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Biomedical subjects

A Alonso

Publications and source records attributed to A Alonso.

At least 145 records · Page 8Linked to original sources
PubMed2000

Induction of cellular genes is mediated by the Bel1 transactivator in foamy virus-infected human cells.

To gain insight into human foamy virus (HFV; also called spumaretrovirus)-induced alterations of cellular genes, the expression profiles of defined genes in HFV-infected primary human cells were analyzed by cDNA array assays. Several distinct cellular genes activated by HFV infection were identified; the identities of the cellular genes were confirmed by RNA blot analyses. Compared with mock-infected controls, the concentrations of cellular Kip2, Egr-1, COUP-TF1, insulin-like growth factor II (IGF-II), and EphB3 mRNAs were significantly increased in HFV-infected cells and showed a gene-specific and time-dependent induction. Immunoblot analyses with antibodies against some of the cellular gene products revealed increased levels of the corresponding proteins. To investigate mechanisms of HFV-induced alterations in cellular gene expression, the capacity of known HFV genes to increase expression of defined cellular genes was analyzed by transient expression experiments. Plasmids that encode the HFV Bel1 transcriptional transactivator were necessary and sufficient to strongly increase expression of p57Kip2, IGF-II, and EphB3 genes in 293T cells. Potential mechanisms and consequences of activation of cellular genes during HFV infection and Bel1 transactivation of the Kip2 gene are discussed.

Blotting, Northern↗
PubMed2000

Properties and role of I(h) in the pacing of subthreshold oscillations in entorhinal cortex layer II neurons.

Various subsets of brain neurons express a hyperpolarization-activated inward current (I(h)) that has been shown to be instrumental in pacing oscillatory activity at both a single-cell and a network level. A characteristic feature of the stellate cells (SCs) of entorhinal cortex (EC) layer II, those neurons giving rise to the main component of the perforant path input to the hippocampal formation, is their ability to generate persistent, Na(+)-dependent rhythmic subthreshold membrane potential oscillations, which are thought to be instrumental in implementing theta rhythmicity in the entorhinal-hippocampal network. The SCs also display a robust time-dependent inward rectification in the hyperpolarizing direction that may contribute to the generation of these oscillations. We performed whole cell recordings of SCs in in vitro slices to investigate the specific biophysical and pharmacological properties of the current underlying this inward rectification and to clarify its potential role in the genesis of the subthreshold oscillations. In voltage-clamp conditions, hyperpolarizing voltage steps evoked a slow, noninactivating inward current, which also deactivated slowly on depolarization. This current was identified as I(h) because it was resistant to extracellular Ba(2+), sensitive to Cs(+), completely and selectively abolished by ZD7288, and carried by both Na(+) and K(+) ions. I(h) in the SCs had an activation threshold and reversal potential at approximately -45 and -20 mV, respectively. Its half-activation voltage was -77 mV. Importantly, bath perfusion with ZD7288, but not Ba(2+), gradually and completely abolished the subthreshold oscillations, thus directly implicating I(h) in their generation. Using experimentally derived biophysical parameters for I(h) and the low-threshold persistent Na(+) current (I(NaP)) present in the SCs, a simplified model of these neurons was constructed and their subthreshold electroresponsiveness simulated. This indicated that the interplay between I(NaP) and I(h) can sustain persistent subthreshold oscillations in SCs. I(NaP) and I(h) operate in a "push-pull" fashion where the delay in the activation/deactivation of I(h) gives rise to the oscillatory process.

Animals↗
PubMed2000

Role of 17beta-estradiol and/or progesterone on insulin sensitivity in the rat: implications during pregnancy.

The mechanism for the development of insulin resistance in normal pregnancy is complex and is associated with serum levels of both progesterone and 17beta-estradiol. However, it remains unclear whether estrogens alone or progestins alone can cause insulin resistance, or whether it is a combination of both which produces this effect. We attempted to determine the role played by progesterone and/or 17beta-estradiol on the phenomena of sensitivity to insulin action that take place during pregnancy in the rat. Ovariectomized rats were treated with different doses of progesterone and/or 17beta-estradiol in order to simulate the plasma levels in normal pregnant rats. A euglycemic/hyperinsulinemic clamp was used to measure insulin sensitivity. At days 6 and 11, vehicle (V)- and progesterone (P)-treated groups were more insulin resistant than 17beta-estradiol (E)- and 17beta-estradiol+progesterone (EP)-treated groups. Nevertheless, at day 16, the V, EP and E groups were more resistant to insulin action than the P group. On the other hand, the V, EP and E groups were more insulin resistant at day 16 than at day 6, whereas the P group was more insulin resistant at day 6 than at day 16. Our results seem to suggest that the absence of female steroid hormones gives rise to a decreased insulin sensitivity. The rise in insulin sensitivity during early pregnancy, when the plasma concentrations of 17beta-estradiol and progesterone are low, could be due to 17beta-estradiol. However, during late pregnancy when the plasma concentrations of 17beta-estradiol and progesterone are high, the role of 17beta-estradiol could be to antagonize the effect of progesterone, diminishing insulin sensitivity.

Analysis of Variance↗
PubMed2000

[Accumulation and efflux of quinolones by clinical isolates of Stenotrophomonas maltophilia].

The presence of quinolone efflux pumps was analyzed in clinical isolates of Stenotrophomonas maltophilia. The presence of the protein Omp54, which is associated with the expression of multidrug resistant systems, was also tested. Western blot analysis of outer membrane proteins demonstrated that Omp54 was expressed in strains with high-level resistance to quinolones, whereas those strains with low MIC values did not express this protein. This result shows that Omp54 has an important role in the phenotype of quinolone resistance in clinical S. maltophilia isolates. Biochemical analyses have shown that clinical S. maltophilia strains are capable of quinolone efflux (namely norfloxacin, ofloxacin and ciprofloxacin). Quinolone efflux is dependent on the integrity of membrane potential. These data indicate the presence of active quinolone efflux pump systems in clinical S. maltophilia isolates.

Anti-Infective Agents↗
PubMed1999

Direct demonstration of persistent Na+ channel activity in dendritic processes of mammalian cortical neurones.

1. Single Na+ channel activity was recorded in patch-clamp, cell-attached experiments performed on dendritic processes of acutely isolated principal neurones from rat entorhinal-cortex layer II. The distances of the recording sites from the soma ranged from approximately 20 to approximately 100 microm. 2. Step depolarisations from holding potentials of -120 to -100 mV to test potentials of -60 to +10 mV elicited Na+ channel openings in all of the recorded patches (n = 16). 3. In 10 patches, besides transient Na+ channel openings clustered within the first few milliseconds of the depolarising pulses, prolonged and/or late Na+ channel openings were also regularly observed. This 'persistent' Na+ channel activity produced net inward, persistent currents in ensemble-average traces, and remained stable over the entire duration of the experiments ( approximately 9 to 30 min). 4. Two of these patches contained < or = 3 channels. In these cases, persistent Na+ channel openings could be attributed to the activity of one single channel. 5. The voltage dependence of persistent-current amplitude in ensemble-average traces closely resembled that of whole-cell, persistent Na+ current expressed by the same neurones, and displayed the same characteristic low threshold of activation. 6. Dendritic, persistent Na+ channel openings had relatively high single-channel conductance ( approximately 20 pS), similar to what is observed for somatic, persistent Na+ channels. 7. We conclude that a stable, persistent Na+ channel activity is expressed by proximal dendrites of entorhinal-cortex layer II principal neurones, and can contribute a significant low-threshold, persistent Na+ current to the dendritic processing of excitatory synaptic inputs.

Algorithms↗
PubMed1999

The human papillomavirus type 16 E5 protein modulates phospholipase C-gamma-1 activity and phosphatidyl inositol turnover in mouse fibroblasts.

The human papillomavirus type 16 E5 (HPV16-E5) protein is a membrane protein that has been associated with malignant growth. The protein affects growth factor-mediated signal transduction in a ligand-dependent manner. We show now that E5 expression in A31 fibroblasts results in an increased level of diacylglycerol (DAG) and inositol phosphates. Immunoprecipitation of phospholipase C-gamma-1 (PLC-gamma-1) with specific antibodies and immunoblotting with anti-phosphotyrosine antibodies reveal a large increase in tyrosine phosphorylation of the enzyme in E5-expressing cells compared to control vector-transfected cells. This activation of tyrosine phosphorylation is growth factor independent. In addition, an enhanced formation of phosphatidic acid (PA) was observed in E5 cells. This increase did not result from activation of phospholipase D (PLD), although the enzyme was activatable by treatment with phorbol ester Thus, a phosphohydrolase-mediated DAG synthesis from PLD-produced PA can be excluded. The observed effects were not further enhanced by EGF showing that the presence of the growth factor is not necessary for maintaining permanent activation of PLC-gamma-1 in E5-expressing cells. The DAG- and inositol phosphate-mediated signal cascade within the cells is thus effectively uncoupled from external control via EGF and its receptor in the presence of E5 protein.

Animals↗
PubMed1999

EGFR family-mediated signal transduction in the human keratinocyte cell line HaCaT.

We analyzed the formation of homo- and heterodimers between EGFR, ErbB2, and ErbB3 (members of the EGF receptor family) in the human skin keratinocyte cell line HaCaT, in dependence of the added ligand. ErbB4 was not unambiguously identified. By immunoprecipitation and Western blots, we showed the formation of heterodimers between all members of the family. Whereas EGF and TGF-alpha strongly induced heterodimerization, no effect was observed with heregulin. At the concentrations used all ligands elicited a similar, differentiation-independent activation of erk1/2 MAP kinase, with the exception of heregulin which activated p42/44 only marginally. We also found that different ligands triggered different transcription patterns of "early genes," with the exception of heregulin which did not modulate transcription. TGF-alpha was the most efficient ligand in promoting incorporation of tritiated thymidine into the DNA.

Cell Line↗
PubMed1999

Effect of Sublytic Concentrations of Sodium Cholate on Phospholipase C Hydrolysis of Phospholipid Bilayers.

Phospholipase C activity has been assayed with phosphatidylcholine as substrate in the presence of sodium cholate at concentrations well below those producing lipid solubilization. With short-chain phosphatidylcholine, which exists in monomeric form in aqueous solution, cholate has little or no effect. However, when the substrate is egg phosphatidylcholine in the form of bilayers, small cholate concentrations (below 1 mM, corresponding to an effective surfactant:lipid ratio below 0.05) increase the maximum enzyme rates by about threefold, while decreasing drastically the latency periods of enzyme activity. Previous studies from this laboratory have associated the phospholipase enhancing activity of a variety of amphiphiles to their ability to facilitate the formation of inverted hexagonal phospholipid structures, yet sodium cholate has the opposite effect, stabilizing the lamellar versus the inverted hexagonal phase. This suggests that cholate is activating phospholipase C through a hitherto undescribed mechanism. Sodium cholate concentrations above 1 mM decrease further the enzyme lag time, but they are less effective in enhancing enzyme rates. These observations may be pertinent in the analysis of biochemical data with purified lipases, as well as in physiological studies of biliary function. Copyright 1999 Academic Press.

Journal Article↗
PubMed1999

The nuclear pore complex protein Nup88 is overexpressed in tumor cells.

It has been previously shown (J. Schneider et al., Br. J. Cancer, 77: 1015-1020, 1998) that an antibody directed against Candida albicans proteins (C6) recognizes specifically a protein in human ovarian tumors. We have now performed an immunoscreening of a human cDNA library using this antibody and identified the antigen as Nup88, a protein localized preferentially at the nuclear membrane and probably implicated in nucleocytoplasmic transport. Our results show that Nup88 is strongly expressed in a series of human tumor cell lines compared with nontransformed cell lines at the RNA and the protein levels. Furthermore, we observed that in 76% of 21 ovarian tumors analyzed, the protein is also overexpressed in malignant tissue when compared with healthy adjacent tissue. Nup88 may, therefore, be considered as a putative marker for tumor growth and is probably related to increased cell cycling.

Antibodies, Monoclonal↗
PubMed1999

High conductance sustained single-channel activity responsible for the low-threshold persistent Na(+) current in entorhinal cortex neurons.

Stellate cells from entorhinal cortex (EC) layer II express both a transient Na(+) current (I(Na)) and a low-threshold persistent Na(+) current (I(NaP)) that helps to generate intrinsic theta-like oscillatory activity. We have used single-channel patch-clamp recording to investigate the Na(+) channels responsible for I(NaP) in EC stellate cells. Macropatch (more than six channels) recordings showed high levels of transient Na(+) channel activity, consisting of brief openings near the beginning of depolarizing pulses, and lower levels of persistent Na(+) channel activity, characterized by prolonged openings throughout 500 msec long depolarizations. The persistent activity contributed a noninactivating component to averaged macropatch recordings that was comparable with whole-cell I(NaP) in both voltage dependence of activation (10 mV negative to the transient current) and amplitude (1% of the transient current at -20 mV). In 14 oligochannel (less than six channels) patches, the ratio of transient to persistent channel activity varied from patch to patch, with 10 patches exhibiting exclusively transient openings and one patch showing exclusively persistent openings. In two patches containing only a single persistent channel, prolonged openings were observed in >50% of test depolarizations. Moreover, persistent openings had a significantly higher single-channel conductance (19.7 pS) than transient openings (15.6 pS). We conclude that this stable high-conductance persistent channel activity is responsible for I(NaP) in EC stellate cells. This persistent channel behavior is more enduring and has a higher conductance than the infrequent and short-lived transitions to persistent gating modes that have been described previously in brain neurons.

Animals↗
PubMed1999

Towards the in vitro reconstitution of caveolae. Asymmetric incorporation of glycosylphosphatidylinositol (GPI) and gangliosides into liposomal membranes.

Large unilamellar vesicles consisting of phospholipids with or without cholesterol have been prepared containing GPI and/or gangliosides asymmetrically located in the outer leaflet of the bilayer. Such asymmetric distribution of GPI and gangliosides is found in 'rafts' and caveolae. Using these vesicles, GPI can be readily hydrolysed by phospholipases. Both cholesterol and ganglioside are seen to inhibit, in an additive way, the hydrolytic activity of GPI-specific phospholipase D.

Animals↗
PubMed1999

Genotoxicity testing of six insecticides in two crosses of the Drosophila wing spot test.

Among the great variety of genotoxicity assays available, the wing spot test in Drosophila melanogaster has some characteristics that make it very suited for the screening of genotoxic activity, i.e., it is an easy and inexpensive assay using a eukaryotic organism in vivo. One of the most interesting characteristics of the assay is its capacity to detect genotoxic activity of promutagens without the necessity of an exogenous metabolic activation system. In this paper we present results obtained with a recently developed high bioactivation cross of the wing spot test (NORR cross). The positive results obtained with the five well-known procarcinogens 7, 12-dimethylbenz[a]anthracene, N-nitrosopyrrolidine, p-dimethylaminoazobenzene, diethylnitrosamine and urethane clearly show that the NORR strains are similar to the other high bioactivation strains previously described, but they lack their methodological disadvantages. We have tested six insecticides, which are characterised by having contradictory results in other genotoxicity tests, using both the standard and the high bioactivation (NORR) cross. The six insecticides analysed are the pyrethroid allethrin, the methylenedioxyphenolic compound piperonyl butoxide, the chlorinated hydrocarbons dieldrin and endrin, and the organophosphates dimethoate and malathion. We obtained negative results for all six compounds. Our results show the suitability of the wing spot test for the evaluation of compounds at the first level of genotoxicity testing.

Animals↗
PubMed1999

Spanish population data on the four STR loci D8S1179, D16S539, D18S51 and D21S11.

Population data were generated for four tetrameric short tandem repeat loci systems (D8S1179, D16S539, D18S51 and D21S11) for a Spanish Caucasian population sample (n = 218-219 individuals) using PCR. All loci were highly polymorphic, met Hardy-Weinberg expectations and the results demonstrated the assumption of independence of the loci analysed. The allele frequency data can be used in identity testing to estimate the frequency of a multiple PCR-based DNA profile in the Spanish population.

Alleles↗
PubMed1999

Pump prime only aprotinin inhibits cardiopulmonary bypass-induced neutrophil CD11b up-regulation.

BACKGROUND: The expression of neutrophil integrin CD11b is up-regulated after cardiopulmonary bypass (CPB) and is the neutrophil adhesive molecule of most importance in neutrophil- endothelial adherence. This neutrophil-endothelial adherence is responsible for post-CPB neutrophil-induced reperfusion injury. Low-dose aprotinin protocols inhibit the CPB-induced neutrophil CD11b up-regulation. This investigation was undertaken to evaluate the effects of pump prime only aprotinin (280 mg) on the CPB-induced up-regulation of this neutrophil integrin. METHODS: Twenty-two patients scheduled for elective myocardial revascularization were randomized into two groups: (1) control (n = 12), or (2) pump prime only aprotinin (280 mg) (n = 10). Neutrophils were isolated at baseline, 50 minutes of CPB, and 30 minutes after CPB and neutrophil CD11b expression was measured. RESULTS: The control group demonstrated a significant (p < 0.05) increase in neutrophil CD11b immunofluorescent staining at 50 minutes of CPB and at 30 minutes after CPB when compared to same group baseline and to the pump prime only aprotinin group at similar time intervals. CONCLUSIONS: These results indicate that pump prime only aprotinin modulates the CPB-induced up-regulation of neutrophil CD11b integrin, an important indicator of the systemic inflammatory response to CPB. In addition to blunting of the CPB-induced up-regulation of this neutrophil integrin expression, this pump prime only dose of aprotinin is also reported to be effective at reducing post-CPB bleeding and transfusion requirements. This salutary effect of pump prime only aprotinin suggests that such low-dose regimens can be both therapeutically effective and cost effective.

Aged↗
PubMed1999

Ceramides in phospholipid membranes: effects on bilayer stability and transition to nonlamellar phases.

The effects of ceramides of natural origin on the gel-fluid and lamellar-inverted hexagonal phase transitions of phospholipids (mainly dielaidoylphosphatidylethanolamine) have been studied by differential scanning calorimetry, with additional support from infrared and 31P nuclear magnetic resonance (NMR) spectroscopy. In the lamellar phase, ceramides do not mix ideally with phospholipids, giving rise to the coexistence of domains that undergo the gel-fluid transition at different temperatures. The combination of differential scanning calorimetry and infrared spectroscopy, together with the use of deuterated lipids, allows the demonstration of independent melting temperatures for phospholipid and ceramide in the mixtures. In the lamellar-hexagonal phase transitions, ceramides (up to 15 mol %) decrease the transition temperature, without significantly modifying the transition enthalpy, thus facilitating the inverted hexagonal phase formation. 31P-NMR indicates the coexistence, within a certain range of temperatures, of lamellar and hexagonal phases, or hexagonal phase precursors. Ceramides from egg or from bovine brain are very similar in their effects on the lamellar-hexagonal transition. They are also comparable to diacylglycerides in this respect, although ceramides are less potent. These results are relevant in the interpretation of certain forms of interfacial enzyme activation and in the regulation and dynamics of the bilayer structure of cell membranes.

Animals↗