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M E Fuentes

Publications and source records attributed to M E Fuentes.

At least 19 recordsLinked to original sources
PubMed2006

Expressions for the fractional modification in different monocyclic enzyme cascade systems: analysis of their validity tested by numerical integration.

This paper presents the derivation, under a minimal set of assumptions, of a general expression for the steady-state fractional modification of an interconvertible protein involved in four different schemes of monocyclic enzyme cascade systems. From this general expression we derive, as particular cases, other, simpler expressions by applying additional assumptions and which have, therefore, a smaller range of validity. Some of these particular expressions coincide with those already obtained in previous contributions on individualised analyses. We discuss the relationships between the kinetic parameters and the concentrations needed for the fulfilment of the additional assumptions. The goodness of the analysis was tested by reference to the shape in the steady-state of the simulated time progress curves obtained by numerical integration.

Algorithms↗
PubMed2001

Rapid up-regulation of CXC chemokines in the airways after Ag-specific CD4+ T cell activation.

Ag-specific activation of CD4(+) T cells is known to be causative for the cytokine production associated with lung allergy. Chemokine-induced leukocyte recruitment potentially represents a critical early event in Ag-induced lung inflammation. Whether Ag-specific, lung CD4(+) T cell activation is important in lung chemokine production is currently not clear. Using alphabeta-TCR transgenic BALB/c DO11.10 mice, we investigated the ability of Ag-specific CD4(+) T cell activation to induce lung chemokine production and leukocyte recruitment. Within 1 h of exposure of DO11. 10 mice to OVA aerosol, lung mRNA and protein for the neutrophil chemokines KC and macrophage inflammatory protein (MIP)-2 were greatly increased. Accordingly, neutrophils in the airways increased by >50-fold, and KC and MIP-2 proved to be functional because their neutralization significantly reduced airway neutrophilia. CD4(+) T cell activation was critical because CD4(+) but not CD8(+) T cell depletion reduced KC production, which correlated well with the previously observed inhibition of neutrophil influx after CD4(+) T cell depletion. In vitro studies confirmed that OVA-induced KC and MIP-2 production was conditional upon the interaction of CD4(+) T cells with APCs. A likely secondary mediator was TNF-alpha, and a probable source of these chemokines in the lung was alveolar macrophages. Thus, Ag-specific CD4(+) T cell activation in the lung leads to rapid up-regulation of neutrophil chemokines and the recruitment of neutrophils to the site of Ag exposure. This may be a key early event in the pathogenesis of Ag-induced lung inflammation.

Administration, Inhalation↗
PubMed2001

Sulfation of L-selectin ligands by an HEV-restricted sulfotransferase regulates lymphocyte homing to lymph nodes.

Lymphocytes home to lymph nodes, using L-selectin to bind specific ligands on high endothelial venules (HEV). In vitro studies implicate GlcNAc-6-sulfate as an essential posttranslational modification for ligand activity. Here, we show that genetic deletion of HEC-GlcNAc6ST, a sulfotransferase that is highly restricted to HEV, results in the loss of the binding of recombinant L-selectin to the luminal aspect of HEV, elimination of lymphocyte binding in vitro, and markedly reduced in vivo homing. Reactivity with MECA 79, an adhesion-blocking mAb that stains HEV in lymph nodes and vessels in chronic inflammatory sites, is also lost from the luminal aspects of HEV. These results establish a critical role for HEC-GlcNAc6ST in lymphocyte trafficking and suggest it as an important therapeutic target.

Animals↗
PubMed1999

[Feasibility and safety of intracoronary ultrasound. Experience of a single center].

BACKGROUND AND OBJECTIVES: Intracoronary ultrasound provides a number of advantages in the quantification and characterization of coronary stenoses with regard to contrast angiography. However, previous studies have reported a 3.5 to 11% complication rate, and a 10-30% failure rate in performing this technique. The purpose of the study is to analyze the feasibility of performing intracoronary ultrasound and the incidence of complications associated with the use of contemporary, state of the art equipment. MATERIAL AND METHODS: The feasibility of performing intracoronary ultrasound, analyzed as the percentage of successes and failures in performing the examination was reviewed, as well as the complication rate associated with the technique in all the procedures carried out between July 1, 1994 and February 29, 1996 in which intravascular ultrasound was attempted. Complications were categorized as related, non-related and uncertainly related to the ultrasound study. RESULTS: 239 vessels were studied with intravascular ultrasound in 209 procedures (74% interventional) performed on 139 patients. Ultrasound examination was feasible in all the diagnostic studies and in 96% of the interventional procedures. The major and minor procedural complication rate was 2.4 and 10.5% respectively. No major complication was related to the ultrasound examination. Three patients experienced minor complications (1.4%) related to the ultrasound study. All three complications occurred in baseline studies during interventional procedures. CONCLUSIONS: Intracoronary ultrasound is feasible and safe in the vast majority of the procedures. Improvements in smaller catheter size and design and larger operator expertise have significantly reduced the complication rate, particularly the most frequent coronary spasm so far. Complications are associated with baseline studies during interventional procedures and with less operator expertise.

Aged↗
PubMed1999

Embryonic lethality, liver degeneration, and impaired NF-kappa B activation in IKK-beta-deficient mice.

IkappaB kinase-alpha and -beta (IKK-alpha and IKK-beta), the catalytic subunits of the IKK complex, phosphorylate IkappaB proteins on specific serine residues, thus targeting IkappaB for degradation and activating the transcription factor NF-kappaB. To elucidate the in vivo function of IKK-beta, we generated IKK-beta-deficient mice. The homozygous mouse embryo dies at approximately 14.5 days of gestation due to liver degeneration and apoptosis. IKK-beta-deficient embryonic fibroblasts have both reduced basal NF-kappaB activity and impaired cytokine-induced NF-kappaB activation. Similarly, basal and cytokine-inducible kinase activities of the IKK complex are greatly reduced in IKK-beta-deficient cells. These results indicate that IKK-beta is crucial for liver development and regulation of NF-kappaB activity and that IKK-alpha can only partially compensate for the loss of IKK-beta.

Animals↗
PubMed1998

Electric lead field for a piecewise homogeneous volume conductor model of the head.

A new method is presented for computing the electric lead field of a realistic head shape model which has piecewise homogeneous conductivity. The basic formulae are derived using the well-known reciprocity theorem. Previously described methods are also based upon this theorem, but these first calculate the electric potential inside the head by a scalar boundary element method (BEM), and then approximate the ohmic current density by some sort of gradient. In contrast, this paper proposes the direct evaluation of the ohmic current density by discretizing the vector Green's second identity which leads to a vector version of BEM. This approach also allows the derivation of the same equations for the three concentric spheres model as obtained by Rush and Driscoll [8]. The results of simulations on a spherical head model indicate that the use of a vector BEM leads to an improvement of accuracy in the computation of the ohmic current density with respect to those reported previously, in term of different measures of error.

Algorithms↗
PubMed1996

Neutrophil infiltration, glial reaction, and neurological disease in transgenic mice expressing the chemokine N51/KC in oligodendrocytes.

Chemokines (pro-inflammatory chemoattractant cytokines) are expressed in pathological conditions of the central nervous system (CNS). Previous studies suggested that the CNS is relatively resistant to leukocyte diapedesis after chemokine injection, leaving their functional role unresolved. The CNS function of N51/KC, a neutrophil-selective chemokine, was addressed by expressing N51/KC under control of the myelin basic protein (MBP) promoter in transgenic (tg) mice (MBP-N51/KC mice). CNS-specific N51/KC expression produced remarkable neutrophil infiltration into perivascular, meningeal, and parenchymal sites, demonstrating that this chemokine exerts the multiple functions in vivo required to recruit leukocytes into the CNS. MBP-N5 1/KC mice represent an incisive model for the molecular dissection of neutrophil entry into the CNS. Unexpectedly, MBP-N51/KC mice developed a neurological syndrome of pronounced postural instability and rigidity at high frequency beginning at 40 days of age, well after peak chemokine expression. 68/182 mice in one tg fine were found dead before one year of age, with prominent neurological symptoms premortem in 26 (38%). Florid microglial activation and blood-brain barrier disruption without dysmyelination were the major neuropathological alterations. Late-onset neurological symptoms in MBP-N51/KC mice may indicate unanticipated consequences of CNS chemokine expression.

Animals↗
PubMed1995

Controlled recruitment of monocytes and macrophages to specific organs through transgenic expression of monocyte chemoattractant protein-1.

Transgenic mice overexpressing the chemokine monocyte chemoattractant protein-1 (MCP-1) in the thymus and central nervous system have a higher number of mononuclear cells in those tissues than do control littermates. In the thymus, there is a modest increase in the number of Mac-1 and F4/80 positive cells, but no apparent change in the number of lymphoid cells. A more pronounced mononuclear infiltrate is detected in transgenic mice expressing MCP-1 in the brain. The vast majority of the recruited cells in the brain are monocytes and macrophages, as defined by light microscopy, and ultrastructural and immunohistochemical criteria. Such cells are found in a perivascular orientation with minimal parenchymal infiltration, possibly as a consequence of the accumulation of MCP-1 in the vessels, as shown by immunohistochemistry. The mononuclear cell infiltrate in the brain can be significantly amplified by LPS treatment, suggesting that the recruitment properties of MCP-1 can be potentiated by additional factors.

Animals↗
PubMed1995

Cadmium-induced expression of metallothionein and suppression of RNA to DNA ratios during molluscan development.

One-week-old larvae of the mollusc Crassostrea virginica, an oyster, were exposed to Cd concentrations ranging from the control with no added Cd to 0.2 microM CdCl2 for 24 hr. Concentration-dependent increases in total cadmium accumulation, cytosolic and metallothionein-bound Cd concentrations, and levels of the MT mRNA were detectable over this range of exposure concentrations. Increases in these measures were apparent at the lowest exposure concentration of 0.005 microM, with dramatic increases observed between 0.04 and 0.2 microM Cd. Although the concentrations of MT-bound Cd increased, the fraction of total Cd that was bound to MTs declined in larvae treated with 0.04 and 0.2 microM Cd, presumably due to the inability of MT biosynthesis to keep up with the higher rates of Cd uptake at the higher concentrations. This decreased effectiveness of MTs in sequestering Cd in relation to the total Cd load and the associated increase in the nonthionein Cd fraction at these higher treatment conditions coincided with a decline in the RNA to DNA ratio and an increase in a nonacetylated variant of the MT. The former is indicative of an inhibition of larval metabolism and growth. The latter is an unexpected form for this MT and is believed to signify disruption of normal cotranslational modification of the N-terminal amino acid sequence.

Animals↗
PubMed1994

Expression of the chemokine N51/KC in the thymus and epidermis of transgenic mice results in marked infiltration of a single class of inflammatory cells.

Transgenic mice expressing the chemokine N51/KC in thymus, skin, and tongue showed a marked infiltration of a single class of inflammatory cells (neutrophils) in the sites of transgene expression. In the thymus, neutrophils were most numerous in the cortex and juxta-medullary regions, often forming aggregates or clusters. A similar, but less intense, neutrophilic infiltrate occurred in close proximity to the epidermal basal layer of the tongue and skin. No morphologic evidence of injury was observed in the thymus, skin, or tongue of these transgenic mice, indicating that N51/KC expression induces recruitment but not inflammatory activation of neutrophils. The lack of activation in the thymus resulted in a large senescent neutrophilic population that was phagocytosed by thymic macrophages and epithelial-reticular cells. These results indicate that N51/KC is a neutrophil chemoattractant in vivo and establish these transgenic mice as effective models to study the phenomena of recruitment and clearance of neutrophils, events that are critical for the initiation and resolution of the inflammatory response.

Aging↗
PubMed1994

Regulation of acetylcholinesterase mRNA stability by calcium during differentiation from myoblasts to myotubes.

The expression of acetylcholinesterase (AChE), nicotinic acetylcholine receptors (nAChR), and their corresponding mRNAs increases dramatically during the conversion of myoblasts to myotubes in C2-C12 cells. The increase in expression of nAChR arises from transcriptional activation of the genes encoding the receptor subunits, whereas stabilization of labile transcripts is primarily responsible for enhanced AChE expression. In a search for the signaling pathways responsible for stabilization of the AChE mRNA, we found that ryanodine, synthetic ryanodine receptor antagonists and L-type, but not N-type, Ca2+ channel blockers inhibit the differentiation-induced expression of AChE mRNA, but not the nAChR mRNA. Selective inhibition of increased expression of AChE is also evident. Inhibition by ryanodine and nifedipine is additive suggesting different target sites for the two Ca2+ channel ligands. Ryanodine binding sites can be detected in both myoblasts and myotubes, but they increase substantially during differentiation. Rates of AChE gene transcription are not altered by ryanodine and nifedipine, indicating that decreased Ca2+ availability prevents stabilization of the mRNA normally seen with differentiation. Muscle cells still undergo elongation and fusion in the presence of ryanodine or L-type Ca2+ channel antagonists. Ryanodine block is fully reversible, indicating functional integrity of the cellular expression system after the drug treatment. These findings indicate that intracellular ryanodine-sensitive calcium channels and extracellular L-type Ca2+ channels link to play an important role in stabilizing AChE mRNA and suggest that transient increases in intracellular Ca2+ may be critical for the commitment of AChE expression during myogenesis.

Acetylcholinesterase↗
PubMed1994

Monomeric amphiphilic forms of acetylcholinesterase appear early during brain development and may correspond to biosynthetic precursors of the amphiphilic G4 forms.

We have studied the development of mouse brain acetylcholinesterase (AChE). Only tetrameric (G4) and monomeric (G1) forms were detected both in vivo and in vitro. The amphiphilic G4 form increased continuously during development, whereas an amphiphilic G1 form appears transiently around embryonic day 17. A causal relationship between the monomers and tetramers was established using pulse-chase experiments with paraoxon, a reversible AChE inhibitor. We report here, for the first time, the presence of an amphiphilic monomer possibly involved in the assembly of the amphiphilic G4 AChE form during mouse brain development.

Acetylcholinesterase↗
PubMed1994

Expression and ligand specificity of acetylcholinesterase and the nicotinic receptor: a tale of two cholinergic sites.

The functional design of the nAChR and AChE rather than their recognition capacities requires divergence in structure of the two binding sites. The receptor requires co-operativity to link ligand occupation to the response, rapid conformational transitions of activation, and slower transitions of desensitization. Hence, its binding sites have evolved at subunit interfaces. By contrast, AChE functions with a large kcat and a comparatively large Km. To do so, it must force acetylcholine through a low-energy transition site that features tetrahedral rather than the ground-state, trigonal conformation around the carbonyl carbon. This requires a high affinity (KD approximately 10(-17) M) for the enzyme complex of the transient transition state. Interestingly, the three-finger peptide toxins (alpha-bungarotoxin and fasciculin), though closely homologous, use different interaction sites on the receptor (the agonist recognition site) and AChE (a peripheral site). Finally, although the two proteins show co-ordinated expression during muscle differentiation, the receptor relies primarily on transcriptional control while AChE expression is post-transcriptional, being controlled by mRNA stability.

Acetylcholinesterase↗
PubMed1993

Structure and regulation of expression of the acetylcholinesterase gene.

Acetylcholinesterase, an enzyme essential for the termination of the action of acetylcholine, is encoded by a single gene. Alternative mRNA processing gives rise to the expression of enzyme forms with three distinct carboxyl-termini. These structural differences govern the cellular disposition of the expressed enzyme but do not influence catalytic activity. Alternative polyadenylation signals give rise to distinct 3' non-coding regions which are likely to affect mRNA stability. Alternative splicing also occurs at the 5' end of the gene where two promoter regions can be identified. Hence, regulation of expression of the gene occurs at 3 levels, transcriptional through alternative promoters, translational by affecting mRNA stability and processing of distinct mRNAs and post-translationally by giving rise to distinct peptide chains which are processed differently. Recombinant DNA studies have also been extended to modifying protein structure through site-specific mutagenesis and studying the function of the mutant enzymes.

Acetylcholinesterase↗
PubMed1993

Control of acetylcholinesterase gene expression during myogenesis.

Acetylcholinesterase (AChE) and the nicotinic acetylcholine receptor show enhanced expression upon differentiation of C2-C12 myoblasts into myotubes. Northern blots and RNAase protection show substantial increases in the mRNAs encoding both proteins. In contrast to transcriptional control of gene expression of acetylcholine receptor subunits during differentiation, Ache mRNA levels increase through stabilization of the transcript. The transcriptional rate remains unchanged during Ache mRNA accumulation, whereas transcription rate increase for the receptor subunits. Inhibition of protein synthesis produces a dramatic superinduction of Ache mRNA in undifferentiated cells without affecting transcription rates, suggesting the presence of labile proteins that uniquely destabilize the mRNA. Hence, Ache mRNA in myoblasts shows rapid turnover, and upon differentiation, its level is increased by slowing the rate of mRNA degradation.

Acetylcholinesterase↗
PubMed1992

Amphiphilic behavior of a brain tetrameric acetylcholinesterase form lacking the plasma membrane anchoring domain.

We have studied the behavior of a mammalian brain tetrameric acetylcholinesterase (AChE) form released by proteinase K from a crude membrane fraction of bovine caudate nucleus. The solubilization of active AChE indicated the presence of a protease-sensitive site in the anchored protein. Unexpectedly, the solubilized AChE maintained its capacity to form aggregates in detergent-free gradients. We demonstrate here that this property was due neither to the presence of the hydrophobic membrane-anchoring domain still linked to the enzyme, nor to the presence of AChE activity trapped in small plasma membrane vesicles. Moreover, we found that the proteinase K-treated extract, devoid of AChE activity, induced the aggregation of purified hydrophilic AChE which usually does not form aggregates. Our results suggest the presence of an AChE aggregating factor in bovine brain extracts prepared in the presence of proteinase K. It is possible that this aggregation may reflect a process of AChE clustering on neurons.

Acetylcholinesterase↗