[Show abstract][Hide abstract] ABSTRACT: The correlations of genotypic and phenotypic tests with treatment, clinical history and the significance of mutations in viruses of HIV-infected patients are used to establish resistance mutations to protease inhibitors (PIs). Emerging mutations in human immunodeficiency virus type 1 (HIV-1) protease confer resistance to PIs by inducing structural changes at the ligand interaction site. The aim of this study was to establish an in silico structural relationship between natural HIV-1 polymorphisms and unusual HIV-1 mutations that confer resistance to PIs.
Protease sequences isolated from 151 Mexican HIV-1 patients that were naive to, or subjected to antiretroviral therapy, were examined. We identified 41 unrelated resistance mutations with a prevalence greater than 1%. Among these mutations, nine exhibited positive selection, three were natural polymorphisms (L63S/V/H) in a codon associated with drug resistance, and six were unusual mutations (L5F, D29V, L63R/G, P79L and T91V). The D29V mutation, with a prevalence of 1.32% in the studied population, was only found in patients treated with antiretroviral drugs. Using in silico modelling, we observed that D29V formed unstable protease complexes when were docked with lopinavir, saquinavir, darunavir, tipranavir, indinavir and atazanavir.
The structural correlation of natural polymorphisms and unusual mutations with drug resistance is useful for the identification of HIV-1 variants with potential resistance to PIs. The D29V mutation likely confers a selection advantage in viruses; however, in silico, presence of this mutation results in unstable enzyme/PI complexes, that possibly induce resistance to PIs.
[Show abstract][Hide abstract] ABSTRACT: Systemic administration of kainic acid (KA) in rodents triggers limbic seizures following selective neuronal loss in the hippocampus attributed to the excitotoxic process. Lipid peroxidation products, such as 4-hydroxynonenal, are produced by oxidative stress and are present on the hippocampus, which contribute to neuronal death in the KA excitotoxicity model. Several antioxidants are neuroprotective agents. The aim of the present study was to analyse whether pirfenidone (PFD, 5-methyl-1-phenyl-2-(1H)-pyridone), an antioxidant drug, protects the neurons in the hippocampus of pubescent rats administered with KA. We evaluated the neuroprotective effect of PFD by quantifying the surviving neurons under hematoxilin-eosin staining after using three different doses of 100, 250, and 325 mg/kg administered via an orogastric tube 90 min after KA intraperitoneal injection (12 mg/kg). Only 325 mg/kg of PFD-attenuated neuronal loss in the hippocampal areas cornu ammonis field 1 (CA1) and cornu ammonis field 3 (CA3c) was observed; therefore, this dose was used in our subsequent studies. Later, we established that PFD reduces neuronal degeneration using Fluoro-Jade B stain in the CA3c but not in the CA1, and PFD reduces the presence of 4-hydroxynonenal, a lipid peroxidation product, in the CA3 by tissue immunohistochemistry. We concluded that only a single 325 mg/kg PFD dose had a neuroprotective effect after KA brain injury. This treatment may be advantageous because adequate pharmacological therapy with PFD can be developed to protect the neuron even after an acute neuronal disorder such as seizures or hypoxic/ischemic damage.
Journal of Molecular Neuroscience 10/2013; · 2.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: IntroductionNicotinic acetylcholine receptors (nAChRs) are widely expressed throughout several brain regions. Formation of the α4β2 and α7 subtypes in particular is involved in the organisation of different types of memory. Furthermore, due to their location, these receptors can control the release of various types of neurotransmitters and contribute to synaptic plasticity.Methods
Rats were divided into three groups, an experimental group (E), a sham-operated group, (S) and an intact group (T). In group E, stereotactic guidance was used to induce a chemical lesion with 1 μ/μL of 5,7-dihydroxytryptamine (5,7-DHT) in the anteroventral part of the dorsal raphe nucleus (DRN). In the sham-operated group (S), animals underwent surgery including delivery of the same excipient solution to the same site. The intact group (T) received no treatment whatsoever. Twenty days after surgery, animals in all groups were euthanised by decapitation to evaluate the expression of α4 and α7 nAChRs by means of molecular biology techniques.Results5-HT denervation of the rat PFC differentially modified the expression of α4 and α7 receptors: while α4 receptor expression increased, α7 expression decreased.Conclusion
Expression differences observed between the two subtypes may be due to their separate locations. The α4 subtype is found in postsynaptic locations and may be related to adaptive changes in postsynaptic cells, while the location of α7 is presynaptic. This explains why the lesion and the elimination of 5-HT fibres in the CPF would cause a decrease in α7 expression.
[Show abstract][Hide abstract] ABSTRACT: Glutamic acid (Glu) is the major excitatory neurotransmitter in the central nervous system, and interacts with two classes of receptor: metabotropic and ionotropic receptors. Ionotropic receptors are divided according to the affinity of their specific agonists: N-methyl-D-aspartate (NMDA), amino acid-3-hydroxy-5-methyl-4-isoxazole acid (AMPA) and kainic acid (KA). NMDA receptors (NMDA-R) are macromolecular structures that are formed by different combinations of subunits, NMDAR1 (NR1), NMDAR2 (NR2) and NMDAR3 (NR3). The study of this receptor has aroused great interest, partly due to its role in synaptic plasticity but mainly because of its permeability to the Ca2+ ion. This review examines the molecular composition of NMDA-R and the variants of NR1 subunit editing in association with NR2 subunit dimers, which form the main components of this receptor. Their composition, structure, function and distinct temporal and spatial expression patterns demonstrate the versatility and diversity of functionally different isoforms of NR1 subunits and the various pharmacological properties of the NR2 subunit. Finally, the involvement of NMDA-R in the excitotoxicity phenomenon, as well as, its expression changes under these conditions as neuronal response is also showed.
Current pharmaceutical design 03/2013; · 4.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Decreased Choline Acetyltransferase (ChAT) brain level is one of the main biochemical disorders in Alzheimer's Disease (AD). In rodents, recent data show that the CHAT gene can be regulated by a neural restrictive silencer factor (NRSF). The aim of the present work was to evaluate the gene and protein expression of CHAT and NRSF in frontal, temporal, entorhinal and parietal cortices of AD patient brains. Four brains from patients with AD and four brains from subjects without dementia were studied. Cerebral tissues were obtained and processed by the guanidine isothiocyanate method for RNA extraction. CHAT and NRSF gene and protein expression were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. CHAT gene expression levels were 39% lower in AD patients as compared to the control group (p < 0.05, U test). ChAT protein levels were reduced by 17% (p = 0.02, U test). NRSF gene expression levels were 86% higher in the AD group (p = 0.001, U test) as compared to the control group. In the AD subjects, the NRSF protein levels were 57% higher (p > 0.05, U test) than in the control subjects. These findings suggest for the first time that in the brain of AD patients high NRSF protein levels are related to low CHAT gene expression levels.
Genetics and Molecular Biology 03/2013; 36(1):28-36. · 0.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cognitive impairment or its recovery has been associated with the absence or reestablishment of estrogenic actions in the central nervous system of female experimental animals or women. It has been proposed that these cognitive phenomena are related to estrogen-mediated modulatory activity of synaptic transmission in brain structures involved in cognitive functions. In the present work a morphological study was conducted in adult female ovariectomized rats to evaluate estradiol-dependent dendritic spine sprouting in hippocampal pyramidal neurons, and changes in the presynaptic marker synaptophysin. Three or ten days after estradiol treatment (10 μg/day, twice) in the ovariectomized rats, a significant increase of synaptophysin was observed, which was coincident with a significant higher numerical density of thin (22%), stubby (36%), mushroom (47%) and double spines (125%), at day 3, without significant changes of spine density at day 10, after treatment. These results may be interpreted as evidence of pre- and postsynaptic plastic events that may be involved in the modulation of cognitive-related behavioral performance after estrogen replacement therapy.
Brain research 06/2012; 1470:1-10. · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION: Nicotinic acetylcholine receptors (nAChRs) are widely expressed throughout several brain regions. Formation of the α4β2 and α7 subtypes in particular is involved in the organisation of different types of memory. Furthermore, due to their location, these receptors can control the release of various types of neurotransmitters and contribute to synaptic plasticity. METHODS: Rats were divided into three groups, an experimental group (E), sham-operated group (S) and a intact group (T). In group E, stereotactic guidance was used to induce a chemical lesion with 1 μ/μl of 5,7-dihydroxytryptamine (5,7-DHT) in the anteroventral part of the dorsal raphe nucleus (DRN). In the sham-operated group (S), animals underwent surgery including delivery of the same excipient solution to the same site. The intact group (T) received no treatment whatsoever. Twenty days after surgery, animals in all groups were euthanised by decapitation to evaluate the expression of α4 and α7 nAChRs by means of molecular biology techniques. RESULTS: 5-HT denervation of the rat PFC differentially modified the expression of α4 and α7 receptors: while α4 receptor expression increased, α7 expression decreased CONCLUSION: Expression differences observed between the two subtypes may be due to their separate locations. The α4 subtype is found in postsynaptic locations and may be related to adaptive changes in postsynaptic cells, while the location of α7 is presynaptic. This explains why the lesion and the elimination of 5-HT fibres in the CPF would cause a decrease in α7 expression.
[Show abstract][Hide abstract] ABSTRACT: To review the physiology of the glutamate receptor subunits such as N-methyl-D-aspartate (NMDA).
Glutamic acid (Glu) is the major excitatory neurotransmitter in the central nervous system which interacts with two types classified into two types: metabotropic and ionotropic. Ionotropic receptors are classified according to the affinity of their specific agonists: N-methyl-D-aspartate (NMDA), α-amino acid-3-hydroxy-5-methyl-4-isoxazole (AMPA) and kainic acid (KA). NMDA receptors are macromolecular structures that are formed by different combinations of subunits, NMDAR1 (NR1), NMDAR2 (NR2) and NMDAR3 (NR3)
The study of this receptor has been of great interest due to its role in synaptic plasticity, but mainly due to the permeability it has to Ca(++) ion. This review examines the molecular composition of NMDA receptor and the variants of NR1 subunit edition in association with NR2 subunit dimer, the main form of this receptor. The composition, structure and function and their distinct expression patterns in both time and space, has shown the versatility and diversity of functionally different isoforms of the NR1 subunit and various pharmacological properties of the NR2 subunit.
[Show abstract][Hide abstract] ABSTRACT: Hypoxia-inducible factor-1 alpha (HIF-1α) is a master transcription factor that regulates the response to hypoxia and ischemia and induces the expression of various genes, including vascular endothelial growth factor (VEGF) and erythropoietin (EPO). This study shows the systemic response of increased HIF-1α, EPO, and VEGF mRNA and protein. In addition, VEGF expression was increased in neurons and over-expressed in glial cells in a model of neuroexcitotoxicity in the hippocampus, in which rats were neonatally exposed to high glutamate concentrations. Simultaneous increases in HIF-1α, EPO and VEGF mRNA in peritoneal macrophages were also observed. Our study is consistent with the hypothesis that these genes exert a protective effect in response to neurotoxicity.
Journal of neuroimmunology 07/2011; 238(1-2):12-8. · 2.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: HIF-1 alpha (hypoxia-inducible factor-1 alpha) mediates the responses of mammalian cells to hypoxia/ischemia by inducing the expression of adaptive gene products (e.g., vascular endothelial growth factor (VEGF) and erythropoietin (EPO)). Persistent pulmonary hypertension of the newborn (PPHN) and cyanotic congenital heart disease (CCHD) are common neonatal diseases considered as paradigms of hypoxemia. Since the expression HIF-1 alpha, VEGF and EPO in newborns diagnosed with these diseases has yet to be studied, we set out to define the expression of these genes in peripheral blood from newborn infants diagnosed with PPHN and CCHD.
The mRNA transcripts encoding HIF-1 alpha, VEGF and EPO were measured by RT-PCR in healthy newborn infants and infants diagnosed with PPHN and CCHD.
An important increase in HIF-1 alpha expression was observed in both pathological conditions, accompanied by significant increases in VEGF and EPO expression when compared to healthy infants.
HIF-1 alpha mRNA expression increases in newborn infants with PPHN or CCHD, as does the expression of its target genes VEGF and EPO.
[Show abstract][Hide abstract] ABSTRACT: Monosodium glutamate (MSG) administered to neonatal rats during the first week of life induces a neurodegenerative process, which is represented by several neurochemical alterations of surviving neurons in the brain, where signalling mediated by GABA is essential for excitation threshold maintenance. GABA-positive cells, [3H]-GABA uptake, expression of mRNA for GABA transporters GAT-1 and GAT-3, and expression of mRNA and protein for two main GABA synthesizing enzymes, GAD65 and GAD67, were measured at postnatal day 60, after MSG neonatal treatment in two critical cerebral regions, cerebral cortex and hippocampus. GABA-positive cells, [3H]-GABA uptake, and mRNA for GAT-1, were significantly diminished in both cerebral regions. In the cerebral cortex, MSG neonatal treatment also decreased the mRNA for GAD67 and protein for GAD65 without significant changes in its corresponding protein and mRNA, respectively. Moreover in the hippocampus, mRNA and protein for GAD65 were increased, whilst GAD67 protein was elevated without significant changes in its mRNA. Clearly these results confirm the GABA cells loss after MSG neonatal treatment in both cerebral regions. As most of the GABAergic markers measured were reduced in the cerebral cortex, this region seems to be more sensitive than hippocampus, where interesting compensatory changes over GAD65 and GAD67 proteins were observed. However, it is possible that others neurotransmission systems are also compensating the GABA-positive cells loss in the cerebral cortex, and that elevations in two main forms of GAD in the hippocampus are not sufficient to maintain the neural excitation threshold for this region.
International journal of developmental neuroscience: the official journal of the International Society for Developmental Neuroscience 01/2009; · 2.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Overactivation of NMDA-Rs may mediate excitotoxic cell death associated with epileptic seizures, and hypoxic-ischemic conditions. We assessed whether repeated subcutaneous administration of l-glutamate to neonatal rats affects the subunit composition of NMDA-Rs. Accordingly, cortical and hippocampal tissue from 14-day-old rats was analyzed by Western blotting and RT-PCR to quantify the protein and mRNA expression of different NMDA-R subunits. In addition, tissue sections were Nissl stained to assess the cell damage in this tissue. Early exposure of neonatal rats to L-glutamate differentially affects the expression of mRNA transcripts for NMDA-R subunits in the cerebral cortex and hippocampus. In the cerebral cortex, a decrease in NR2B subunit mRNA expression was observed, as well as a loss of NR1 and NR2A protein. By contrast, neonatal L-glutamate administration augmented the transcripts encoding the NR1, NR2B, and NR2C subunits in the hippocampal formation. The expression of mRNA encoding the NR2A subunit was not affected by neonatal L-glutamate administration in either of the brain regions examined. This differential expression of NMDA-R subunits following neonatal exposure to L-glutamate may represent an adaptive response of the glutamate receptors to overactivation in order to reduce the effect of high L-glutamate during the early period of life when the animal is more vulnerable to excitotoxicity.
International journal of developmental neuroscience: the official journal of the International Society for Developmental Neuroscience 10/2008; 27(2):197-204. · 2.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pro-inflammatory cytokines TNF-alpha, IL-1beta and IL-6 rises significantly during neuronal damage and activate the signaling p38 MAPK pathway, which is involved in the apoptotic (AP) neuronal death. Systemic administration of glutamate as monosodium salt (MSG) to newborn animals induces neuronal death, however whether neurons die by AP or necrosis through MAPK p38 pathway activation it is unknown. In this study, TNF-alpha, IL-1beta and IL-6 expression levels, AP neuronal death and cellular type that produces TNF-alpha was also identified in the cerebral cortex (CC) and striatum (St) of rats at 8, 10, and 14 days of age after neonatal exposure to MSG. TNF-alpha production and AP neuronal death was significantly increased in the CC at PD8-10, and in the St in all ages studied by excitotoxicity effect induced with MSG. This effect was completely inhibited by SB203580 (p38 inhibitor) in both regions studied. TNF-alpha, IL-1beta and IL-6 RNAm increased after MSG administration, whereas SB203580 did not modify their expression. These data indicates that neuronal death induced by excitotoxicity appears to be mediated through p38 signaling pathway activated by TNF-alpha and their inhibition may have an important neuroprotective role as part of anti-inflammatory therapeutic strategy.
International Journal of Developmental Neuroscience 09/2008; 26(5):487-95. · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sparteine was administered subcutaneously to male neonate rats, and the effects on muscarinic subunit receptor types m-m 4 from different brain regions were studied. A semi-quantitative reverse transcription-polymerase chain reaction and western blot was used to measure expression levels in the cerebral cortex and hippocampus. Sparteine (25 mg/kg body weight, on postnatal days 1 and 3) produced an important reduction of m 1 , m 2 and m 3 of mRNA and protein during the first three weeks of age in those animals under sparteine treatment compared to control group in cerebral cortex and hippocampus. Changes in the expression of the subunit m 4 were increased during the first two weeks of age in those animals under sparteine treatment compared to control group in cerebral cortex and hippocampus. We propose that an early excessive activation of muscarinic receptors could modify subunit expression and its structural composition on postnatal development. This, as part of a compensatory response by an altered neuronal circuit, suggests that these subunits will have an important role in the mechanisms of neuronal death.
[Show abstract][Hide abstract] ABSTRACT: Kainic acid receptor (KA-R) subunits are differentially expressed during brain development, and they modulate both neural growth and survival. High concentrations of glutamate in the brain can induce neuronal injury through these receptors, altering normal development. However, it is unclear whether KAR subunit expression itself is also modified by neonatal exposure to high glutamate. To analyze this, monosodium glutamate (4mg/g of body weight) was subcutaneously administered on postnatal days 1, 3, 5 and 7, and the expression of GluR5, GluR6, KA1 and KA2, as well as [(3)H]-kainic acid (KA-R) binding, was evaluated on postnatal days 14, 21, 30 and 60 in different regions of rat brain. As a result, high levels of GluR5 expression associated with strong [(3)H]-kainic acid binding were observed on postnatal days 30 and 60 in the cerebral cortex of rats exposed to glutamate. Similarly, the changes induced by glutamate administration in the expression of the KA1 and KA2 subunits were paralleled by those of [(3)H]-kainic acid binding in the striatum at postnatal days 21 and 30. In contrast, while KAR subunits were over expressed in the hippocampus, no changes were observed in [(3)H]-kainic acid binding in adult rats that had been exposed to glutamate. Therefore, glutamate modifies both the expression of kainic acid receptor subunits and kainic acid binding in a determined spatial and temporal manner, which may be indicative of a regional susceptibility to glutamate neurotoxicity.
International Journal of Developmental Neuroscience 03/2007; 25(1):53-61. · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sparteine is a quinolizidine alkaloid (QA) produced by Lupine species that has generated much interest due to its anti-hypertensive, anti-pyretic, and anti-inflammatory properties. In the nervous system, sparteine has been shown to display anti-cholinergic and depressive activity, although how sparteine exerts its toxic effects in the brain remains unclear. We have addressed this issue by administering subcutaneous injections of sparteine (25 mg/kg of body weight) to rats on postnatal days 1 and 3, and then examining the expression of the muscarinic acetylcholine receptor (mAChR) subunits m1-m4 in the brains of the neonatal rats 14-60 days later. Administration of sparteine to neonatal rats caused neuronal damage in the cerebral motor cortex accompanied by transient changes in the expression of m1-m4 mAChR subunits as revealed by both RT-PCR and Western blotting. This effect could be prevented by pre-treatment with atropine (10 mg/kg) 1 h prior to the injection of sparteine, suggesting that the cytotoxic activity of sparteine is mediated through mAChRs.
International Journal of Developmental Neuroscience 11/2006; 24(6):401-10. · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The proinflammatory cytokines TNF-alpha, IL-1beta, and IL-6 rise during neuronal damage and activate the apoptotic mitogen-activated protein kinase p38. We studied apoptosis, the levels of TNF-alpha, IL-1beta, and IL-6, and the cell type producing TNF-alpha in rats at 8, 10, and 14 days of age after neonatal exposure to glutamate, which induces neuronal damage. TNF-alpha production was significantly increased by glutamate, but inhibited by SB203580 (a p38 inhibitor). TNF-alpha, IL-1beta, and IL-6 mRNA levels increased, but SB203580 did not modify their expression. Thus, the p38 signaling pathway influences the expression of inflammatory genes and its inhibition may offer anti-inflammatory therapy.
Journal of Neuroimmunology 09/2005; 165(1-2):53-62. · 3.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Monosodium glutamate (MSG) was administered subcutaneously to male neonate rats, and the effects on N-methyl-D-asparatate (NMDA) subunit receptor types NR2C and NR2D from different brain regions were studied. A semi-quantitative reverse transcription-polymerase chain reaction was used to measure NR2C and NR2D expression levels in the cerebral cortex, hippocampus and striatum. MSG treatment (4 mg/g body weight, on postnatal days 1, 3, 5, and 7) produced an important increase of NR2C and NR2D subunit gene expression levels in the hippocampus and striatum of adults rats. No change was observed in the cerebral cortex. We propose that an early excessive activation of glutamate receptors could modify NMDA subunit expression and its structural composition on postnatal development. This, as part of a compensatory response by an altered neuronal circuitry, mainly in the hippocampus and striatum, suggests that the NMDA receptor could be a determinant factor to modulate the dendritic arrangement and the synaptogenesis.