Biomedical subjects

Maria Moreno

Publications and source records attributed to Maria Moreno.

15 recordsLinked to original sources

PubMed2007

Triiodothyronine modulates the expression of aquaporin-8 in rat liver mitochondria.

The recent identification of aquaporin-8 (AQP8), an aquaporin (AQP) channel permeable to water and ammonia, in the inner membrane (IMM) of rat liver mitochondria suggested a role for such AQP in the hydration state and the metabolic function of mitochondria. Since thyroid hormone triiodothyronine (T3) is known to modulate both the shape and the metabolic activities of liver mitochondria, it was interesting to investigate the expression and distribution of AQP8 as well as the osmotic water permeability of the IMM in liver mitochondria from rats in different thyroid states. By semi-quantitative reverse transcriptase (RT)-PCR, when compared with the euthyroid counterpart, the levels of hepatic AQP8 mRNA significantly increased in the hypothyroid state, whereas they were strongly decreased after administration of T3. A similar pattern was seen at the protein level by immunoblotting mitochondrial membranes. The upregulation of mitochondrial AQP8 in the hypothyroid liver was confirmed by immunogold electron microscopy. Stopped-flow light scattering with IMM vesicles showed no significant differences in terms of osmotic water permeability among the IMMs in the various thyroid states. Overall, our data indicate that the T3 modulation of the AQP8 gene is a rapid downregulation of transcription. Modulation of hepatic AQP8 expression may be relevant to the regulation of mitochondrial metabolism by thyroid hormones.

Animals↗

PubMed2006

Sequential changes in the signal transduction responses of skeletal muscle following food deprivation.

Coping with reduced energy sources entails drastic morphological and functional changes in skeletal muscle, but the sequence of events required classification. We found that gastrocnemius muscle from food-deprived rats shows acute rises in peroxisome proliferator activated receptor (PPAR) gamma coactivator (PGC) -1alpha/PPAR delta nuclear protein and myosin heavy chain (MHC) Ib protein, while type I fibers accumulate and the muscle tissue appears redder. AMP levels, phosphorylation of both AMP-activated protein kinase (AMPK) and its downstream target acetyl coenzyme A carboxylase (ACC) are induced within 6 h. Rapidly increased MyoD mRNA levels are followed by an increase in uncoupling protein (UCP) 3 (UCP3) transcription. Increased serum fatty acid levels coincide with increases in mitochondrial UCP3 protein levels and fatty acid oxidation. Accompanying this is a decrease in AMPK phosphorylation, reversible upon nicotinic acid treatment, indicating that fatty acids may modulate this kinase's activity after the metabolic challenges posed by food deprivation.

AMP-Activated Protein Kinases↗

PubMed2006

Fenofibrate activates the biochemical pathways and the de novo expression of genes related to lipid handling and uncoupling protein-3 functions in liver of normal rats.

Fibrates (anti-hyperlipidemic agents) enhance the mRNA expression of uncoupling protein 2 (UCP2) in the liver and that of uncoupling protein 3 (UCP3) in skeletal muscle in standard-diet-fed rats and induce a de novo expression of UCP3 (mRNA and protein) in the liver of high-fat-fed rats. Here, we report that in the liver of normal rats, fenofibrate induces a de novo expression of UCP3 and a 6-fold increase in UCP2 mRNA, whereas UCP2 protein was not detectable. Indeed, we evidenced an ORF in UCP2 exon 2 potentially able to inhibit the expression of the protein. Fenofibrate increases the expression and activity of hepatic enzymes and cofactors involved in lipid handling and UCP3 activity and, as is the case for UCP3, induces other muscle-specific genes (e.g., Carnitine palmitoyl transferase 1b and Ubiquinone biosynthesis protein COQ7 homolog). In addition, we demonstrated that in mitochondria from fenofibrate-treated rats a palmitoyl-carnitine-induced GDP-sensitive uncoupling takes place, involving UCP3 rather than other uncouplers (i.e., UCP2 and Adenine Nucleotide Translocase). Thus, the liver of fenofibrate-treated standard-diet- fed rat is a useful model for investigations of the biochemical functions of UCP3 and allowed us to demonstrate that fenofibrate programs a gene-expression pattern able to modulate lipid handling and UCP3 activation.

Amino Acid Sequence↗

PubMed2006

A proteomics approach to identify protein expression changes in rat liver following administration of 3,5,3'-triiodo-L-thyronine.

We analyzed whole cell protein content of rat liver following T3 administration. Fourteen differentially expressed proteins were unambiguously identified and were involved in substrates and lipid metabolism, energy metabolism, detoxification of cytotoxic products, calcium homeostasis, amino acid catabolism, and the urea cycle. This study represents the first systematic identification of T3-induced changes in liver protein expression profile and provides novel information at the molecular, cellular, and tissue level of T3 action.

Animals↗

PubMed2005

3,5-diiodo-L-thyronine powerfully reduces adiposity in rats by increasing the burning of fats.

The effect of thyroid hormones on metabolism has long supported their potential as drugs to stimulate fat reduction, but the concomitant induction of a thyrotoxic state has greatly limited their use. Recent evidence suggests that 3,5-diiodo-L-thyronine (T2), a naturally occurring iodothyronine, stimulates metabolic rate via mechanisms involving the mitochondrial apparatus. We examined whether this effect would result in reduced energy storage. Here, we show that T2 administration to rats receiving a high-fat diet (HFD) reduces both adiposity and body weight gain without inducing thyrotoxicity. Rats receiving HFD + T2 showed (when compared with rats receiving HFD alone) a 13% lower body weight, a 42% higher liver fatty acid oxidation rate, appoximately 50% less fat mass, a complete disappearance of fat from the liver, and significant reductions in the serum triglyceride and cholesterol levels (-52% and -18%, respectively). Thyroid hormones and thyroid-stimulating hormone (TSH) serum levels were not influenced by T2 administration. The biochemical mechanism underlying the effects of T2 on liver metabolism involves the carnitine palmitoyl-transferase system and mitochondrial uncoupling. If the results hold true for humans, pharmacological administration of T2 might serve to counteract the problems associated with overweight, such as accumulation of lipids in liver and serum, without inducing thyrotoxicity. However, the results reported here do not exclude deleterious effects of T2 on a longer time scale as well as do not show that T2 acts in the same way in humans.

AMP-Activated Protein Kinases↗

PubMed2005

Comparison of six human anti-transglutaminase ELISA-tests in the diagnosis of celiac disease in the Saharawi population.

AIM: Celiac disease (CD) is an enteropathic disorder very prevalent in Saharawi people. Our aim was to investigate the diagnostic accuracy of six human tissue transglutaminase (tTG) based ELISA tests in Saharawi CD patients. METHODS: Fifty-two CD patients and 23 controls were selected from the Saharawi refugee camps in Tinduf. CD patients were divided into two groups according to their anti-endomysium (EmA) status: 41 EmA positive and 11 EmA negative. Sera from patients and controls were tested for human tTG using six commercial ELISA kits. We used receiver operating characteristics (ROC) curves and areas under the curve to compare the diagnostic accuracies of the six assays. RESULTS: In general, there are differences in the sensitivity and specificity of the human tTG ELISA assays used. Diagnostic accuracy of tests was significantly improved by adjusting the cut-off thresholds according to ROC plot analysis; the correction of the cut-off with the employment of the ROC curve analysis modifies the decision limit in more than 50% in five of the six kits evaluated. CONCLUSION: Some of the human tTG ELISAs used in this study have a diagnostic accuracy similar to EmA determination for diagnosis of CD in Saharawi people. However, it is necessary to select the assay with a higher sensitivity and specificity, and recalculate the cut-off threshold using samples from the referral population.

Adolescent↗

PubMed2005

Optimal methods to characterize the G93A mouse model of ALS.

In the present study, we used the SOD1 (G93A) mutant transgenic mice as a model of amyotrophic lateral sclerosis (ALS). This model is widely used as a laboratory tool to study experimental treatments in vivo for ALS to investigate new therapeutic strategies for this neurodegenerative disease. Such studies require the objective quantification of different parameters while mice develop the disease. We have applied a battery of different and specific tests: scoring of motor deficits by a trained observer, weighing, survival measure, hanging wire test, rotarod task and electromyography, most of them commonly used to evaluate G93A animals. We have critically compared these methods, showing the significant influence of gender on the onset of symptoms, and the optimal moment to apply each test. These results should be taken into account in future therapeutic assays on this ALS model.

Age Factors↗

PubMed2004

Interactions of EGFR and caveolin-1 in human glioblastoma cells: evidence that tyrosine phosphorylation regulates EGFR association with caveolae.

Epidermal growth factor receptor (EGFR) amplification and type III mutation (EGFRvIII), associated with constitutive tyrosine kinase activation and high malignancy, are commonly observed in glioblastoma tumors. The association of EGFR and EGFRvIII with caveolins was investigated in human glioblastoma cell lines, U87MG and U87MG-EGFRvIII. Caveolin-1 expression, determined by RT-PCR, real-time quantitative PCR and Western blot, was upregulated in glioblastoma cell lines (two-fold) and tumors (20-300-fold) compared to primary human astrocytes and nonmalignant brain tissue, respectively. U87MG-EGFRvIII expressed higher levels of caveolin-1 than U87MG. In contrast, the expression of caveolin-2 and -3 were downregulated in glioblastoma cells compared to astrocytes. A colocalization of EGFR, but not of EGFRvIII, with lipid rafts and caveolin-1 was observed by immunocytochemistry. Association of EGFR and EGFRvIII with caveolae, assessed in vitro by binding to caveolin scaffolding domain peptides and in vivo by immunocolocalization studies in cells and caveolae-enriched cellular fraction, was phosphorylation-dependent: ligand-induced phosphorylation of EGFR resulted in dissociation of EGFR from caveolae. In contrast, inhibition of the EGFRvIII constitutive tyrosine phosphorylation by AG1478 increased association of EGFRvIII with caveolin-1. AG1478 also increased caveolin-1 expression and reduced glioblastoma cell growth in a semi-solid agar. The evidence suggests that the phosphorylation-regulated sequestration of EGFR in caveolae may be involved in arresting constitutive or ligand-induced signaling through EGFR responsible for glial cell transformation.

Caveolin 1↗

PubMed2004

An invertebrate histocompatibility complex.

We have developed defined genetic lines of the hydroid Hydractinia symbiolongicarpus and confirmed earlier results showing that allorecognition is controlled by a single chromosomal region within these lines. In a large backcross population, we detected recombinants that display a fusibility phenotype distinct from typical fusion and rejection. We show that this transitory fusion phenotype segregates in a fashion expected of a single Mendelian trait, establishing that the chromosomal interval contains at least two genes that interact to determine fusibility. Using bulked segregant analysis, we have identified amplified fragment length polymorphisms (AFLP) cosegregating with fusibility, used these markers to independently confirm linkage of the two loci, and constructed a 3.4-cM map of an invertebrate histocompatibility complex.

Alleles↗

PubMed2003

Combined cDNA array/RT-PCR analysis of gene expression profile in rat gastrocnemius muscle: relation to its adaptive function in energy metabolism during fasting.

We evaluated the effects of fasting on the gene expression profile in rat gastrocnemius muscle using a combined cDNA array and RT-PCR approach. Of the 1176 distinct rat genes analyzed on the cDNA array, 114 were up-regulated more than twofold in response to fasting, including all 17 genes related to lipid metabolism present on the membranes and all 10 analyzed components of the proteasome machinery. Only 7 genes were down-regulated more than twofold. On the basis of our analysis of genes on the cDNA array plus the data from our RT-PCR assays, the metabolic adaptations shown by rat gastrocnemius muscle during fasting are reflected by i) increased transcription both of myosin heavy chain (MHC) Ib (associated with type I fibers) and of at least three factors involved in the shift toward type I fibers [p27kip1, muscle LIM protein (MLP), cystein rich protein-2], of which one (MLP) has been shown to enhance the activity of MyoD, which would explain the known increase in the expression of skeletal muscle uncoupling protein-3 (UCP3); ii) increased lipoprotein lipase (LPL) expression, known to trigger UCP3 transcription, which tends, together with the first point, to underline the suggested role of UCP3 in mitochondrial lipid handling (the variations under the first point and this one have not been observed in mice, indicating a species-specific regulation of these mechanisms); iii) reduced expression of the muscle-specific coenzyme Q (CoQ)7 gene, which is necessary for mitochondrial CoQ synthesis, together with an increased expression of mitochondrial adenylate kinase 3, which inactivates the resident key enzyme for CoQ synthesis, 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), the mRNA level for which fell during fasting; and iv) increased transcription of components of the proteasomal pathways involved in protein degradation/turnover.

Animals↗

PubMed2003

A vascular endothelial growth factor high affinity receptor 1-specific peptide with antiangiogenic activity identified using a phage display peptide library.

Vascular endothelial growth factor (VEGF) is known to play a predominant role in tumor angiogenesis and metastasis formation that is mediated by its interactions with two tyrosine kinase receptors, VEGFRI (Flt-1) and VEGFRII (KDR). Inhibition of VEGF-dependent events in tumor tissues is known to enhance apoptosis and to suppress tumor growth. A novel peptide, SP5.2, which selectively binds Flt-1 and inhibits a broad range of VEGF-mediated events, was identified using a phage-display library screening. The fluorescein-labeled SP5.2 specifically bound to VEGF-stimulated primary human cerebral endothelial cells (HCECs), whereas non-stimulated HCECs, as well as human neuroblastoma cells (ShyY) did not show any interaction with the peptide. SP5.2 prevented proliferation of cultured primary human umbilical vein endothelial cells induced by recombinant human VEGF165 with an IC50 of 5 microm. SP5.2 was also shown to antagonize VEGF- and PLGF-induced, but not basic fibroblast growth factor-induced proliferation of HCECs. In contrast to "scrambled" peptide, SP5.2 was also found to selectively inhibit VEGF-stimulated migration of HCECs. The in vitro analysis of antiangiogenic activity of SP5.2 using a capillary-like tube formation assay showed that VEGF-induced angiogenesis of HCECs grown on Matrigel was completely inhibited in the presence of 10 microm SP5.2. Further studies demonstrated that SP5.2 prevented VEGF-induced permeability increase in HCECs monolayers. To explore whether SP5.2 can be used as a targeting agent, chemical and recombinant conjugates of SP5.2 with reporter proteins (peroxidase and beta-galactosidase) were produced. The resulting products showed significant increases (200-fold for SP5.2-beta-gal and 400-fold for SP5.2-peroxidase) in binding affinity to recombinant Flt-1 compared with the original synthetic SP5.2, suggesting that conjugate with therapeutic activity in nanomolar range could potentially be developed based on SP5.2 structure.

Amino Acid Sequence↗

PubMed2003

Fasting, lipid metabolism, and triiodothyronine in rat gastrocnemius muscle: interrelated roles of uncoupling protein 3, mitochondrial thioesterase, and coenzyme Q.

We investigated the role of uncoupling protein 3 (UCP3) during fasting and examined the effect of triiodothyronine (T3) administration in such a condition. The possible involvement of mitochondrial thioesterase (MTE I) and the role of putative cofactors, such as coenzyme Q (CoQ), was also examined. Here, we report that fasting induced a more than twofold elevation in the expression and activity of MTE I, and an increase in UCP3 expression, without any associated uncoupling activity. Administration of T3 to fasting rats further up-regulated UCP3 as well as MTE I expression, markedly enhanced MTE I enzyme activity and prevented the impairment of the uncoupling activity of UCP3 normally seen during fasting. Indeed, T3-treatment induced an UCP3-dependent decrease in mitochondrial membrane potential, which was abolished by the addition of either GDP or superoxide dismutase (SOD). T3 administration also prevented the marked decrease of CoQ levels observed in fasting rats and this provides evidence that also, in vivo, CoQ represents an essential cofactor for the UCP3-mediated uncoupling. The data also show that MTE I and UCP3 are likely involved in the same biochemical mechanism and that UCP3 postulated functions, such as lipid handling and uncoupling, are not mutually exclusive but may coexist in vivo.

Animals↗

PubMed2003

Comparison of the disinfection efficacy of chlorine-based products for inactivation of viral indicators and pathogenic bacteria in produce wash water.

Outbreaks of pathogenic bacteria infections associated with the consumption of fresh produce has occurred with increased frequency in recent years. This study was undertaken to determine the efficacy of three commonly used disinfectants in packing-houses of Culiacan, Mexico (sodium hypochlorite [NaOCl], trichlor-s-triazinetrione [TST] and thrichlormelamine [TCM]) for inactivation of viral indicators and pathogenic bacteria inoculated onto produce wash water. Each microbial challenge consisted of 2 L of water containing approximately 8 log10 bacterial CFU ml(-1), and 8 log10 viral PFU ml(-1) treated with 100 and 300 mg l(-1) of total chlorine with modified turbidity. Water samples were taken after 2 min of contact with chlorine-based products and assayed for the particular microorganisms. TST and NaOCl were found to effectively reduce for bacterial pathogens and viral indicators 8 log10 and 7 log10, respectively (alpha=0.05). The highest inactivation rate was observed when the turbidity was low and the disinfectant was applied at 300 mg l(-1). TCM did not show effective results when compared with the TST and NaOCl (P<0.05). These findings suggest that turbidity created by the organic and inorganic material present in the water tanks carried by the fresh produce may affect the efficacy of the chlorine-based products.

Chlorine Compounds↗

PubMed2003

Identification of 3,5-diiodo-L-thyronine-binding proteins in rat liver cytosol by photoaffinity labeling.

In this study, we obtained evidence for the presence of cytosolic-binding proteins for 3,5-diiodo-L-thyronine (3,5-T(2)). UV irradiation of rat liver cytosol with [(125)I]3,5-T(2) resulted in specific covalent attachment of (125)I to three polypeptides with apparent molecular masses of 86, 66, and 38 kDa. The photoaffinity labeling of all three proteins was strongly inhibited (by about 90%) when the reaction was carried out in the presence of a 10-fold excess of unlabeled 3,5-T(2) or T(3). However, whereas inhibition by 3,5-T(2) was nicotinamide adenine dinucleotide phosphate reduced (NADPH) independent, T(3) inhibited only in the presence of NADPH. The 38-kDa protein, which showed the greatest affinity for 3,5-T(2), was partially purified by preparative fast-performance liquid chromatography. Its binding activity was optimal at pH 7.4, stable between 0 and 37 C, and already maximal after 5-10 min of incubation. The finding that a 38-kDa cytosolic-binding protein binds 3,5-T(2) in the absence of NADPH, but T(3) only in a NADPH-dependent manner, suggests that it may serve to regulate intracellular T(3)/3,5-T(2) translocation in a way that depends on the nicotinamide adenine dinucleotide phosphate/NADPH ratio.

Animals↗

PubMed2002

Are the effects of T3 on resting metabolic rate in euthyroid rats entirely caused by T3 itself?

Because we previously reported that T3 and 3,5-diiodo-L-thyronine (3,5-T2) both increase resting metabolic rate (RMR), 3,5-T2 could be another thyroidal regulator of energy metabolism. This effect of 3,5-T2 is evident in rats made hypothyroid by propylthiouracil and iopanoic acid, not in normal euthyroid (N) rats. Possibly, under euthyroid conditions, active 3,5-T2 may need to be formed intracellularly from a precursor such as T3. We tested this hypothesis by giving a single injection of T3 to N rats and comparing the time course of the variations in RMR with those of the changes in the serum and hepatic levels of 3,5-T2. Acute injection had an evident effect on RMR, 25 h earlier, in N rats than in rats made hypothyroid by propylthiouracil and iopanoic acid, maximal values (+40%) being reached in the former at 24-26 h. In N rats, the simultaneous injection of actinomycin D with the T3 inhibited the late part of the effect (after 24 h) more strongly than the early part (14-24 h). In serum and liver, 3,5-T2 levels were increased significantly at 12-24 h after T3 injection into N rats, a time at which RMR was rising rapidly to peak. These results seem to indicate that when T3 is injected into N animals, not all the effects on RMR are attributable to T3 itself, the early effect presumably being largely because of its in vivo deiodination to 3,5-T2. Because the effects of T3 and 3,5-T2 are additive, in N rats, the two iodothyronines probably cooperate in vivo to determine the total metabolic rate.

Animals↗