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Biomedical subjects

A Fernandez

Publications and source records attributed to A Fernandez.

At least 127 records · Page 7Linked to original sources
PubMed1995

Two nuclear localization signals present in the basic-helix 1 domains of MyoD promote its active nuclear translocation and can function independently.

MyoD, a member of the family of helix-loop-helix myogenic factors that plays a crucial role in skeletal muscle differentiation, is a nuclear phosphoprotein. Using microinjection of purified MyoD protein into rat fibroblasts, we show that the nuclear import of MyoD is a rapid and active process, being ATP and temperature dependent. Two nuclear localization signals (NLSs), one present in the basic region and the other in the helix 1 domain of MyoD protein, are demonstrated to be functional in promoting the active nuclear transport of MyoD. Synthetic peptides spanning these two NLSs and biochemically coupled to IgGs can promote the nuclear import of microinjected IgG conjugates in muscle and nonmuscle cells. Deletion analysis reveals that each sequence can function independently within the MyoD protein since concomittant deletion of both sequences is required to alter the nuclear import of this myogenic factor. In addition, the complete cytoplasmic retention of a beta-galactosidase-MyoD fusion mutant protein, double deleted at these two NLSs, argues against the existence of another functional NLS motif in MyoD.

Amino Acid Sequence↗
PubMed1995

Degradation of lamin B1 precedes oligonucleosomal DNA fragmentation in apoptotic thymocytes and isolated thymocyte nuclei.

Chromatin condensation and nuclear envelope breakdown are characteristic features of apoptotic cell death, but the mechanisms underlying these phenomena have not been identified. Solubilization of nuclear lamin is responsible for both events in mitosis. In this work, we report that glucocorticoids stimulate rapid degradation of lamin B1 that occurs before oligonucleosomal DNA fragmentation in apoptotic thymocytes. Protease inhibitors and the Ca2+ buffering agent BAPTA-AM block lamin degradation and DNA fragmentation, indicating that the processes are regulated by similar or identical mechanisms. Incubation of isolated thymocyte nuclei with Ca2+ stimulates lamin degradation before the detection of oligonucleosomal DNA fragments. However, in contrast to lamin dissolution during mitosis and some other forms of apoptosis, glucocorticoid-induced degradation of lamin B1 in thymocytes is not accompanied by dephosphorylation-mediated activation of cdc2. Our results demonstrate that lamin degradation is an early feature of apoptosis in thymocytes and suggest that chromatin condensation and breakdown of the nuclear envelope may occur as a result of disruption of nuclear lamina architecture.

Animals↗
PubMed1995

Differential regulation of endogenous endonuclease activation in isolated murine fibroblast nuclei by ras and bcl-2.

Transfection of a murine fibroblast cell line with an activated form of the Harvey ras oncogene conferred sensitivity to apoptosis induced by various agents. This intrinsic sensitivity to apoptosis correlated with the expression of endogenous endonuclease activity in isolated nuclei that was undetectable in the untransfected parental cell line. Subsequent transfection with the human bcl-2 oncogene prevented the morphological and biochemical features of apoptosis in whole cells, although it failed to confer complete protection against cell death. Furthermore, transfection of the bcl-2 oncogene also inhibited the enhanced endonuclease activity in isolated nuclei. Our results indicate that some of the effects of Ha-ras and bcl-2 and potentially other oncogenes, are exerted on the biochemical machinery of apoptosis at the level of the nucleus.

Animals↗
PubMed1995

Implications for cAMP-dependent protein kinase in the maintenance of the interphase state.

The cAMP dependent protein kinase (A-kinase) is one of the first and best studied kinases in mammalian cells. There is extensive evidence that A-kinase activity acts antagonistically toward mitotic entry both in oocyte and somatic cells. Firstly, A-kinase seems to directly compromise the activation process of the cdc2 cyclin B mitotic kinase. Secondly, as shown by specific in vivo inhibition of A-kinase using microinjection of a stable form of its inhibitor peptide PKI, A-kinase modulates several key interphase cellular processes including cytoskeletal dynamics, transcription, chromatin structure and nuclear localization. We discuss the potential mechanisms involved in the down regulation of A-kinase activity at the interphase/mitosis transition.

Animals↗
PubMed1995

Characterization of neurotensin-like immunoreactivity in human basal ganglia: increased neurotensin levels in substantia nigra in Parkinson's disease.

A method that combines high performance liquid chromatography with radioimmunoassay (HPLC/RIA) has been used to characterize neurotensin-like immunoreactivity (NT-IR) in the basal ganglia from control subjects and Parkinson's disease (PD) patients. In samples from the caudate nucleus and putamen, NT-IR eluted as two HPLC peaks. One was indistinguishable from the synthetic tridecapeptide, while the other peak corresponded to oxidized NT, as judged by its chromatographic behaviour and its reaction with the antiserum employed. There were marked discrepancies between the IR detected in crude extracts and that in HPLC purified samples. NT levels (HPLC/RIA) were unaltered in the caudate nucleus, putamen and both segments of the globus pallidus in the parkinsonian brain. In contrast, there was a two-fold increase in NT content in both zona compacta and zona reticulata of the substantia nigra in PD patients compared to controls. Degeneration of the nigrostriatal pathway and/or prolonged antiparkinsonian treatment in PD appears to alter neurotensin levels in an attempt to activate the dopaminergic nigrostriatal pathway.

Aged↗
PubMed1995

Retrograde-delivered cardioplegia is not distributed equally to the right ventricular free wall and septum.

Right ventricular myocardial protection during cardiac surgery continues to be a challenge. Retrograde delivery of cardioplegia has been shown to perfuse left ventricular regions subtended by critical coronary stenosis and not adequately protected by antegrade delivery. However, the distribution of cardioplegia from the coronary sinus to the right ventricle remains in question. A reliable means for assessing such flow distribution intraoperatively is provided by contrast echocardiography. It was hypothesized that conventional use of coronary sinus catheters for retrograde cardioplegia delivery does not reliably perfuse the myocardial region subtended by the right coronary artery. Six patients scheduled to undergo elective coronary artery bypass surgery were evaluated with contrast echocardiography to determine the distribution of retrograde-delivered cardioplegia into the right ventricle. Sonicated Renografin-76 (Squibb Diagnostics, Princeton, NJ) was injected during retrograde delivery of cold crystalloid cardioplegia solution and continuous two-dimensional ultrasound imaging of the heart. On-line videodensitometric analysis was performed with a digital ultrasound system. The area under the curve and peak pixel intensity were determined for the anterior septum, the posterior septum, and the right ventricular free wall for each contrast injection. Recorded VHS videotape images of contrast-enhanced perfusion patterns were also reviewed and scored. On-line acoustic-densitometric analysis showed that right ventricular posterior and anterior septal peak pixel intensities were 4.8 +/- 3.2 and 7.3 +/- 1.5, respectively, compared with only 1.6 +/- 1.2 (p < or = 0.05) in the right ventricular free wall.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1995

Residual cavities after surgery for hepatic hydatid cysts--an ultrasonographic evaluation.

Ultrasonography has made it easier to diagnose precisely hepatic hydatid cysts, but the interpretation of images of residual cavities persisting long after the operation can be difficult. We reviewed the clinical histories of 22 children treated surgically for hepatic hydatid cysts at our hospital (1985-1992) to find clues to the proper attitude toward residual cavities. The children's mean age was 7.4 +/- 2.5 years. Fourteen children had single cysts (11 right and 3 left) and 8 children had multiple cysts, one of them 30 cysts (16 right and 14 left). The mean ultrasonographic diameter of the 44 cysts was 6.1 +/- 3 cm. Cystectomy with partial pericystectomy was performed in every case. Six months after operation, 29 cavities had disappeared, became calcified, or were substituted with fibrous scar tissue. The remaining 15 residual cavities had a mean diameter of 3 +/- 1 cm. The residual cavities exhibited little change at 12 months (2.8 +/- 1 cm), but at 18 months 9 had disappeared, 2 that increased in size and 4 remained the same size. Of these 4 stable lesions, surgery performed in 2 found no cysts and 2 have been followed-up ultrasonographically for 5 years. This short series shows that ultrasonography is useful in the diagnosis and follow-up of residual cavities after surgery for hepatic hydatid cysts. Most residual cavities disappeared by 18 months. In the cavities that persisted, our findings suggest that surgical treatment is justified only when the cyst becomes larger on ultrasonography.

Child↗
PubMed1995

Changes in brain, plasma and cerebrospinal fluid contents of beta-endorphin in dogs at the moment of death.

To investigate the possible participation of endogenous opioids in the cerebral events that take place at the moment of death we studied brain, cerebrospinal fluid and serum contents of beta-endorphin in dogs that were either conscious or unconscious at the moment of sudden death induced by cardiac arrest. Although with great interindividual variations, the animals that were conscious at the moment of cardiac arrest, presented a significant increase in beta-endorphin when compared with their own previous values (p < 0.05) or with the values found in animals that were deeply anaesthetized at the time of cardiac arrest. There seems to be a sudden increment of beta-endorphin in brain tissue and body fluids of dogs who are conscious at the moment of sudden death, this change was not observed in dogs that were anaesthetized prior to death. Brain opioids could participate in the sensations narrated by subjects in the so called near-death experience.

Animals↗
PubMed1995

Differential methylation and altered conformation of cytoplasmic and nuclear forms of protein phosphatase 2A during cell cycle progression.

Protein phosphatase 2A (PP2A) appears to be involved in the regulation of many cellular processes. Control mechanisms that lead to the activation (and deactivation) of the various holoenzymes to initiate appropriate dephosphorylation events remain obscure. The core components of all PP2A holoenzymes are the catalytic (PP2Ac) and 63-65-kD regulatory (PR65) subunits. Monospecific and affinity-purified antibodies against both PP2Ac and PR65 show that these proteins are ubiquitously localized in the cytoplasm and the nucleus in nontransformed fibroblasts. As determined by quantitative immunofluorescence the core subunits of PP2A are twofold more concentrated in the nucleus than in the cytoplasm. Detailed analysis of synchronized cells reveals striking changes in the nuclear to cytoplasmic ratio of PP2Ac-specific immunoreactivity albeit the total amounts of neither PP2Ac nor PR65 in each compartment alters significantly during the cell cycle. Our results imply that differential methylation of PP2Ac occurs at the G0/G1 and G1/S boundaries. Specifically a demethylated form of PP2Ac is found in the cytoplasm of G1 cells, and in the nucleus of S and G2 cells. In addition nuclear PP2A holoenzymes appear to undergo conformational changes at the G0/G1 and G1/S boundaries. During mitosis PP2A is lost from the nuclear compartment, and unlike protein phosphatase 1 shows no specific association with the condensed chromatin.

Amino Acid Sequence↗
PubMed1995

Measurement of general health status in obstructive sleep apnea hypopnea patients.

Sleep apnea hypopnea syndrome (SAHS) is an entity that occurs frequently in the population and produces an elevated morbidity and mortality, especially at an apnea index greater than 20 events per hour. To our knowledge there are only a few studies available addressing the general health status of sleep apnea hypopnea patients. Such information may be useful for both clinical management and better understanding of the physiopathological mechanisms of the disease, particularly with the consideration that not infrequently the physiological disturbances found in such patients do not always agree with their own health perception. Therefore, the aim of the present study was to assess the general health status and the degree of daytime somnolence, as a major symptom in SAHS patients, and relate them to the number of respiratory events per hour. Measurements of general health status and the degree of daytime somnolence were assessed in 103 consecutive patients 50.4 +/- 12 years old [mean +/- standard deviation (SD)] with an apnea/hypopnea index (AHI) of 38 +/- 27 events per hour. Forty nonsnoring healthy subjects were used as the control group. During the afternoon preceding the full polysomnography, a medical history was taken; basic anthropometric data and the presence of other diseases were recorded. The Nottingham Health Profile (NHP) questionnaire and a questionnaire assessing the degree of daytime somnolence were administered to the patients. No significant differences were found in the general health status and the degree of daytime hypersomnolence when patients were divided into three groups according to the severity of the respiratory events during the night, but there were significant differences between SAHS patients and control subjects. It was concluded that in spite of the fact that SAHS patients showed a deterioration of general health status parameters in comparison with healthy subjects, these parameters do not correlate with the physiological disturbances of SAHS, expressed as the number of respiratory events per hour.

Adult↗
PubMed1995

Protection by almagate of ethanol-induced gastric mucosal damage in rats.

The study was designed to analyse the protective effects of almagate on a model of gastric injury, ethanol-induced mucosal damage, in which acid plays little, if any, role. Pretreatment with almagate dose-dependently reduced the level of gastric damage induced by oral administration of 1 mL 100% ethanol. Administration of 12 mumol kg-1 alamagate 30 min before ethanol significantly reduced the area of mucosal damage by 65 +/- 10%, and the maximum level of inhibition (74 +/- 11%) was obtained with 150 mumol kg-1 almagate. Administration of higher doses of almagate (200-250 mumol kg-1) did not result in any further increase in the level of protection against ethanol-induced gastric damage. Administration of 1 mL 100% ethanol induces substantial damage to the gastric mucosa, with nearly 40% of the length of the section evaluated exhibiting deep necrotic and haemorrhagic damage. Pretreatment with almagate caused a significant diminution in all parameters of histological damage, whereas damage to the epithelial cell layer was only significantly reduced by pretreatment with the highest doses evaluated (25, 50 and 150 mumol kg-1). Administration of aluminium hydroxide did not modify ethanol-induced mucosal damage, even at doses containing concentrations of aluminium higher than those present in gastroprotective doses of almagate. Pretreatment with sucralfate, another aluminium containing compound, at doses of 250 mumol kg-1 protected the mucosa, although lower doses did not. The present study has shown that almagate prevents ethanol-induced gastric mucosal damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗
PubMed1995

The serum response factor nuclear localization signal: general implications for cyclic AMP-dependent protein kinase activity in control of nuclear translocation.

We have identified a basic sequence in the N-terminal region of the 67-kDa serum response factor (p67SRF or SRF) responsible for its nuclear localization. A peptide containing this nuclear localization signal (NLS) translocates rabbit immunoglobulin G (IgG) into the nucleus as efficiently as a peptide encoding the simian virus 40 NLS. This effect is abolished by substituting any two of the four basic residues in this NLS. Overexpression of a modified form of SRF in which these basic residues have been mutated confirms the absolute requirement for this sequence, and not the other basic amino acid sequences adjacent to it, in the nuclear localization of SRF. Since this NLS is in close proximity to potential phosphorylation sites for the cAMP-dependent protein kinase (A-kinase), we further investigated if A-kinase plays a role in the nuclear location of SRF. The nuclear transport of SRF proteins requires basal A-kinase activity, since inhibition of A-kinase by using either the specific inhibitory peptide PKIm or type II regulatory subunits (RII) completely prevents the nuclear localization of plasmid-expressed tagged SRF or an SRF-NLS-IgG conjugate. Direct phosphorylation of SRF by A-kinase can be discounted in this effect, since mutation of the putative phosphorylation sites in either the NLS peptide or the encoded full-length SRF protein had no effect on nuclear transport of the mutants. Finally, in support of an implication of A-kinase-dependent phosphorylation in a more general mechanism affecting nuclear import, we show that the nuclear transport of a simian virus 40-NLS-conjugated IgG or purified cyclin A protein is also blocked by inhibition of A-kinase, even though neither contains any potential sites for phosphorylation by A-kinase or can be phosphorylated by A-kinase in vitro.

Amino Acid Sequence↗
PubMed1995

Delayed cyclin A and B1 degradation in non-transformed mammalian cells.

Cyclins A and B are known to exhibit significant differences in their function, cellular distribution and timing of degradation at mitosis. On the basis of observations in marine invertebrates and Xenopus, it was proposed that cyclin destruction triggers cdc2 kinase inactivation and anaphase onset. However, this model has recently been questioned, both in Xenopus and in budding yeast. In this report, we present evidence for delayed degradation of both cyclins A and B1 in non-transformed mammalian cells. Indeed, by means of indirect immunofluorescence and confocal microscopy, we show that cyclins A and B1 are present up to anaphase in REF52, Hs68, human primary fibroblasts and NRK epithelial cells. In marked contrast, cyclin A is shown to be degraded within metaphase and cyclin B just at the transition to anaphase in HeLa and two transformed cell lines, derivatives of normal NRK and REF52. These results further support the notion that cyclin destruction might be not correlated with anaphase onset in normal cells and highlight a significant difference in the fate of mitotic cyclins between transformed and non-transformed cells.

Animals↗
PubMed1994

Differential beta-adrenergic regulation and phenotypic modulation of voltage-gated calcium currents in rat aortic myocytes.

1. We studied the beta-adrenergic regulation of voltage-gated Ca2+ channel currents using the whole-cell patch-clamp technique (18-22 degrees C) in freshly isolated and in cultured (1-20 days) rat aortic vascular smooth muscle cells (VSMCs). These currents include a transient low-voltage-activated (LVA) current and two L-type-related high-voltage-activated currents (HVA1 and HVA2, respectively). 2. At 10 microM, the beta-adrenergic agonist, isoprenaline, increased the HVA2 current (65 +/- 30%, n = 10) but had no effect on LVA and HVA1 currents. This potentiation was dose dependent in the range 0.01-10 microM, developed with a slow time course and was mimicked by elevating intracellular cyclic AMP using the permeant analogue dibutyryl cyclic AMP (100 microM). 3. In the well-differentiated freshly isolated myocytes, only the HVA1 current was recorded. In cultured cells, a predominant frequency of occurrence of LVA and HVA1 currents was observed in modulated and differentiated myocytes, respectively. The occurrence of the HVA2 current was stable during culture but this current disappeared when the cells were confluent. It was retrieved when the confluent cells were dispersed and subcultured. 4. In conclusion, we present evidence for a differential beta-adrenergic regulation of three types of Ca2+ channel current in adult rat aortic VSMCs. The differential expression of these currents, associated with marked changes in cell phenotypes in vitro, suggests that they serve distinct physiological functions.

Animals↗
PubMed1994

Nuclear localization of p85s6k: functional requirement for entry into S phase.

Immunolocalization of a newly described isoform of p70s6k, termed p85s6k, demonstrated a predominantly nuclear location in rat embryo fibroblasts (REF-52), a compartment in which growth factor-mediated phosphorylation of S6 has recently been reported. Microinjection of expression vectors encoding either p85s6k or a fusion protein containing only the putative nuclear localization motifs led to the exclusive accumulation of both products in the nucleus. Consistent with such a localization, microinjection of affinity-purified anti-p85s6k IgG into the nucleus, but not the cytoplasm, blocked serum-induced initiation of DNA synthesis. Co-injection into the nucleus of the anti-p85s6k IgG with activated p70s6k, which lacks the antigenic epitope, rescued the S phase block, arguing that the antibody exerts its effects through inhibiting p85s6k function. The results indicate a novel role for S6 phosphorylation in the nucleus distinct from that in the cytoplasm, a role essential for mitogenesis.

Amino Acid Sequence↗
PubMed1994

Cloning and characterization of AKAP 95, a nuclear protein that associates with the regulatory subunit of type II cAMP-dependent protein kinase.

The subcellular location of the type II cAMP-dependent protein kinase is dictated by the interaction of the regulatory subunit (RII) with A-kinase anchor proteins (AKAPs). Using an interaction cloning strategy with RII alpha as a probe, we have isolated cDNAs encoding a novel 761-amino acid protein (named AKAP 95) that contains both RII- and DNA-binding domains. Deletion analysis and peptide studies revealed that the RII-binding domain of AKAP 95 is located between residues 642 and 659 and includes a predicted amphipathic helix. Zinc overlay and DNA binding studies suggest that the DNA-binding domain is composed of two CC/HH-type zinc fingers between residues 464 and 486 and residues 553 and 576. The AKAP was detected in a nuclear matrix fraction, and immunofluorescence using purified anti-AKAP 95 antibodies revealed a distinct nuclear staining in a variety of cell types. Direct overlay of fluorescein isothiocyanate-labeled RII alpha onto fixed rat embryo fibroblasts showed that high-affinity binding sites for RII exist in the nucleus and that these sites are blocked by an anchoring inhibitor peptide. Furthermore, AKAP 95 was detected in preparations of RII that were purified from cellular extracts using cAMP-agarose. The results suggest that AKAP 95 could play a role in targeting type II cAMP-dependent protein kinase for cAMP-responsive nuclear events.

A Kinase Anchor Proteins↗
PubMed1994

Activation of p34cdc2 protein kinase by microinjection of human cdc25C into mammalian cells. Requirement for prior phosphorylation of cdc25C by p34cdc2 on sites phosphorylated at mitosis.

Human cdc25C protein, a specific tyrosine phosphatase that activates the p34cdc2 protein kinase at mitosis, is itself a phosphoprotein that shows increased phosphorylation during the G2-M transition. In vitro, cdc25C protein is substantially phosphorylated by purified p34cdc2-cyclin B protein kinase. Of seven putative phosphorylation sites for p34cdc2 protein kinase present in human cdc25C, five are phosphorylated by p34cdc2 protein kinase in vitro, as assessed by tryptic phosphopeptide mapping and peptide sequencing. These same sites are also phosphorylated in vivo during the G2-M transition in normal mammalian fibroblasts and have been precisely mapped. The cdc25C phosphorylated in vitro by p34cdc2 protein kinase exhibits a 2-3-fold higher activity than the nonphosphorylated cdc25C, as assayed by activation of inactive cdc2 prokinase. Microinjection of purified cdc25C proteins into living fibroblasts reveals that only the phosphorylated form of cdc25 is highly effective in activating G2 cells into premature prophase in a manner similar to microinjection of purified active p34cdc2 protein kinase. Together these data show that multisite phosphorylation of cdc25C by p34cdc2-cyclin B protein kinase occurs at the G2-M transition and is sufficient to induce the autoamplification of cdc2/M-phase promoting factor necessary to drive somatic mammalian cells into mitosis.

Amino Acid Sequence↗