Biomedical subjects

Angel Alonso

Publications and source records attributed to Angel Alonso.

At least 37 records · Page 2Linked to original sources

PubMed2004

Causes of death and mortality risk factors.

BACKGROUND: Patient death continues to be a leading cause of renal transplant failure. This mortality is mainly due to cardiovascular, infectious and tumoural diseases. The aim of our study was to analyse the evolution of the mortality and its causes after renal transplantation (RT). METHODS: We studied 3365 adult renal transplant recipients, surviving at least 1 year, and transplanted in Spain in the years 1990, 1994 and 1998. Risk factors for all-cause and specific-cause mortality were analysed employing simple and multivariate Cox regression. RESULTS: 300 patients (8.9%) died after the first post-transplantation year. The follow-up was shorter (maximum 2.5 years) in recipients transplanted in 1998. When we consider an identical follow-up (2.5 years) for all patients, we did not find statistical differences in mortality rates or its causes, in the three analysed periods. Cardiovascular diseases (CVDs) and neoplasia were the most frequent causes of death. Mortality was higher for males and patients > 60 years. Renal function, evaluated by creatinine level or proteinuria range at 3 months and its increase in the first year post-transplantation were significant factors associated with high risk for cardiovascular and infectious death. CONCLUSIONS: During the last decade, in Spain, the mortality after RT (2.5 years follow-up) remains stable. Recipient age (> 60 years), male gender and renal function in the first year were associated with higher risk of death, especially for CVD.

Adult↗

PubMed2004

Identification of membrane proteins differentially expressed in human papillomavirus type 16 E5-transfected human keratinocytes by nanoelectrospray ionization mass spectrometry.

Membrane proteins differentially expressed in human papillomavirus type 16 (HPV-16) E5-transfected HaCaT cells have been identified. Membrane proteins were isolated and separated by two-dimensional gel electrophoresis. Spots showing quantitative differences between E5-transfected and control cells were extracted and the proteins were identified by nanoelectrospray ionization mass spectrometry. A total of 24 spots was analysed. Among the proteins showing differential expression, a decreased amount of calnexin and increased expression of hsp70, proteins both involved in maturation and transport of MHC class I complexes to the plasma membrane, were noticed. These findings correlate with the decreased surface expression of MHC class I molecules described in E5-expressing cells, HPV-positive cervical lesions and cervical carcinomas. These results stress the value of the proteomic approach, as used here in the experimental design, which allows the correlation of changes in host gene expression with biological functions of viral genes.

Histocompatibility Antigens↗

PubMed2004

Mucosal human papillomaviruses encode four different E5 proteins whose chemistry and phylogeny correlate with malignant or benign growth.

We performed a phylogenetic study of the E2-L2 region of human mucosal papillomaviruses (PVs) and of the proteins therein encoded. Hitherto, proteins codified in this region were known as E5 proteins. We show that many of these proteins could be spurious translations, according to phylogenetic and chemical coherence criteria between similar protein sequences. We show that there are four separate families of E5 proteins, with different characteristics of phylogeny, chemistry, and rate of evolution. For the sake of clarity, we propose a change in the present nomenclature. E5alpha is present in groups A5, A6, A7, A9, and A11, PVs highly associated with malignant carcinomas of the cervix and penis. E5beta is present in groups A2, A3, A4, and A12, i.e., viruses associated with certain warts. E5gamma is present in group A10, and E5delta is encoded in groups A1, A8, and A10, which are associated with benign transformations. The phylogenetic relationships between mucosal human PVs are the same when considering the oncoproteins E6 and E7 and the E5 proteins and differ from the phylogeny estimated for the structural proteins L1 and L2. Besides, the protein divergence rate is higher in early proteins than in late proteins, increasing in the order L1 < L2 < E6 approximately E7 < E5. Moreover, the same proteins have diverged more rapidly in viruses associated with malignant transformations than in viruses associated with benign transformations. The E5 proteins display, therefore, evolutionary characteristics similar to those of the E6 and E7 oncoproteins. This could reflect a differential involvement of the E5 types in the transformation processes.

Amino Acid Sequence↗

PubMed2004

The HPV-16 E5 protein inhibits TRAIL- and FasL-mediated apoptosis in human keratinocyte raft cultures.

By using raft cultures of the polyclonal HaCaT cell lines stably transfected either with E5 (HaCaT/E5) or the empty vector (HaCaT/pMSG) as reference, we investigated the effect of the human papillomavirus type 16 (HPV-16) E5 protein on apoptosis. In comparison to conventional monolayer cultures this model system allows analysis of apoptosis under more tissue-like conditions by mimicking the stratified organization of a normal surface epithelium. Apoptosis was triggered either by FasL or TRAIL. Execution of the death program was checked at early and late stages by monitoring procaspase-3 cleavage and DNA fragmentation, respectively. Rafts of E5-expressing keratinocytes were completely protected from apoptosis and showed a background of apoptotic cells as low as the untreated cultures. In contrast, the HaCaT/pMSG cultures revealed a dramatic increase in apoptotic cells upon ligand treatment throughout the epithelial compartment. We conclude that the presence of the HPV-16 E5 protein in our tissue-like model prevents FasL- or TRAIL-mediated apoptosis.

Apoptosis↗

PubMed2004

Activation of dendritic cells and induction of T cell responses by HPV 16 L1/E7 chimeric virus-like particles are enhanced by CpG ODN or sorbitol.

Chimeric human papillomavirus-like particles, consisting of human papillomavirus (HPV) 16 L1-E7 fusion proteins [HPV 16 L1/E7 chimeric virus-like particles (CVLP)], are a vaccine candidate for treatment and prevention of cervical cancer. Although in preclinical studies CVLPs were shown to induce neutralizing antibodies and L1- and E7-specific T cell responses, the results of a recent clinical trial emphasized the need of improved immunogenicity of CVLPs. Here we studied the interaction of HPV 16 L1/E7 CVLPs with mouse bone marrow-derived dendritic cells (BMDCs) activated with different immune adjuvants. We found that lipopolysaccharides (LPS), unmethylated CpG motifs (CpG ODN) and sorbitol enhanced CVLP-induced stimulation of C57BL/6 mouse BMDCs as revealed by increased levels of CD40, CD80, MHC II and CD54 at the cell surface. CpG ODN and sorbitol also enhanced the presentation of Db-restricted cytotoxic T lymphocyte epitopes to HPV 16 L1- or E7-specific T lymphocytes after loading of CVLPs onto BMDCs. Treatment of BMDCs with CpG ODN in combination with CVLPs improved in vitro priming of naive T lymphocytes by CVLP-loaded BMDCs. In vivo, CVLP-loaded BMDCs were more immunogenic as compared with injection of CVLPs alone. CpG ODN and sorbitol further enhanced priming of antigen-specific T cell responses. Our data demonstrate that CpG ODN- or sorbitol-activated BMDCs substantially increase the immunogenicity of CVLPs. Implementing our results in clinical trial protocols may lead to improved activity of therapeutic HPV vaccines for the treatment of HPV-induced cancer.

Adjuvants, Immunologic↗

PubMed2003

Functional dissection of the mouse tyrosinase locus control region identifies a new putative boundary activity.

Locus control regions (LCRs) are complex high-order chromatin structures harbouring several regulatory elements, including enhancers and boundaries. We have analysed the mouse tyrosinase LCR functions, in vitro, in cell lines and, in vivo, in transgenic mice and flies. The LCR-core (2.1 kb), located at -15 kb and carrying a previously described tissue-specific DNase I hypersensitive site, operates as a transcriptional enhancer that efficiently transactivates heterologous promoters in a cell-specific orientation-independent manner. Furthermore, we have investigated the boundary activity of these sequences in transgenic animals and cells. In mice, the LCR fragment (3.7 kb) rescued a weakly expressed reference construct that displays position effects. In Drosophila, the LCR fragment and its core insulated the expression of a white minigene reporter construct from chromosomal position effects. In cells, sequences located 5' from the LCR-core displayed putative boundary activities. We have obtained genomic sequences surrounding the LCR fragment and found a LINE1 repeated element at 5'. In B16 melanoma and L929 fibroblast mouse cells, this element was found heavily methylated, supporting the existence of putative boundary elements that could prevent the spreading of condensed chromatin from the LINE1 sequences into the LCR fragment, experimentally shown to be in an open chromatin structure.

Animals↗

PubMed2003

The human papillomavirus HPV2a E5 protein localizes to the Golgi apparatus and modulates signal transduction.

The low-risk human papillomavirus type 2a (HPV2a) has been found associated with benign skin epithelial tumors and has only been very rarely identified in malignized epithelia. Here we report the identification of the E5 gene of HPV2a and demonstrate that the protein is mainly expressed in the Golgi apparatus of transfected cells, similar to the known high-risk types E5 proteins. Further, we present experimental evidence demonstrating that HPV2a E5, similar to HPV16 E5, is able to modulate EGF-mediated erk1/2 MAP kinase activation and to down-regulate the expression of MHC class I molecules at the plasma membrane. Thus, the E5 gene of at least one cutaneous low-risk HPV type displays similar biological characteristics to those described for the high-risk type HPV16.

Amino Acid Sequence↗

PubMed2003

Analysis of protein tyrosine phosphorylation by nanoelectrospray ionization high-resolution tandem mass spectrometry and tyrosine-targeted product ion scanning.

A novel highly sensitive strategy is introduced for analysis of tyrosine phosphorylation in previously identified proteins channelling for this aim all analytical and sequence information available. Nanoelectrospray high-resolution MS/MS analysis is targeted to precalculated m/z values corresponding to phosphotyrosine-containing tryptic peptides. Identification of these peptides is supported by the occurrence of the phosphotyrosine immonium ion at m/z 216, neutral loss of 79.97/z (= loss of HPO3), and similarity of the fragmentation patterns of phosphotyrosine-containing peptides with their nonphosphorylated analogues. This tyrosine-targeted tandem mass spectrometry strategy is demonstrated for epidermal growth factor receptor showing that phosphotyrosine-containing tryptic peptides invisible in the survey spectrum can be safely identified.

Amino Acid Sequence↗

PubMed2003

Kinetic diversity of single-channel burst openings underlying persistent Na(+) current in entorhinal cortex neurons.

The kinetic diversity of burst openings responsible for the persistent Na(+) current (I(NaP)) in entorhinal cortex neurons was examined by separately analyzing single bursts. Although remarkable kinetic variability was observed among bursts in terms of intraburst opening probability and mean open and closed times, the values of time constants describing intraburst open times (tau(o(b))s) and closed times (tau(c(b))s) were distributed around well-defined peaks. At -40 mV, tau(o(b)) peaks were found at approximately 0.34 (tau(o(b))1) and 0.77 (tau(o(b))2) ms, and major tau(c(b)) peaks were found at approximately 0.24 (tau(c(b))1) and 0.54 (tau(c(b))2) ms. In approximately 80% of the bursts two preferential gating modes were found that consisted of a combination of either tau(o(b))1 and tau(c(b))2 ("intraburst mode 1"), or tau(o(b))2 and tau(c(b))1 ("intraburst mode 2"). Individual channels could switch between different gating modalities, but normally tended to maintain a specific gating mode for long periods. Mean burst duration also displayed considerable variability. At least three time constants were found to describe burst duration, and the frequencies at which each of the corresponding "bursting states" occurred varied in different channels. Short-lasting bursting states were preferentially associated with intraburst mode 1, whereas very-long-lasting bursts tended to gate according to mode 2 only or other modes that included considerably longer mean open times. These results show that I(NaP) channels can generate multiple intraburst open and closed states and bursting states, but these different kinetic states tend to combine in definite ways to produce a limited number of prevalent, well-defined gating modalities. Modulation of distinct gating modalities in individual Na(+) channels may be a powerful form of plasticity to influence neuronal excitability and function.

Action Potentials↗

PubMed2003

Increased persistent sodium currents in rat entorhinal cortex layer V neurons in a post-status epilepticus model of temporal lobe epilepsy.

PURPOSE: Spontaneous seizures in rats emerge several weeks after induction of status epilepticus with pharmacologic treatment or electrical stimulation, providing an animal model for human temporal lobe epilepsy. In this study, we investigated whether status epilepticus caused changes in the function of voltage-gated sodium channels in entorhinal cortex layer V neurons, a cellular group important for the genesis of limbic seizures. METHODS: We induced status epilepticus in rats, by using lithium-pilocarpine, and then 2-12 weeks later, used whole-cell voltage-clamp to examine voltage-activated sodium currents of acutely dissociated layer V neurons. RESULTS: Transient sodium currents of entorhinal cortex layer V neurons isolated from 9- to 12-week post-status epilepticus rats were similar to currents in age-matched controls; however, low-threshold persistent sodium currents were significantly larger. This increase in persistent activity was not seen 2-3 weeks after pilocarpine treatment; thus it occurred after a delay comparable to the delay in the appearance of spontaneous seizures. CONCLUSIONS: Increased persistent currents are expected to accentuate neuronal excitability and thus may contribute to the genesis of spontaneous seizures after status epilepticus.

Animals↗

PubMed2003

Rhythmically discharging basal forebrain units comprise cholinergic, GABAergic, and putative glutamatergic cells.

The basal forebrain plays important roles in arousal, learning, and memory by stimulating cortical activation characterized by rhythmic slow theta and high-frequency beta-gamma activities. Although cholinergic neurons play a significant part in these roles, other, including GABAergic, neurons appear to contribute. Using juxtacellular labeling with neurobiotin of neurons recorded within the magnocellular preoptic-substantia innominata area in urethan-anesthetized rats, we show that in addition to cells that are cholinergic or GABAergic, other cells that are neither fire rhythmically in correlation with stimulation-induced rhythmic slow activity on the cortex. Neurons with the characteristics of the noncholinergic/nonGABAergic cells contain phosphate-activated glutaminase (PAG), the synthetic enzyme for transmitter glutamate and may thus be glutamatergic. Within their oscillatory spike trains, putative glutamatergic neurons fire at a lower frequency (~20 Hz) than the GABAergic neurons (~40 Hz) and the cholinergic neurons (average: 75 Hz), whose spike trains include high-frequency bursts. The three groups all discharge rhythmically at a slow frequency in correlation with rhythmic slow activity recorded on the prefrontal, entorhinal, piriform and olfactory bulb cortices. The predominant slow frequency corresponds to the respiratory-olfactory rhythm, which is commonly slower than, yet can be as fast as, the hippocampal theta rhythm during certain coordinated behaviors, such as sniffing-whisking. While stimulating higher frequency beta-gamma activities, putative glutamatergic together with GABAergic and cholinergic cells may thus collectively modulate rhythmic slow activity and thereby promote coherent processing and plasticity across distributed cortical networks during coordinated behaviors and states.

Acetylcholine↗

PubMed2003

Immunochemical expression of proliferative cell nuclear antigen in aging cultured astrocytes.

Cell differentiation degree and mitotic activity were sequentially assessed by immunoperoxidase labeling of glial fibrillary acidic protein (GFAP) and proliferative cell nuclear antigen (PCNA), respectively, in rat brain cultured astrocytes maintained up to 60 days in vitro (DIV) of first subculture, or weekly passaged until their 12th subculture. Cell count was performed through a 0.01 mm2 section reticule and morphometric analysis with a stereological grid. The number of double immunoreactive cells peaked by 2 DIV to achieve its lowest value at 60 DIV. At 24 hs of cell seeding of successive passages, such values peaked by the 6th subculture to gradually decrease thereafter. Increasing cell hypertrophy was found during the long-term first subculture but not after passaging. At the end of the observation period, doubly immunolabeled astrocytes were still recorded, thus evidencing retention of proliferative potential despite aging.

Aging↗

PubMed2002

Phosphorylation and subcellular distribution of connexin43 in normal and stressed cells.

We have developed polyclonal antibodies (SA226P) to a peptide of the human connexin43 (Cx43) protein between amino acids 271 and 288 containing phosphorylated S279 and S282. Antibodies specific for the phosphorylated form of the peptide were isolated by double immunoaffinity chromatography and were characterised using proteins of the cell line WB-F344, known to contain large amounts of Cx43. SA226P recognises specifically the slowest migrating Cx43 band in immunoblots of proteins isolated from untreated cells. In immunofluorescence experiments SA226P scarcely stains the plasma membrane in untreated cells in contrast to a commercial antibody recognising all isoforms of the Cx43 protein. EGF or stress treatment of the cells results in a rapid increase in the phosphorylated forms of Cx43 as revealed by immunoblotting. Immunofluorescence experiments reveal that both phosphorylated and non-phosphorylated Cx43 could be found at the plasma membrane. Whether phosphorylation of S279/S282 takes place before or after incorporation of Cx43 into the membranes is so far unknown. More interestingly, confocal microscopy using our antibodies and a commercial antibody recognising all isoforms of Cx43 shows the coexistence of differentially phosphorylated forms of the protein at the plasma membrane. Our results indicate that MAP kinases erk1/2 are mainly responsible for this phosphorylation, as already published. Nevertheless, treatment of the cells with anisomycin, known to activate stress kinase p38 but not erk1/2, also results in a weak but reproducible Cx43 phosphorylation.

Animals↗

PubMed2002

Modelling of the human papillomavirus type 16 E5 protein.

The product of the E5 oncogene in human papillomaviruses (HPVs) participates in cellular transformation. The sequences of E5 from high-risk HPV types are closely related, and the ability to transform is thought to be associated with their structure. Structural determination by standard biophysical methods has proved impossible due to the extreme hydrophobicity of the gene product. We have achieved limited solubility by dividing the sequence into three, structurally distinct domains. Synthetic peptides corresponding to these domains have been examined using circular dichroism (CD) spectroscopy, a method that can detect secondary structure elements in highly dilute protein solutions. Using data on the secondary structure content of these domains under different conditions and in systematic combination to detect constructive domain interactions, a model of HPV E5 structure and position in the membrane is proposed that is consistent with what is known of the larger family of leucine-rich repeat (LRR) proteins to which it belongs.

Amino Acid Sequence↗

PubMed2002

Signaling mechanisms underlying reversible, activity-dependent dendrite formation.

Neuronal activity and neurotrophins play a central role in the formation, maintenance, and plasticity of dendritic arbors. Here, we show that neuronal activity, mediated by electrical stimulation, KCl depolarization, or cholinergic receptor activation, promotes reversible dendrite formation in sympathetic neurons and that this effect is enhanced by NGF. Activity-dependent dendrite formation is accompanied by increased association of HMW MAP2 with microtubules and increased microtubule stability. Inhibition of either CaMKII or the MEK-ERK pathway, both of which phosphorylate MAP2, inhibits dendrite formation, but inhibition of both pathways simultaneously is required for dendrites to retract. These data indicate that neuronal activity signals via CamKII and the ERKs to regulate MAP2:microtubule interactions and hence reversible dendrite stability, and to provide a mechanism whereby activity and neurotrophins converge intracellularly to dynamically regulate dendritic morphology.

Adrenergic Fibers↗

PubMed2002

The human papillomavirus type 16 (HPV-16) E5 protein sensitizes human keratinocytes to apoptosis induced by osmotic stress.

We have studied the role of the HPV-16 E5 protein in apoptosis, using HaCaT cell lines stably transfected with either E5 (HaCaT/E5) or the empty vector (HaCaT/pMSG) as control. When subjected to a hyperosmolar concentration of sorbitol, HaCaT/E5 cells respond with cytochrome c release, activation of caspase-3, -8, and -9, and PARP-cleavage, showing that the mitochondria and death-receptor mediated apoptotic pathways are involved in subsequent cell death. Similar effects are observed for the control cells only after extended sorbitol treatment. Thus, E5-expressing cells are more sensitive to osmotic stress, perhaps because of modifications of the cellular membranes caused by this strongly hydrophobic molecule.

Animals↗

PubMed2002

Stress kinase p38 mediates EGFR transactivation by hyperosmolar concentrations of sorbitol.

Activation of the epidermal growth factor receptor (EGFR) has been shown to occur by ligand-dependent and ligand-independent mechanisms. Different molecular mechanisms have been found to be responsible for ligand-independent receptor transactivation. Here, we show that hyperosmolar concentrations of sorbitol activate the EGFR in human keratinocytes. Experiments using specific inhibitors of EGFR phosphorylation show that the increased amount of activated receptors is the result of a decreased rate of dephosphorylation. Furthermore, sorbitol treatment results in a strong activation of stress kinase p38. Treatment of the cells with SB203580, a known inhibitor of p38 alpha and beta kinases, results in impairment of receptor activation, indicating that the stress kinase is involved in receptor activation modulation. This is further reinforced by experiments showing that addition of Toxin B, known to be an inhibitor of the small Rho GTPases rac1, cdc42, and Rho A/B, to the cells results in a strong induction of EGFR activation. Our results point, therefore, to a mechanism by which osmotic shock activates EGFR through the small Rho GTPases-p38 stress kinase pathway.

Bacterial Toxins↗

PubMed2002

Hyperosmotic stress induces phosphorylation of cytosolic phospholipase A(2) in HaCaT cells by an epidermal growth factor receptor-mediated process.

Cytosolic phospholipase A(2) (cPLA(2)) is an enzyme involved in the formation of proinflammatory mediators by catalyzing the release of arachidonic acid, thereby mediating eicosanoid biosynthesis. Using HaCaT keratinocytes as a model system, we present experimental evidence that in these cells, cPLA(2) is constitutively phosphorylated and that the degree of phosphorylation dramatically increases in cells under hyperosmotic stress induced by sorbitol. In parallel, a rapid release of arachidonic acid followed by prostaglandin E(2) formation was detected. Elucidating the mechanism of cPLA(2) upregulation, we observed that it is mediated via epidermal growth factor receptor (EGFR) activation, since tyrphostin AG1478, a selective inhibitor of EGFR tyrosine kinase, completely inhibited cPLA(2) phosphorylation. Furthermore, addition of PD98059, which is an inhibitor of MEK1 activation, but not of SB203580, which is an inhibitor of p38 stress kinase, inhibited cPLA(2) phosphorylation, indicating that the ras-raf-MEK cascade is the major signalling pathway involved in cPLA(2) phosphorylation. In addition, depletion of the cells from intracellular calcium does not prevent sorbitol-elicited cPLA(2) phosphorylation, suggesting that this process is independent of the presence of calcium. Together, our results demonstrate that hyperosmotic stress phosphorylates cPLA(2) in human keratinocytes by an EGFR-mediated process.

Arachidonic Acid↗