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Background:Evidence that periodontal disease is a possible risk factor for cognitive impairment may be explained by the inflammatory hypothesis. The aim of this study is to determine whether periodontitis is related to the amyloid β (Aβ) load in blood and the role of any such relationship in the association between Aβ and cognitive impairment. Methods:A case‐control study was performed in elderly people diagnosed with cognitive impairment with or without dementia (cases group) and cognitively healthy elderly people (control group); data were collected on the medical and dental history of participants, and blood samples were drawn to determine Aβ levels using enzyme‐linked immunosorbent assay. Results:The study included 166 patients and 122 control participants. Higher blood Aβ1‐42levels (P= 0.01) and higher Aβ42:40ratio (P= 0.06) were observed in participants with severe attachment loss than in other participants. Periodontitis was a significant interaction variable, given that the association between Aβ1‐42and Aβ1‐40and cognitive impairment was only observed in patients with severe periodontitis. According to these data, periodontitis may be a modulating variable of the association between Aβ and cognitive impairment. Conclusions:Plasma Aβ1‐42levels are higher in individuals who have severe periodontal disease. The presence of periodontitis may modify the association between Aβ and cognitive impairment. more...

AbstractN-Heterocyclic carbene (NHC) ligands have attracted great interest over the last decade for their use in the design of homogenous catalysts. NHC-based metal complexes have interesting potential biomedical applications, such as in antimicrobial and cancer therapy, which are beginning to be explored more fully. We have studied here the oxidant activities of a series of Ru(II) complexes in vitroand zebrafish (Danio rerio) have been used as a model in vivoto investigate and characterize the toxicity of some of these compounds. Dual behavior was observed for the NHC-based complexes as they behaved as antioxidants at low concentrations but showed pro-oxidant capacity at higher concentrations. Zebrafish embryos were exposed to Ru(II) complexes under several different conditions (0 or 24 h postfertilization, with or without the chorion) and various parameters, such as viability, edema, heart rate, blood coagulation, pigmentation, scoliosis, malformation, and hatching, were tested. In general, zebrafish embryos were not harmed by exposure to Ru(II) complexes whatever the experimental conditions. Several toxicity profiles were observed depending upon the chemical structure of the compound in question. Their characteristics as pro-oxidant and/or antioxidant agents together with their biosafety may point to their having biomedical applications as antitumoral or neuroprotective drugs. more...

AbstractThe zebrafish (Danio rerio) is rapidly gaining ground as a disease model. However, until now, the use of this species with human pathogens has been restricted to just three bacteria; no studies involving viruses that infect humans are recorded. In this study, the zebrafish was used as a model of herpes simplex virus type 1 (HSV-1) infection of the nervous system. Fish infected using viral culture supernatants showed detectable HSV-1 DNA concentrations 1–4 days after inoculation, indicating that this virus can experimentally infect and persist in this host. The kinetics of infection was dose dependent, especially in the head. Histological immunodetection of HSV-1 glycoproteins confirmed the presence of HSV-1 in the organs studied; infection led to histopathological changes. Moreover, the suppression of the immune system by cyclophosphamide and the antiviral effect of acyclovir were demonstrated. The infection of the encephalon was studied in detail, and the time course of viral colonization recorded. Immunofluorescence studies provided immunoreactive evidence of viral antigens in the encephalon and spinal cord. Viruses cleared from infected brains showed the ability to infect human neuroblastoma cells. This study is the first to demonstrate HSV-1 infection in the zebrafish and manifests the potential use of this species in herpesvirus studies. more...

Abstract: The aim of this study was to discuss the relevance of the Epstein-Barr virus (EBV) in the nasopharyngeal carcinoma (NPC), analyzing the variations of several molecules potentially involved in the pathogenesis of this cancer. EBV was detected in all the NPC samples by several techniques including PCR, in situ hybridization, and immunohistochemical methodologies. CD21 membrane receptor was absent after EBV infection, being a differential morphological feature of the tumoral cells. Latent membrane protein-1 (LMP1), an oncogenic viral product, was detected in a high percentage of samples, supporting a role for EBV in the pathogenesis of NPC. Bcl-2, an anti-apoptotic protein up-regulated by LMP1, was also overexpressed, coinciding with the majority of samples positive for LMP1. Finally, p53 presented abnormalities only in a low percentage of samples. These results reinforce the role of the EBV in the NPC discussing several potential mechanisms of pathogenesis in this neoplasia. more...

The objective of this study was to analyse the relationships between the Epstein–Barr virus (EBV) infection and the molecular abnormalities of the p53 oncoprotein in the nasopharyngeal carcinoma (NPC). Fifty-five NPC paraffin samples from a group of Spanish patients with EBV demonstrated presence by nested-PCR and non-isotopic in situhybridisation, were analysed for p53 expression using immunohistochemistry. The positive samples by immunohistochemistry were studied for p53 gene alterations in the exon 5 by single strand conformation polymorphism (SSCP). Among the 55 specimens, 14 (25.5%) showed expression of p53 protein. All these positive samples corresponded to the late stage of the tumour. Of the 14 samples, p53 gene alterations were found only in three cases using SSCP. These results indicate that the p53 mutations are an infrequent event in NPC in Spanish patients needing exogenous factors as the EBV infection for the development of this malignancy. more...

ABSTRACTApolipoprotein E (ApoE), a constituent of the lipoproteins, may be relevant in herpes simplex virus type 1 (HSV-1) infection of the central nervous system (CNS), since HSV-1 binds to human serum ApoE lipoproteins. This study demonstrates the involvement of ApoE in the hematogenous route of HSV-1 to the CNS. more...

Objectives/HypothesisThe aim of this study was to analyze the relevance of the CD21 membrane receptor in nasopharyngeal carcinoma (NPC). CD21 is implicated in the introduction of the Epstein‐Barr virus (EBV) genome into epithelial cells and B lymphocytes. more...

The aim of this study was to show that Cl−dependent excitotoxicity, with its characteristic cell swelling, involves actual water influx into the intracellular compartment. Taking advantage of the Ca2omission paradigm of Cl−-dependent excitotoxicity, in the chick embryonic neural retina ex vivo, which is associated with toxicity levels (lactate dehydrogenase (LDH) release) considerably higher than those seen after simple exposure of the retinas to glutamate agonists, we have demonstrated that an intracellular water intake of 4.2 μl into retinal cells is associated with 13.3 total retinal LDH release. The fact that mannitol blocks both water inflow and LDH release appears to link both events from a pathogenic point of view. more...

Ex vivo preparations of chick neural retina have been successfully used in the assessment of excitotoxicity and in the evaluation of the protective effects of glutamate antagonists. Using a variation of this approach, and measuring the acute and delayed toxic effects of kainate (KA) in terms of lactate dehydrogenase release, we have shown that guanine nucleotides behave as effective neuroprotecting agents. The anti‐excitotoxic potency of guanine nucleotides (in the case of GMP and GDPβS it is about 100 times lower than that of DNQX, a powerful kainate antagonist) correlates well with their ability to displace KA from retinal KA receptors. more...

The prevalence of acute renal failure (ARF) in the elderly is much greater than in the general population. Important structural and functional changes are present in the aging kidneys and predispose the elderly patient to multiple types of acute renal disease. Prerenal failure, hemodynamically-mediated acute tubular necrosis, nephrotoxic ARF, ARF of vascular origin and obstructive ARF are of special importance in the geriatric population. In the present review we analyze some aspects of interest for the nephrologist and urologist regarding diagnosis and management of these severe but often reversible diseases. Elderly patients with ARF appear to have a moderately worse prognosis than younger patients, but age should not be used as a discriminant factor in therapeutic decisions concerning ARF. Instead, early clinical features such as hypotension, assisted respiration or coma are decidedly related to mortality in ARF patients of any age. Although elderly patients who survive after an ARF episode appear to need more time for total recovery and exhibit a lower level of renal function than younger survivors, long-term prognosis is quite good. more...

Serum levels of sICAM-1 and sVCAM-1 adhesion molecules as well as TNF cytokine were measured in 50 kidney transplant recipients [14 patients with stable graft function, 18 patients with acute rejection, 10 patients with CsA toxicity, 6 patients with ATN, and 2 patients with cytomegalovirus (CMV) disease] during the first 3 postoperative weeks. All the patients studied showed, on the first posttransplant day, elevated serum levels of sICAM-1 (260 ± 53 ng/mL). Interestingly, all the patients with good graft function presented thereafter a reduction of slCAM-1 serum levels close to the normal values (185.2 ± 40 ng/ mL). In contrast, the group of patients with acute allograft rejection showed significantly increased serum levels of slCAM-1 (371.5 ± 86 ng/mL; p < 0.001), 3-4 days before diagnosis of acute rejection. Although sVCAM-1 levels were increased in both acute graft rejection (2263 ± 106) and CsA toxicity (1650 ± 315) patients, such increase was not significant among either group of patients when the group with CsA toxicity was compared with either ATN (1320 ± 204) or stable renal function (1089 ± 167). No statistical differences in the levels of TNF were demonstrated between the different groups of patients studied. Our findings demonstrate that quantitative determination of serum slCAM-1 may be of predictive clinical value in transplanted patients with acute renal allograft rejection. more...

Apolipoprotein E (ApoE) plays a relevant role in herpes simplex type 1 (HSV-1) infection of the CNS; after infection by the hematogenous route, the viral neuroinvasiveness directly depends on the APOE gene dose. To analyze the effect of ApoE isoforms on the HSV-1 infectivity to the brain, we have used a model of hematogenous infection of mice humanized for the ApoE3 or the ApoE4 alleles, and we have analyzed the presence of viral DNA in several organs by real time quantitative PCR. We have found that animals expressing human ApoE4 present very high levels of virus in the brain when compared to those expressing the ApoE3 allele; in contrast, there were no significant differences in the viral levels found in peripheral organs. Apolipoprotein E4 seems to facilitate the entry andor spread of HSV-1 in the brain much more efficiently than E3, pointing to a novel potential mechanism underlying the susceptibility to neurodegenerative processes associated with the ApoE4 allele. more...

The chick embryonic neural retina ex vivohas been singled out as a unique example of Cl−-dependent/Ca2-independent excitotoxicity. However, after continuous incubation with 100 μM kainate, we have demonstrated the susceptibility of the chick retina to Ca2-mediated damage, which becomes apparent after 12 h of exposure to the agonist in the absence of Cl−. Of the 20.8 lactate dehydrogenase released after 24 h incubation with kainate, some 11 is Cl−-dependent and the rest (9.8) is presumably Ca2-dependent. Upon omission of both Cl−and Ca2, a 5 residual toxicity can still be detected after 24 h. This can be overcome by inclusion of EGTA in the incubation medium to neutralize Ca2released during incubation. A Ca2-dependent toxicity mechanism is then operative in the embryonic chick retina ex vivo. more...

AMPA (EC50= 1.0 × 10−6M) and NMDA (EC50= 1.3 × 10−4M) trigger 45Ca2influx in 13-day chick embryonic retinal explants. This agonist-driven cationic flux is specifically inhibited by typical competitive antagonists, such as 6, 7-dinitroquinoxaline-2, 3-dione (DNQX) and 2-amino-7-phospho-noheptanoate (AP7), respectively. Guanine nucleotides, with different degrees of phosphorylation, namely 5'-GMP, guanosine 5'-O-(2-thiodiphosphate) (GDPβS), guanosine 5'-O-(3-thiotriphosphate) (GTPβS) and 5'-guanylyl-imidodiphosphate (GppNHp), are also efficient blockers of 45Ca2influx. These results confirm the antagonistic behavior of guanine nucleotides towards ionotropic glutamate receptors and suggest a convenient experimental approach for screening of novel agonists and antagonists. more...

CHICK kainate binding protein was solubilized from cerebellar membranes and purified (×19) by use of two chromatographic steps. Measurements of [3H]kainate binding and GTPase activity in the different fractions reveal a consistent decrease of GTPase activity as the purification proceeds so that no GTPase is detectable after the final purification step. This fact, in the context of the differential involvement in nucleotide recognition of some critical amino acid residues in the p-loop motif of GTPases and in the guanine nucleotide-binding sequence of ionotropic glutamate receptors, together with significant discrepancies concerning the activity of individual nucleotides, suggests that both guanine nucleotide-recognizing sequences are unlikely to be alternative expressions of the same functional domain. more...

Argania spinosais an example of an avoider tree growing under semi-arid conditions in Morocco. To assess what are the physiological strategies of this species, different variables were measured through an annual cycle in two populations located in the species’ main distribution area. Results show the expected decrease of leaf water potential (Ψ) with an increase of water-use efficiency (A/gs) with the onset of the dry season. In summer, leaf conductance (gs) was sensitive to vapour pressure deficit (VPD), and stomatal closure occurred over 30 mbar of VPD. Surprisingly, carbon isotope discrimination (δ13C) maintained very low values over the year, with almost no relationship with any physiological or morphological variable. Hence Argania spinosapresents a complex set of mechanisms to avoid water deficit, but δ13C cannot be used as an ecological tracer of long term WUE. more...

MS-based quantitative proteomics plays an increasingly important role in biological and medical research and the development of these techniques remains one of the most important challenges in mass spectrometry. Numerous stable isotope labeling approaches have been proposed. However, and particularly in the case of 18O-labeling, a standard protocol of general applicability is still lacking, and statistical issues associated to these methods remain to be investigated. In this work we present an improved high-throughput quantitative proteomics method based on whole proteome concentration by SDS-PAGE, optimized in-gel digestion, peptide 18O-labeling, and separation by off-gel isoelectric focusing followed by liquid chromatography-LIT-MS. We demonstrate that the off-gel technique is fully compatible with 18O peptide labeling in any pH range. A recently developed statistical model indicated that partial digestions and methionine oxidation do not alter protein quantification and that variances at the scan, peptide, and protein levels are stable and reproducible in a variety of proteomes of different origin. We have also analyzed the dynamic range of quantification and demonstrated the practical utility of the method by detecting expression changes in a model of activation of Jurkat T-cells. Our protocol provides a general approach to perform quantitative proteomics by 18O-labeling in high-throughput studies, with the added value that it has a validated statistical model for the null hypothesis. To the best of our knowledge, this is the first report where a general protocol for stable isotope labeling is tested in practice using a collection of samples and analyzed at this degree of statistical detail. more...

MS-based quantitative proteomics plays an increasingly important role in biological and medical research and the development of these techniques remains one of the most important challenges in mass spectrometry. Numerous stable isotope labeling approaches have been proposed. However, and particularly in the case of 18O-labeling, a standard protocol of general applicability is still lacking, and statistical issues associated to these methods remain to be investigated. In this work we present an improved high-throughput quantitative proteomics method based on whole proteome concentration by SDS-PAGE, optimized in-gel digestion, peptide 18O-labeling, and separation by off-gel isoelectric focusing followed by liquid chromatography-LIT-MS. We demonstrate that the off-gel technique is fully compatible with 18O peptide labeling in any pH range. A recently developed statistical model indicated that partial digestions and methionine oxidation do not alter protein quantification and that variances at the scan, peptide, and protein levels are stable and reproducible in a variety of proteomes of different origin. We have also analyzed the dynamic range of quantification and demonstrated the practical utility of the method by detecting expression changes in a model of activation of Jurkat T-cells. Our protocol provides a general approach to perform quantitative proteomics by 18O-labeling in high-throughput studies, with the added value that it has a validated statistical model for the null hypothesis. To the best of our knowledge, this is the first report where a general protocol for stable isotope labeling is tested in practice using a collection of samples and analyzed at this degree of statistical detail. more...

In vitro hypocholesterolemic and neuroprotection assays show that novel monacolin J derivate NST0037 (Id) has a very low 3‐hydroxy‐3‐methylglutaryl CoA reductase IC50and high protection rate for oxidative‐stress‐induced neuron cell death. more...