chain were consistent with mixing of the sphingolipid and glycerolipid components, in fluid-phase membranes. In the gel phase of egg sphingomyelin and its mixtures with phosphatidylcholine, the outer hyperfine splittings of sphingomyelin spin-labeled at C-14 of the acyl chain of sphingomyelin are smaller than those of the corresponding sn-2 chain spin-labeled phosphatidylcholine. This is in contrast to the situation with sphingomyelin and phosphatidylcholine spin-labeled at C-5, for which the outer hyperfine splitting is always greater for the spin-labeled sphingomyelin. The behavior of the C-14 spin-labels is attributed to a different geometry of the acyl chain attachments of the sphingolipids and glycerolipids that is consistent with their respective crystal structures. The two-component ESR spectra of sphingomyelin and phosphatidylcholine spin-labeled at C-14 of the acyl chain directly demonstrate a broad two-phase region with coexisting gel and fluid domains in sphingolipid mixtures with phosphatidylcholine. Domain formation in membranes composed of sphingolipids and glycerolipids alone is related primarily to the higher chain-melting transition temperature of the sphingolipid component.

Electron Spin Resonance Spectroscopy↗
PubMed2000

Binding of human papillomavirus 16 E5 to the 16 kDa subunit c (proteolipid) of the vacuolar H+-ATPase can be dissociated from the E5-mediated epidermal growth factor receptor overactivation.

Human papillomavirus type 16 E5 protein (HPV16 E5) upregulates ligand-mediated activation of the epidermal growth factor receptor (EGFR) in transfected human keratinocytes. HPV16 E5 binds to the 16 kDa proteolipid (subunit c) of the vacuolar H+-ATPase (16K), responsible for endosomal acidification, and this binding has been suggested to be responsible for increased recycling of the EGFRs. Using mutant deletions we show here that amino acids 54-78, but not 79-83 are necessary for binding to the 16K proteolipid. EGF treatment of cells expressing wild type or mutants of the E5 protein show that deletion of the last carboxy terminal 5 amino acids results in loss of E5-mediated EGFR overactivation. Thus, our results show that the binding capacity of HPV16 E5 to 16K can be dissociated from the effect of the viral protein on EGFR activation.

Cell Line↗
PubMed2000

Molecular cytogenetic analysis of eight inversion duplications of human chromosome 13q that each contain a neocentromere.

Neocentromeres are fully functional centromeres that have arisen in previously noncentromeric chromosomal locations on rearranged chromosomes. The formation of neocentromeres results in the mitotic stability of chromosomal fragments that do not contain endogenous centromeres and that would normally be lost. Here we describe a unique collection of eight independent patient-derived cell lines, each of which contains a neocentromere on a supernumerary inversion duplication of a portion of human chromosome 13q. Findings in these patients reveal insight into the clinical manifestations associated with polysomy for portions of chromosome 13q. The results of FISH and immunofluorescent analysis of the neocentromeres in these chromosomes confirm the lack of alpha-satellite DNA and the presence of CENtromere proteins (CENP)-C, -E, and hMAD2. The positions of the inversion breakpoints in these chromosomes have been placed onto the physical map of chromosome 13, by means of FISH mapping with cosmid probes. These cell lines define, within chromosome 13q, at least three distinct locations where neocentromeres have formed, with five independent neocentromeres in band 13q32, two in band 13q21, and one in band 13q31. The results of examination of the set of 40 neocentromere-containing chromosomes that have thus far been described, including the 8 neocentromere-containing chromosomes from chromosome 13q that are described in the present study, suggest that chromosome 13q has an increased propensity for neocentromere formation, relative to some other human chromosomes. These neocentromeres will provide the means for testing hypotheses about sequence requirements for human centromere formation.

Aneuploidy↗
PubMed2000

Morphological and electrophysiological characteristics of layer V neurons of the rat medial entorhinal cortex.

This study aimed to characterize the morphological and electrophysiological properties of neurons in layer V of the entorhinal cortex in the rat brain. Using the in vitro slice preparation and sharp electrode techniques, we recorded from layer V neurons located in the medial entorhinal cortex. Recorded cells were also labeled with biocytin. Based on morphological criteria, layer V of the entorhinal cortex is comprised of three categories of neurons: pyramidal cells, horizontal cells, and polymorphic cells. Horizontal cells could be easily distinguished from the pyramidal cells because the bulk of their dendritic plexus extended horizontally within layer V. Polymorphic cells vary in size and shape. Interestingly, they typically do not have apical dendrites, and some of them have dendrites that extend into the subiculum. Based on electrophysiological criteria alone, it was not possible to unequivocally distinguish the morphological cell types because they were somewhat heterogeneous with respect to several parameters including inward rectification, spike-frequency adaptation, and intrinsic oscillations. Nevertheless, although most horizontal cells displayed time-dependent inward rectification, most pyramidal cells displayed fast inward rectification exclusively. None of the entorhinal cortex layer V cells displayed oscillatory activity like that of neocortical layer V "bursting" cells, although neurons from all groups displayed rhythmic subthreshold membrane potential oscillations. In summary, we have found that layer V of the rat medial entorhinal cortex consists of three morphologically distinct neuronal subtypes that cannot be clearly distinguished from each other by traditional electrophysiological measures.

Animals↗
PubMed2000

Stratum corneum protein mobility as evaluated by a spin label maleimide derivative.

The molecular dynamics in the vicinity of sulfhydryl groups of stratum corneum (SC) proteins has been studied by electron paramagnetic resonance (EPR) spectroscopy of maleimide spin labels covalently bound to the proteins. The total amount of bound maleimide was around 4 nmol per mg of SC. We have interpreted the coexistence of two spectral components in the EPR spectra by a two-state model with a fraction of label hydrogen bonded to proteins and another fraction exposed to the aqueous environment. We showed that the relative populations among these two states, determined by spectral simulation, are in thermodynamic equilibrium. The calculated energetic gain for the nitroxide to form hydrogen bond with SC proteins rather than to be dissolved in the buffer was approximately 12 kcal/mol in the temperature range of 2-30 degrees C and approximately 5 kcal/mol in the range of 30-86 degrees C. Temperature profiles of other EPR parameters related to the rotational diffusion of the probe also showed changes in the temperature interval of 26-42 degrees C, suggesting alterations in the vibration modes of SC proteins which are sensitive to higher motional freedom above 26-42 degrees C. We also compared samples of intact and lipid-depleted SC and we found that the delipidization process does not alter significantly the backbone mobility in the SH group regions, but the data suggest that the protein cavity is more open in the case of the delipidized samples. These results contribute to the understanding of the protein participation in the barrier function of SC, and can be useful to improve the spectral analysis of site-directed spin labeling, particularly for a more quantitative description of the dynamic modes of the nitroxide side chains.

Electron Spin Resonance Spectroscopy↗
PubMed2000

Discharge properties of juxtacellularly labeled and immunohistochemically identified cholinergic basal forebrain neurons recorded in association with the electroencephalogram in anesthetized rats.

Multiple lines of evidence indicate that cholinergic basal forebrain neurons play an important role in the regulation of cortical activity and state. However, the discharge properties of cholinergic cells in relation to the electroencephalogram (EEG) are not yet known. In the present study, cells were recorded in the basal forebrain in association with cortical EEG activity in urethane-anesthetized rats, and their discharge was examined during EEG irregular slow activity and during stimulation-induced cortical activation, characterized by rhythmic slow (theta) and high-frequency (gamma) activities. Recorded cells were labeled with Neurobiotin (Nb), using the juxtacellular technique and identified as cholinergic by immunohistochemical staining for choline acetyltransferase (ChAT). Nb-positive/ChAT-positive neurons were distinctive and significantly different from Nb-positive/ChAT-negative neurons, which were heterogeneous in their discharge properties. All Nb(+)/ChAT(+) cells increased their discharge rate with stimulation, and most shifted from an irregular tonic discharge during EEG slow irregular activity to a rhythmic burst discharge during rhythmic slow activity. The stimulation-induced rhythmic discharge was cross-correlated with the EEG rhythmic slow activity. In some units the rhythmic discharge matched the rhythmic slow activity of the retrosplenial cortex; in others, it matched that of the prefrontal cortex, which occurred at a slower frequency, suggesting that subsets of cholinergic neurons may influence their cortical target areas rhythmically at particular frequencies. Cholinergic basal forebrain neurons thus may evoke and enhance cortical activation via both an increase in rate and a change in pattern to rhythmic bursting that would stimulate rhythmic slow (theta-like) activity in cortical fields during active waking and paradoxical sleep states.

Action Potentials↗
PubMed2000

Subcellular localization of intracellular protein tyrosine phosphatases in T cells.

A high protein tyrosine phosphatase (PTPase) activity is required to maintain circulating T lymphocytes in a resting phenotype, and to limit the initiation of T cell activation. We report that 15 of the currently known 24 intracellular PTPases are expressed in T cells, namely HePTP, TCPTP, SHP1, SHP2, PEP, PTP-PEST, PTP-MEG2, PTEN, PTPH1, PTP-MEG1, PTP36, PTP-BAS, LMPTP, PRL-1 and OV-1. Most were found in the cytosol and many were enriched at the plasma membrane. Only TCPTP and PTP-MEG2 had subcellular localizations that essentially excludes them from a direct role in early T cell antigen receptor signaling events. Overexpression of 6 of the PTPases reduced IL-2 gene activation, 3 of them thereby identified as novel candidates for negative regulators of TCR signaling. Our findings expand the repertoire of PTPases that should be considered for a regulatory role in T cell activation.

Cell Membrane↗
PubMed2000

A randomized field trial of ACINDES: a child-centered training model for children with chronic illnesses (asthma and epilepsy).

UNLABELLED: A randomized field trial of a child-centered model of training for self-management of chronic illnesses was conducted of 355 Spanish-speaking school-aged children, between 6 and 15 years old, with moderate to severe asthma and epilepsy, in Buenos Aires, Argentina. The model, based on play techniques, consists of five weekly meetings of 8-10 families, with children's and parents' groups held simultaneously, coordinated by specially trained teachers and outside the hospital environment. Children are trained to assume a leading role in the management of their health; parents learn to be facilitators; and physicians provide guidance, acting as counselors. Group activities include games, drawings, stories, videos, and role-playing. Children and parents were interviewed at home before the program and 6 and 12 months after the program, and medical and school records were monitored for emergency and routine visits, hospitalizations, and school absenteeism. In asthma and epilepsy, children in the experiment showed significant improvements in knowledge, beliefs, attitudes, and behaviors compared to controls (probability of experimental gain over controls = .69 for epilepsy and .56 for asthma, with sigma2 = .007 and .016, respectively). Parent participants in the experiment had improved knowledge of asthma (39% before vs. 58% after) and epilepsy (22% before vs. 56% after), with a probability of gain = .62 (sigma2 = .0026) with respect to the control group. Similar positive outcomes were found in fears of child death (experimental 39% before vs. 4% after for asthma, 69% before vs. 30% after for epilepsy), as well as in disruption of family life and patient-physician relationship, while controls showed no change. Regarding clinical variables, for both asthma and epilepsy, children in the experimental group had significantly fewer crises than the controls after the groups (P = .036 and P = .026). Visits to physicians showed a significant decrease for those with asthma (P = .048), and emergency visits decreased for those with epilepsy (P = .046). An 18-item Children Health Locus of Control Scale (CHLCS) showed a significant increase in internality in experimental group children with asthma and epilepsy (P < .01), while controls did not change or performed worse 12 months after the program. School absenteeism was reduced significantly for those with asthma and epilepsy (for the group with asthma, fall/winter P = .006, and spring P = .029; for the group with epilepsy, P = .011). CONCLUSION: The program was successful in improving the health, activity, and quality of life of children with asthma and epilepsy. The data suggested that an autonomous (Piagetian) model of training is a key to this success, reinforcing children's autonomous decision making.

Absenteeism↗
PubMed2000

Connexin 43 expression is downregulated in raft cultures of human keratinocytes expressing the human papillomavirus type 16 E5 protein.

A decrease in gap junction-mediated cell-to-cell communication has previously been observed in monolayer cultures of human keratinocytes (HaCaT cells) expressing the human papillomavirus type 16 E5 (HPV16 E5) gene and attributed to the reduced phosphorylation of connexin 43, the most abundant connexin in HaCaT cells. In line with this observation, we have now analyzed the effect of HPV16 E5 on connexin 43 expression in raft cultures produced by transfected HaCaT cells. These keratinocytes transcribe HPV16 E5 under the control of a dexamethasone-inducible promoter. Our results show that treatment with dexamethasone leads to an almost complete disappearance of connexin 43 in rafts expressing the E5 gene but not in control rafts. In our study we discuss the possible effects of this downregulation on cell-cell communication and cellular malignant transformation.

Cell Culture Techniques↗