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Biomedical subjects

Angel Alonso

Publications and source records attributed to Angel Alonso.

At least 19 recordsLinked to original sources
PubMed2007

Effects of serotonin on the intrinsic membrane properties of layer II medial entorhinal cortex neurons.

Although serotonin (5-HT) is an important neuromodulator in the superficial layers of the medial entorhinal cortex (mEC), there is some disagreement concerning its influences upon the membrane properties of neurons within this region. We performed whole cell recordings of mEC Layer II projection neurons in rat brain slices in order to characterize the intrinsic influences of 5-HT. In current clamp, 5-HT evoked a biphasic response consisting of a moderately short latency and large amplitude hyperpolarization followed by a slowly developing, long lasting, and small amplitude depolarization. Correspondingly, in voltage clamp, 5-HT evoked a robust outward followed by a smaller inward shift of holding current. The outward current evoked by 5-HT showed a consistent current/voltage (I/V) relationship across cells with inward rectification, and demonstrating a reversal potential that was systematically dependent upon the extracellular concentration of K(+), suggesting that it was predominantly carried by potassium ions. However, the inward current showed a less consistent I/V relationship across different cells, suggesting multiple independent ionic mechanisms. The outward current was mediated through activation of 5-HT(1A) receptors via a G-protein dependent mechanism while inward currents were evoked in a 5-HT(1A)-independent fashion. A significant proportion of the inward current was blocked by the I(h) inhibitor ZD7288 and appeared to be due to 5-HT modulation of I(h) as 5-HT shifted the activation curve of I(h) in a depolarizing fashion. Serotonin is thus likely to influence, in a composite fashion, the information processing of Layer II neurons in the mEC and thus, the passage of neocortical information via the perforant pathway to the hippocampus.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed2006

Ubiquitin protein ligase Nedd4 binds to connexin43 by a phosphorylation-modulated process.

Connexin43 is degraded by the proteasomal as well as the lysosomal pathway with ubiquitin playing a role in both degradation pathways. So far, no ubiquitin protein ligase has been identified for any of the connexins. By using pull-down assays, here we show binding of a ubiquitin protein ligase, Nedd4, to the C-terminus of connexin43. This observation was confirmed in vivo by coimmunoprecipitation and immunofluorescence, showing colocalization of Nedd4 and connexin43. Binding of Nedd4 to its interaction partners is generally carried out by its WW domains. Our results indicate that the interaction with connexin43 occurs through all three WW domains of Nedd4. Furthermore, whereas WW1 and WW2 domains mainly interact with the unphosphorylated form of connexin43, WW3 binds phosphorylated and unphosphorylated forms equally. In addition, using the surface plasmon resonance approach we show that only the WW2 domain binds to the PY motif located at the C-terminus of connexin43. Suppression of Nedd4 expression with siRNA resulted in an accumulation of gap junction plaques at the plasma membrane, suggesting an involvement of the ubiquitin protein ligase Nedd4 in gap junction internalization.

Amino Acid Motifs↗
PubMed2006

Phylogeny and evolution of papillomaviruses based on the E1 and E2 proteins.

Papillomaviridae are a family of small double-stranded DNA viruses that infect stratified squamous epithelia in vertebrates. Members of this family are causative agents of malignant tumours, such as cervical cancer while others are associated with benign proliferative lesions. So far, Papillomaviruses (PVs) are classified according to the sequence identity in the capsid gene L1. However, evidence has accumulated indicating a discontinuity in the evolutionary history of the L1 and L2 genes of many PVs, giving rise to differences in the phylogenetic reconstructions of the early and of the late genes. Neither the oncogenes E5, E6 and E7 nor the upstream regulatory region are suitable for phylogenetic inference due to the poor conservation along the Papillomaviridae family. We have analysed here the evolutionary relationships of the PVs with respect to the E1 and E2 proteins, and the results provide both phylogeny and biologic behaviour of the viruses. The hierarchical taxonomic relationships can be structured as an alternative classification system in which mucosal high-risk viruses, mucosal low-risk viruses and viruses associated with cutaneous lesions are grouped separately and do not appear intermingled. Some important trends are also observed: first, evolution of the PVs has not been homogeneous, even in viruses that infect the same host, and second mucosal human PVs have evolved faster than their cutaneous counterparts. The evolutionary analysis based on the E1 and E2 proteins will allow us to better understand the generation of the diversity of the PVs and the development of malignancy associated with these viruses.

Animals↗
PubMed2006

Different papillomaviruses have different repertoires of transcription factor binding sites: convergence and divergence in the upstream regulatory region.

BACKGROUND: Papillomaviruses (PVs) infect stratified squamous epithelia in warm-blooded vertebrates and have undergone a complex evolutionary process. The control of the expression of the early ORFs in PVs depends on the binding of cellular and viral transcription factors to the upstream regulatory region (URR) of the virus. It is believed that there is a core of transcription factor binding sites (TFBS) common to all PVs, with additional individual differences, although most of the available information focuses only on a handful of viruses. RESULTS: We have studied the URR of sixty-one PVs, covering twenty different hosts. We have predicted the TFBS present in the URR and analysed these results by principal component analysis and genetic algorithms. The number and nature of TFBS in the URR might be much broader than thus far described, and different PVs have different repertoires of TFBS. CONCLUSION: There are common fingerprints in the URR in PVs that infect primates, although the ancestors of these viruses diverged a long time ago. Additionally, there are obvious differences between the URR of alpha and beta PVs, despite these PVs infect similar histological cell types in the same host, i.e. human. A thorough analysis of the TFBS in the URR might provide crucial information about the differential biology of cancer-associated PVs.

Binding Sites↗
PubMed2005

Morphological and electrophysiological properties of lateral entorhinal cortex layers II and III principal neurons.

The intrinsic electrophysiology and morphology of neurons from layers II and III of the lateral entorhinal cortex (EC) was investigated in a rat brain slice preparation by intracellular recording and biocytin labeling. Morphologically, we distinguished three groups of layer II principal neurons. The most numerous group included cells with multiple radiating dendrites that spread over layers II and I in a fan-like fashion. While morphologically "fan" neurons were similar to the "stellate" cells of the medial EC, electrophysiologically the fan cells lacked the persistent rhythmic subthreshold oscillations and the very pronounced time-dependent inward rectification typical of the stellate cells. The second group consisted of pyramidal cells that manifested regular spike firing and had a more negative resting potential and a longer spike duration than the fan cells. In the third group we included all those neurons that had diverse multipolar appearances distinct from the fan cells. Neurons in this group had electrophysiological profiles intermediate between those of the fan and pyramidal cells. All neurons recorded in layer III were pyramidal in shape with a basal dendritic tree that could extend into layer V and an axon that could also give off collaterals into layer V. Electrophysiologically, layer III pyramidal cells were very similar to those of layer II. On the basis of these and other data we suggest that in different EC regions layer II neurons may be conducting more input-dependent specialized processing, while cells from layer III may perform a more global or generalized function.

Animals↗
PubMed2005

Papillomaviruses: different genes have different histories.

Papillomaviruses (PVs) infect stratified squamous epithelia in vertebrates. Some PVs are associated with different types of cancer and with certain benign lesions. It has been assumed that PVs coevolved with their hosts. However, recently it has been shown that different regions of the genome have different evolutionary histories. The PV genome has a modular nature and appeared after the addition of pre-existent blocks. This order of appearance in the PV genome is evident today in the different evolutionary rates of the different genes, with new genes--E5, E6 and E7--diverging faster than old genes--E1, E2, L2 and L1. Here, we propose an evolutionary framework aiming to integrate genome evolution, PV biology and epidemiology of PV infections.

Evolution, Molecular↗
PubMed2005

Antigenicity and immunocrossreactivity of orange tree pollen and orange fruit allergenic extracts.

BACKGROUND: It is important to study the crossreactivity between orange tree pollen (OTPE) and orange fruit (OFE) due to the high incidence of pollen/food-related allergies worldwide. The aim of the present study was to determine the antigenic relationship between OTPE and OFE. METHODS: OTPE and OFErabbit antisera as well as sera from patients allergic to OTPE and OFE were comparatively applied in IgG- and/or IgE-specific ELISA inhibition, crossover or inhibition immunoblotting assays using OTPE and OFE allergenic extracts as solid phase. Periodate and proteinase K treatments were used for carbohydrate and protein depletion, respectively. RESULTS: The antigenicity of OTPE and the presence of common structures between OTPE and OFE extracts were demonstrated by rabbit IgG-specific ELISA inhibition and crossover immunoblotting assays. A 30-kDa protein, common target of the IgE response on OTPE, OFE and mandarin extract, but absent in lemon extract, was identified by ELISA and immunoblot inhibition assays in patients suffering from primary sensitization to OTPE in the context of occupational exposure. Moreover, biochemical treatments showed that antigenic epitopes on the 30-kDa protein contain polypeptidic but no carbohydrate moieties. CONCLUSIONS: The antigenicity of OTPE, the presence of common antigenic determinants between pollen and citrus fruits and an IgE-specific crossreactive protein band of 30 kDa sharing carbohydrate-free epitopes were described. After isolation and purification, this common antigen might be useful for allergen immunotherapy in pollen/fruit-related allergic patients.

Adult↗
PubMed2005

Phosphorylation of the gap junction protein Connexin43 in CIN III lesions and cervical carcinomas.

Connexins are proteins that form the connexons, gap junction structures, which allow cells to communicate. Phosphorylation of connexins has been found to impair this communication. Using an antibody specifically recognizing the S279/S282-phosphorylated form of connexin43 (Cx43) for immunohistochemistry, we have analysed Cx43 phosphorylation in normal epithelium, CIN III lesions, and carcinomas of the cervix. We found that in normal epithelium the basal layer was devoid of staining and most of the protein was localized in stratum spinosum and stratum granulosum. In pre-malignant CIN-III lesions Cx43 was strongly phosphorylated, but the basal layer was still negative. In squamous carcinomas, the cells were intensely stained. In these tumours, sites of strong staining were adjacent to less stained regions, suggesting that the tumours are intrinsically heterogeneous. Immunoblotting of proteins extracted from carcinomas with the specific antibody showed the classical pattern of multiple reacting bands, with the appearance of low migrating forms of the protein. Our results suggest that increased S279/S282 phosphorylation of Cx43 is the result of altered tissue structure rather than of cell malignization.

Cell Communication↗
PubMed2005

Cholinergic basal forebrain neurons burst with theta during waking and paradoxical sleep.

It is known that acetylcholine can stimulate activation and promote plasticity in the cerebral cortex, yet it is not known how the cholinergic basal forebrain neurons, which release acetylcholine in the cortex, discharge in relation to natural cortical activity and sleep-wake states. By recording basal forebrain units in association with electroencephalographic activity across the sleep-wake cycle and labeling individual neurons with Neurobiotin for immunohistochemical identification, we show for the first time that cholinergic neurons discharge in bursts at maximal rates during active waking and paradoxical sleep, when gamma and theta electroencephalographic activity are maximal. They virtually cease firing during slow-wave sleep. Notably, their bursting discharge is synchronized with theta oscillations. Through their maximal firing and rhythmic theta discharge during active waking and paradoxical sleep, the cholinergic neurons can thus modulate the cortex to promote activation along with plasticity during these two states.

Acetylcholine↗
PubMed2005

The phosphorylated form of connexin43 is up-regulated in breast hyperplasias and carcinomas and in their neoformed capillaries.

We applied an antiserum (SA226P) specifically recognizing the phosphorylated form of connexin43 (P-Cx43) to human breast samples including normal breast samples, with fibrocystic disease (FCD), fibroadenomas (FA), in situ and infiltrating carcinomas of all major types, and miscellaneous extramammary tumors. The findings were compared with those obtained with commercial antisera recognizing all Cx43 forms (pan-Cx43). A subset of samples was stained for Her2-neu and p44/42 to mitogen-activated protein kinase. Paraffin step sections were used. Immunoblots were performed on frozen samples of a representative subset of cases. In the normal breast, FCD, and FA, SA226P stained strongly and extensively most myoepithelial cells (MECs); luminal cells remained unstained. In proliferative FCD and some cellular FA, SA226P stained MEC and the capillary endothelium (CE). In ductal and lobular in situ carcinomas, SA226P reacted strongly and diffusely with the remaining MEC, the CE, and the transformed luminal cells. SA226P stained all infiltrating carcinomas except the tubular variant. In all breast carcinomas, the CE within and adjacent to tumors and some myofibroblasts stained with SA226P. By contrast, pan-Cx43 stained weakly and sporadically the MEC and rare samples of invasive carcinomas. Notably, Mab p44/42 reacted in parallel with the samples stained with SA226P, whereas reactions with Her2 were negative. Immunoblot findings paralleled those obtained immunohistochemically. We conclude that P-Cx43, restricted to MEC in the normal breast, is up-regulated in the same cells in hyperplasias and dysplasias and FA and is strongly up-regulated in invasive carcinomas. Notably, in some proliferative FCD and in most in situ and infiltrating carcinomas, P-Cx43 is strongly expressed in CE within and adjacent to the lesions but not away from them. These findings were paralleled by the strong nuclear reactions noted with Mab p44/42. These phenomena, although not exclusive to malignancy, are particularly conspicuous in breast carcinomas and seemingly reflect active proliferation associated with abnormal gap junctional intercellular communication.

Breast↗
PubMed2005

Characterization and differentiation-dependent regulation of secreted phospholipases A in human keratinocytes and in healthy and psoriatic human skin.

Secreted phospholipases A2 (sPLA2) expressed in the skin are thought to be involved in epidermal barrier homeostasis as well as in inflammation. We investigated the expression of the novel sPLA2 subtypes in human skin at mRNA and protein levels in the epidermis and primary keratinocytes from healthy human skin, and in skin sections from patients with psoriasis, where the integrity of the epidermis is drastically affected. Immunofluorescence studies using specific antibodies for the different sPLA2 enzymes show that sPLA2-IB, -IIF, and -X are predominantly expressed in suprabasal layers, whereas sPLA2-V and -IID are detected in the basal and spinous layers. sPLA2-IIA is weakly expressed, and sPLA2-IIE and XIIA are not detectable. Accordingly, in differentiated human primary keratinocyte cultures, the expression of sPLA2-IB, -IIF and -X was increased, whereas that of sPLA2-V and -IID was markedly decreased. In psoriatic skin, sPLA2-X was dramatically downregulated in the epidermis, whereas increased amounts of this enzyme together with sPLA2-IIA, -IID, and -IB appeared in the dermis. An enhanced release of these enzymes with the exception of sPLA2-IID was also observed after treatment of HaCaT keratinocytes with tumor necrosis factor-alpha/interferon-gamma. Treatment of HaCaT cells with sPLA2-X and -IB resulted in an increase in prostaglandin E2 formation, suggesting a proinflammatory role of these enzymes during psoriasis. sPLA2-V completely disappeared. The differential locations of the sPLA2 enzymes propose distinct roles of individual enzymes in skin.

Biopsy↗
PubMed2005

Long-term results and survival rate of implants treated with guided bone regeneration: a 5-year case series prospective study.

One of the key factors for attaining osseointegration is the presence of an adequate osseous volume. In patients with inadequate osseous width or height, a bone augmentation using the guided bone regeneration (GBR) concept may be applied either with a simultaneous or a staged approach. The aim of this multicenter prospective case series study was to evaluate the efficacy and predictability of the GBR technique (simultaneous approach) in patients with peri-implant osseous defects, both dehiscences and fenestrations. Results 5 years post-treatment (survival rates and marginal bone level) were assessed. A total of 19 consecutive patients with 26 peri-implant osseous defects (20 dehiscences and six fenestrations) were treated during the period from September 1992 to June 1993 with a simultaneous GBR approach using non-resorbable membranes combined with autogenous bone grafts or decalcified freeze-dried bone allograft. The mean osseous augmentation was 94.8%. Marginal bone levels at re-entry and 5 years after surgery were calculated from standardized periapical radiographs. One implant was lost 3 months after loading. Thus, the cumulative survival rate was 96.1% after 5 years. The mean marginal bone level after 5 years was 2.03 mm (SD=+/-0.5), without a difference between mesial and distal sites. This study demonstrates that implants with peri-implant defects that are treated with GBR had similar survival rates and crestal bone levels compared with implants in native bone.

Adult↗
PubMed2004

Codon optimization of the human papillomavirus 11 (HPV 11) L1 gene leads to increased gene expression and formation of virus-like particles in mammalian epithelial cells.

The 505 amino acid L1 protein of the human papillomavirus type 11 (HPV 11) is the major capsid polypeptide that has been shown to self-assemble into virus-like particles (VLPs) in vivo and in vitro. While L1 is essential for viral infection, expression studies in mammalian cells have been hampered by different codon preference between the virus and its host. To optimize L1 gene expression in mammalian cells, we converted wild-type HPV 11 L1 (11 L1wt) codons to those more common in human genes. The modified HPV 11 L1 gene (11 L1h) generated protein levels that were at least 100-fold higher than those of wild-type HPV 11 L1, while no obvious differences were seen in the level of mRNA. HPV 11 L1 protein was detected in mammalian epithelial and fibroblast cells, by immunoblotting and indirect immunofluorescence (IIF) techniques. Unlike the situation in situ, IIF revealed the presence of L1 mainly at perinuclear sites. Virus-like particles assembled intranuclearly only to a low extent, as indicated by transmission electron microscopy. DNA vaccination using the HPV 11 L1h gene yielded a drastic increase in L1-specific antibody production in mice as compared to immunization with the wild-type gene.

Animals↗
PubMed2004

Spike patterning by Ca2+-dependent regulation of a muscarinic cation current in entorhinal cortex layer II neurons.

In entorhinal cortex layer II neurons, muscarinic receptor activation promotes depolarization via activation of a nonspecific cation current (I(NCM)). Under muscarinic influence, these neurons also develop changes in excitability that result in activity-dependent induction of delayed firing and bursting activity. To identify the membrane processes underlying these phenomena, we examined whether I(NCM) may undergo activity-dependent regulation. Our voltage-clamp experiments revealed that appropriate depolarizing protocols increased the basal level of inward current activated during muscarinic stimulation and suggested that this effect was due to I(NCM) upregulation. In the presence of low buffering for intracellular Ca(2+), this upregulation was transient, and its decay could be followed by a phase of I(NCM) downregulation. Both up- and downregulation were elicited by depolarizing stimuli able to activate voltage-gated Ca(2+) channels (VGCC); both were sensitive to increasing concentrations of intracellular Ca(2+)-chelating agents with downregulation being abolished at lower Ca(2+)-buffering capacities; both were reduced or suppressed by VGCC block or in the absence of extracellular Ca(2+). These data indicate that relatively small increases in [Ca(2+)](i) driven by firing activity can induce upregulation of a basal muscarinic depolarizing-current level, whereas more pronounced [Ca(2+)](i) elevations can result in I(NCM) downregulation. We propose that the interaction of activity-dependent positive and negative feedback mechanisms on I(NCM) allows entorhinal cortex layer II neurons to exhibit emergent properties, such as delayed firing and enhanced or suppressed responses to repeated stimuli, that may be of importance in the memory functions of the temporal lobe and in the pathophysiology of epilepsy.

Action Potentials↗
PubMed2004

Sleep-wake related discharge properties of basal forebrain neurons recorded with micropipettes in head-fixed rats.

The basal forebrain has been shown to play an important role in cortical activation of wake and paradoxical sleep (PS), yet has also been posited to play a role in slow wave sleep (SWS). In an effort to determine whether these different roles may be fulfilled by different cell groups, including cholinergic and GABAergic cells, we recorded from 123 units in waking-sleeping, head-fixed rats using micropipettes to allow juxtacellular labeling. Functional sets of intermingled cell groups emerged as units whose discharge was as follows: 1) maximum in active wake (aW) and positively or not correlated with EEG gamma activity, while positively correlated with nuchal EMG activity, and thus potentially facilitatory for waking and behavioral arousal (12%); 2) maximum in SWS or SWS-PS and positively correlated with delta EEG activity, while not or negatively correlated with EMG activity, and thus potentially promotive for sleep with cortical slow wave activity and/or accompanying behavioral changes (16%); 3) maximum in PS or PS and aW and positively correlated with gamma and theta EEG activity, while negatively or not correlated with EMG activity, and thus potentially promotive for cortical activation during PS or PS and W (62%); and 4) equivalent across all states and thus not involved in state regulation ( approximately 10%). Units of each group also manifested different firing patterns typified as slow tonic (19.5%), fast tonic (32.5%), or fast phasic (48%), including rhythmic bursting (6%). Through these diverse cell groups, the basal forebrain has the capacity to modulate cortical activity, behavior, and/or related physiological processes across the sleep-waking cycle and thereby regulate the sleep-wake state of the animal.

Animals↗
PubMed2004

Dendritic morphology, local circuitry, and intrinsic electrophysiology of principal neurons in the entorhinal cortex of macaque monkeys.

Little is known about the neuroanatomical or electrophysiological properties of individual neurons in the primate entorhinal cortex. We have used intracellular recording and biocytin-labeling techniques in the entorhinal slice preparation from macaque monkeys to investigate the morphology and intrinsic electrophysiology of principal neurons. These neurons have previously been studied most extensively in rats. In monkeys, layer II neurons are usually stellate cells, as in rats, but they occasionally have a pyramidal shape. They tend to discharge trains, not bursts, of action potentials, and some display subthreshold membrane potential oscillations. Layer III neurons are pyramidal, and they do not appear to display membrane potential oscillations. The distribution of dendrites and of axon collaterals suggests that neurons in layers II and III are interconnected by a network of associational fibers. Layer V and VI neurons are pyramidal and tend to discharge trains of action potentials. The distribution of dendrites and axon collaterals suggests that there is an associative network of principal neurons in layers V and VI, and they also project axon collaterals toward superficial layers. Importantly, entorhinal cortical neurons in monkeys appear to exhibit significant differences from those in rats. Morphologically, neurons in monkey entorhinal layers II and III have more primary dendrites, more dendritic branches, and greater total dendritic length than in rats. Electrophysiologically, layer II neurons in monkeys exhibit less sag, and subthreshold oscillations are less robust and slower. Some monkey layer III neurons discharge bursts of action potentials that are not found in rats. The interspecies differences revealed by this study may influence information processing and pathophysiological processes in the primate entorhinal cortex. J. Comp. Neurol. 470:317-329, 2004.

Animals↗
PubMed2004

Multiple independent kinase cascades are targeted by hyperosmotic stress but only one activates stress kinase p38.

In this report, we analyse the effects of osmotic shock on signal transduction in CHO cells. We demonstrate that at least three different kinase cascades are switched on upon osmotic shock, namely PKA, AMPK, and MLTK. Whereas PKA from cells treated with forskolin activated stress kinase p38, PKA from cells treated with sorbitol did not activate p38, although the enzyme is activated in both cases as analysed in vitro using a specific peptide target. Further, osmolar shock activated AMPK but treatment of the cells with the AMPK activator 5-amino-4-imidazolecarboxamide (AICAr) did not result in p38 activation, strongly suggesting that AMPK is not involved in stress kinase activation. Transfection of CHO cells with dominant negative recombinants of MLTKalpha resulted in inhibition of sorbitol-mediated p38 activation, indicating that the mixed-lineage kinase is involved in the activation of p38 by sorbitol. Finally, in CHO cells overexpressing wild-type MLTKalpha, no activation of AMPK of PKA could be demonstrated, indicating that the activated kinase cascades are not involved in a cross-talk process.

AMP-Activated Protein Kinases↗