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Biomedical subjects

S Lamas

Publications and source records attributed to S Lamas.

At least 19 recordsLinked to original sources
PubMed2007

Paradoxical recovery in a bilingual patient with aphasia after right capsuloputaminal infarction.

We report the case of a bilingual dextral patient, who presented with an uncommon pattern of aphasic deficit following a right capsulo-putaminal infarction. In this patient, the linguistic deficit concerned the use of her mother tongue (Galician, L1) much more than the lesser practised second language (Spanish, L2). Our patient presented spontaneous fluent speech in L2 but not in L1, automatic translation into L2, and impaired repetition in L1, whereas comprehension was spared in both L1 and L2. Reading and writing were less valuable due to educational interference (reduced schooling). Spontaneous speech 16 months after the stroke showed the stability of the impairment. This is the first reporting of a crossed subcortical aphasia in a bilingual patient.

Aged↗
PubMed2006

Testing the use of juvenile Salmo trutta L. as biomonitors of heavy metal pollution in freshwater.

Individual specimens of Salmo trutta were captured, from four sampling sites in Galician rivers (NW Spain) affected by different types of contamination: diffuse urban waste, run-off from an unrestored dump at a copper mine and waste from a fish farm. The ages of the captured trouts were established and only those belonging to the 1+ age class were selected for study. The liver and kidney were removed from each fish and analysed to determine the tissue concentrations of Cu, Fe and Zn. The results obtained showed that: (i) the use of 1+ individuals allowed differentiation of contamination scenarios on the basis of the tissue concentrations of metal; (ii) the use of 1+ individuals allowed standardization of the time of exposure, which was sufficiently long for differential uptake to have taken place; (iii) liver tissue provided the best results as, less effort was required than for processing kidney tissue, and significant differences between sampling sites were detected because the intrapopulational variability in metal levels was lower than for kidney, and (iv) the levels of elements detected were not affected by basal tissue concentrations or residual concentrations due to past contamination, which older trouts may have been exposed to. In addition, the use of 1+ trout may provide better results in annual environmental sampling surveys.

Aging↗
PubMed2006

Validation of the Spanish version of the Addenbrooke's Cognitive Examination in a rural community in Spain.

BACKGROUND: The Addenbrooke's Cognitive Examination (ACE) is a brief cognitive test battery designed to detect and differentiate Alzheimer's disease (AD) and frontotemporal dementia (FTD). Translations of this instrument into French and Malayalam have been recently published OBJECTIVE: To adapt and validate the ACE into Spanish in a rural population of low-educational level. SUBJECTS: A clinical group, composed of 70 patients affected by dementia and 25 patients with memory complaints without dementia, was compared with 72 controls matched for gender, age and educational level METHOD: The clinical group was studied with standard neuropsychological instruments, all patients underwent neuroimaging [Computerized Tomography (CT) or Magnetic Resonance Imaging (MRI), and Single Photon Emission Tomography (SPECT) in all cases of suspected FTD], as well as routine neurological examination. Both groups were studied with the ACE and Clinical Dementia Rating scale (CDR). Sensitivity, specificity, area under curve, reliability and Verbal-Language/ Orientation-Memory (VLOM) ratio were calculated. Subsequently, the sample was stratified regarding educational level in two groups. Receiver Operating Characteristics (ROC) curves were calculated for these conditions. Different cut-off points were calculated addressing educational level. RESULTS: ROC curves demonstrated the superiority of the ACE in the sub sample of patients that finished school at over 14 years old. VLOM ratio confirmed its usefulness for differential diagnosis between AD and FTD CONCLUSIONS: The Spanish version of the ACE is a useful instrument for dementia diagnosis. In our sample VLOM ratio results were useful for differential diagnosis between AD and FTD. Different cut-off points must be used for different educational levels.

Aged↗
PubMed2004

Native and oxidized low density lipoproteins oppositely modulate the effects of insulin-like growth factor I on VSMC.

OBJECTIVE: Changes in the local expression and signaling activity of the insulin-like growth factor-I (IGF-I) axis regulate growth and survival of plaque-derived vascular smooth muscle cells (VSMC) and influence plaque fate. Recent evidence suggests that accumulation of low density lipoproteins (LDL) in VSMC during the progression of atherogenesis is linked to local changes in IGF-I signaling. We investigated the effects of LDL on the biological actions and downstream signaling pathways mediated by this growth factor in A10 VSMC. METHODS AND RESULTS: We first characterized the effects of LDL on the proliferative and anti-apoptotic actions of IGF-I in A10 VSMC. Native LDL were mitogenic and synergistically enhanced DNA synthesis induced by IGF-I from 4-, 9- up to 7.8-fold, while having no effect on its anti-apoptotic actions. In contrast, oxidized LDL, at oxidation levels that did not modify these actions by themselves, significantly reduced the mitogenic and survival effects of IGF-I by 40% and 60%, respectively. These observations correlated with opposite changes exerted by native and oxidized LDL on the insulin receptor substrate-1 (IRS)-associated PI3 kinase/Akt response to IGF-I. The extracellular signal-regulated kinase (ERK) signaling response was not affected. CONCLUSIONS: Our study demonstrates a previously unidentified modulation of the actions of IGF-I on A10 VSMC by LDL, dependent on their extent of oxidative modification. Our findings suggest that the differential modulation of the PI3 kinase/Akt response to IGF-I play a pivotal role.

Animals↗
PubMed2001

Glutathionylation of the p50 subunit of NF-kappaB: a mechanism for redox-induced inhibition of DNA binding.

The cellular redox status can modify the function of NF-kappaB, whose DNA-binding activity can be inhibited by oxidative, nitrosative, and nonphysiological agents such as diamide, iodoacetamide, or N-ethylmaleimide. This inhibitory effect has been proposed to be mediated by the oxidation of a conserved cysteine in its DNA-binding domain (Cys62) through unknown biochemical mechanisms. The aim of this work was to identify new oxidative modifications in Cys62 involved in the redox regulation of the NF-kappaB subunit p50. To address this problem, we exposed p50, both the native form (p50WT) and its corresponding mutant in Cys62 (C62S), to changes in the redox pair glutathione/glutathione disulfide (GSH/GSSG) ratio ranging from 100 to 0.1, which may correspond to intracellular (patho)physiological states. A ratio between 1 and 0.1 resulted in a 40-70% inhibition of the DNA binding of p50WT, having no effect on the C62S mutant. Mass spectrometry studies, molecular modeling, and incorporation of (3)H-glutathione assays were consistent with an S-glutathionylation of p50WT in Cys62. Maximal incorporation of (3)H-glutathione to the p50WT and C62S was of 0.4 and 0.1 mol of (3)H-GSH/mol of protein, respectively. Because this covalent glutathione incorporation did not show a perfect correlation with the observed inhibition in the DNA-binding activity of p50WT, we searched for other modifications contributing to the maximal inhibition. MALDI-TOF and nanospray-QIT studies revealed the formation of sulfenic acid as an alternative or concomitant oxidative modification of p50. In summary, these data are consistent with new oxidative modifications in p50 that could be involved in redox regulatory mechanisms for NF-kappaB. These postranslational modifications could represent a molecular basis for the coupling of pro-oxidative stimuli to gene expression.

Amino Acid Sequence↗
PubMed2001

Nitric oxide as a regulator of gene expression: studies with the transcription factor proteins cJun and p50.

Both oxidative and nitrosative stresses may result in the inactivation of the binding to DNA of redox-sensitive transcription factors. The underlying biochemical mechanisms may involve oxidation or nitrosylation of critical thiols within the DNA binding domains of these proteins. However, S-glutahionylation, the formation of a mixed disulfide between glutathione and the redox-sensitive cysteine residues, has been shown to occur under NO exposure and pro-oxidative conditions in c-Jun, one of the AP-1 constituents. This modification may be functionally important as it is reversible and has been detected in other transcription factors, such as NF-kappaB, by using covalent chromatography with a modified S-nitrosoglutathione sepharose.

Gene Expression↗
PubMed2001

Regulation of cyclooxygenase-2 expression by nitric oxide in cells.

Two different cyclooxygenases (COXs) are functional in mammals: COX-1 and COX-2. COX-2 is mainly an inducible isoform that shares significant features with inducible nitric oxide synthase (iNOS) in terms of its tissue distribution and participation in pathophysiological phenomena. Furthermore, the product of iNOS catalysis, nitric oxide (NO), is an important regulator of COX-2 activity and expression, and the products of COX-1 and COX-2 (diverse prostanoids) may also influence iNOS expression. Both positive and negative effects of NO on COX-2 expression have been encountered in experimental systems, showing that the outcome of the NO-COX-2 interaction is exquisitely dependent upon the temporal frame and the cell type studied. The pathophysiological significance of NO-COX cross-talk also arises from in vivo studies, in which most evidence points to a positive effect of NO on COX-2 activity and/or expression. This emphasizes the need to understand the underlying mechanisms. Among these, the capacity of NO and its effector cyclic GMP to modulate the function of several target proteins, including transcription factors such as nuclear factor-kappaB and activator protein-1, appears as the key pathway by which NO may regulate COX-2 expression. Given the capacity of some prostanoids to modulate the inflammatory response, the interplay between NO synthase and COX pathways stands at the center of the pathophysiological basis of inflammatory diseases.

Animals↗
PubMed2001

Nitrosative stress by cyclosporin A in the endothelium: studies with the NO-sensitive probe diaminofluorescein-2/diacetate using flow cytometry.

BACKGROUND: We have reported previously that exposure of endothelial cells to cyclosporin A (CsA) may result in the regulation of specific genes, such as the endothelial nitric oxide synthase. In the context of endothelial toxicity, whether this represents an adaptive response to injury at the physiological level or contributes further to the generation of nitrosative stress remains to be investigated. The precise reactive species potentially produced by CsA and their capacity to modify proteins functionally are not well known. METHODS: We assessed the possibility of detecting intracellular nitric oxide (NO) by flow cytometry with the cell-permeable probe diaminofluorescein-2 diacetate (DAF-2/DA). RESULTS: CsA increased the intracellular 2 h accumulation of DAF-2T, the oxidized form of DAF-2/DA, in an L-NAME-sensitive process. When CsA was re-added for 2 h to bovine aortic endothelial cells previously pre-incubated with CsA for 16 h, an additional increase in the accumulation of DAF-2T was achieved. CONCLUSIONS: In this work, we provide data showing that intracellular NO can be detected by flow cytometry with DAF-2/DA and suggesting two potential mechanisms (transcriptional and post-translational) for the intracellular accumulation of NO in vascular endothelial cells exposed to the immunosuppressant CSA:

Animals↗
PubMed2001

Posttranscriptional regulation of human iNOS by the NO/cGMP pathway.

Nitric oxide (NO) and cGMP may exert positive or negative effects on inducible NO synthase (iNOS) expression. We have explored the influence of the NO/cGMP pathway on iNOS levels in human mesangial cells. Inhibition of NOS activity during an 8-h stimulation with IL-1beta plus tumor necrosis factor (TNF)-alpha reduced iNOS levels, while NO donors amplified iNOS induction threefold. However, time-course studies revealed a subsequent inhibitory effect of NO donors on iNOS protein and mRNA levels. This suggests that NO may contribute both to iNOS induction and downregulation. Soluble guanylyl cyclase (sGC) activation may be involved in these effects. Inhibition of sGC attenuated IL-1beta/TNF-alpha-elicited iNOS induction and reduced NO-driven amplification. Interestingly, cGMP analogs also modulated iNOS protein and mRNA levels in a biphasic manner. Inhibition of transcription unveiled a negative posttranscriptional modulation of the iNOS transcript by NO and cGMP at late times of induction. Supplementation with 8-bromo-cGMP (8-BrcGMP) reduced iNOS mRNA stability by 50%. These observations evidence a complex feedback regulation of iNOS expression, in which posttranscriptional mechanisms may play an important role.

Cells, Cultured↗
PubMed2001

Cooperation between low density lipoproteins and IGF-I in the promotion of mitogenesis in vascular smooth muscle cells.

Low density lipoproteins (LDL) are an independent risk factor for atherosclerosis and show synergism with some growth factors in vascular smooth muscle cell (VSMC) proliferation. IGF-I has mitogenic actions on VSMC, which, in turn, show enhanced expression of IGF-I and its receptor when exposed to hypercholesterolemic diets in vivo. To investigate the molecular basis of a possible interaction between LDL and the IGF-I signaling system in VSMC, we used A10 cells, where synergism between both factors in DNA synthesis was demonstrated. IGF-I activates phosphatidylinositol 3-kinase (PI3 kinase) and extracellular signal-regulated MAPK pathways in A10 cells, although insulin receptor substrate-1 (IRS-1)-associated PI3 kinase is more closely linked to IGF-I induced proliferation. LDL, in pathophysiological concentrations, affect the IGF-I signaling pathway at multiple levels: 1) they induce phosphorylation of IGF-I receptor beta and IRS-1 in a time- and dose-dependent manner; 2) they up-regulate IRS-1-associated PI3 kinase/Akt activation in response to IGF-I at early times; and 3) they show additive effects with IGF-I on extracellular signal-regulated MAPK 1/2 phosphorylation. These actions are not present in very low density lipoprotein treatments. Taken together, these results indicate specific cooperation between LDL and the IGF-I signaling pathways and may represent a more general mechanism through which proatherogenic lipoproteins modulate VSMC response to growth factors.

Animals↗
PubMed2000

Involvement of Rho GTPases in the transcriptional inhibition of preproendothelin-1 gene expression by simvastatin in vascular endothelial cells.

Endothelial dysfunction is characterized by an impaired vasodilatory response to endothelial agonists as well as by alterations in adhesion and coagulation processes. 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) have been shown to be useful in the reversal of endothelial dysfunction, an effect that may be independent of the reduction in cholesterol levels. Both the L-arginine-nitric oxide-cGMP and endothelin pathways are involved in the regulation of vascular tone. Here, we show that the basal transcription rate of the preproendothelin-1 gene was decreased by simvastatin (10 micromol/L) in bovine aortic endothelial cells. Transfection studies with the preproendothelin-1 gene promoter showed that mevalonate (100 micromol/L) was able to prevent the inhibitory effect mediated by simvastatin. Protein geranylgeranylation, but not farnesylation, proved to be crucial for a correct expression of the preproendothelin-1 gene. The C3 exotoxin from Clostridium botulinum that selectively inactivates Rho GTPases, the processing of which involves geranylgeranylation, reproduced the inhibitory effect of simvastatin on the expression of preproendothelin-1. Overexpression of dominant-negative mutants of RhoA and RhoB led to a significant reduction in the preproendothelin-1 promoter activity, whereas the expression of wild-type and constitutively active forms of these proteins resulted in an increase, in support that Rho proteins are required for the basal expression of the preproendothelin-1 gene. Finally, we show that the Rho-dependent activation of the preproendothelin-1 gene transcription was inhibited by simvastatin. Thus, the control of vascular tone and proliferative response mediated by endothelin-1 is regulated at multiple levels, among which the Rho proteins play an essential role.

Alkyl and Aryl Transferases↗
PubMed2000

Novel application of S-nitrosoglutathione-Sepharose to identify proteins that are potential targets for S-nitrosoglutathione-induced mixed-disulphide formation.

Site-specific S-glutathionylation is emerging as a novel mechanism by which S-nitrosoglutathione (GSNO) may modify functionally important protein thiols. Here, we show that GSNO-Sepharose mimicks site-specific S-glutathionylation of the transcription factors c-Jun and p50 by free GSNO in vitro. Both c-Jun and p50 were found to bind to immobilized GSNO through the formation of a mixed disulphide, involving a conserved cysteine residue located in the DNA-binding domains of these transcription factors. Furthermore, we show that c-Jun, p50, glycogen phosphorylase b, glyceraldehyde-3-phosphate dehydrogenase, creatine kinase, glutaredoxin and caspase-3 can be precipitated from a mixture of purified thiol-containing proteins by the formation of a mixed-disulphide bond with GSNO-Sepharose. With few exceptions, protein binding to this matrix correlated well with the susceptibility of the investigated proteins to undergo GSNO- but not diamide-induced mixed-disulphide formation in vitro. Finally, it is shown that covalent GSNO-Sepharose chromatography of HeLa cell nuclear extracts results in the enrichment of proteins which incorporate glutathione in response to GSNO treatment. As suggested by DNA-binding assays, this group of nuclear proteins include the transcription factors activator protein-1, nuclear factor-kappaB and cAMP-response-element-binding protein. In conclusion, we introduce GSNO-Sepharose as a probe for site-specific S-glutathionylation and as a novel and potentially useful tool to isolate and identify proteins which are candidate targets for GSNO-induced mixed-disulphide formation.

Binding Sites↗
PubMed2000

Transcriptional induction of endothelial nitric oxide gene by cyclosporine A. A role for activator protein-1.

We have previously shown that the immunosuppressant cyclosporine A (CsA) increases the activity, the protein level, and the steady-state levels of the mRNA of the endothelial nitric-oxide synthase (eNOS) gene in bovine aortic endothelial cells (BAEC). We have now investigated the mechanisms responsible for these effects. Preincubation with an inhibitor of RNA polymerase II abolished CsA-induced eNOS up-regulation. Nuclear run-on experiments demonstrated a 1.6-fold increase in the induction of eNOS gene by CsA. In agreement with these results, transient transfections showed that CsA augmented the transactivation of the eNOS promoter. Electrophoretic mobility shift assays showed an increase in the activator protein-1 (AP-1) DNA binding activity in BAEC treated with CsA. An increase in the level of c-fos mRNA and in the nuclear content of c-Fos protein was detected in BAEC treated with CsA. Site-directed mutagenesis of the AP-1 cis-regulatory element in the context of the human eNOS promoter resulted in the abrogation of the induction mediated by CsA. Hence, up-regulation of eNOS mRNA by CsA is a transcriptional phenomenon involving the proximal AP-1 site in the 5'-regulatory region of the human eNOS gene. Furthermore, our data exemplify how immunosuppressive drugs may result in the regulation of specific genes involved in the homeostasis of endothelial function, such as eNOS.

Animals↗
PubMed2000

Regulation of protein function by S-glutathiolation in response to oxidative and nitrosative stress.

Protein S-glutathiolation, the reversible covalent addition of glutathione to cysteine residues on target proteins, is emerging as a candidate mechanism by which both changes in the intracellular redox state and the generation of reactive oxygen and nitrogen species may be transduced into a functional response. This review will provide an introduction to the concepts of oxidative and nitrosative stress and outline the molecular mechanisms of protein regulation by oxidative and nitrosative thiol-group modifications. Special attention will be paid to recently published work supporting a role for S-glutathiolation in stress signalling pathways and in the adaptive cellular response to oxidative and nitrosative stress. Finally, novel insights into the molecular mechanisms of S-glutathiolation as well as methodological problems related to the interpretation of the biological relevance of this post-translational protein modification will be discussed.

Animals↗