Joana B. Melo

Involvement of oxidative stress in the enhancement of acetylcholinesterase activity induced by amyloid beta-peptide

Acetylcholinesterase (AChE) activity is increased within and around amyloid plaques, which are present in Alzheimers disease (AD) patients brain. In this study, using cultured retinal cells as a neuronal model, we analyzed the effect of the synthetic peptide Abeta(25-35) on the activity of AChE, the degradation enzyme of acetylcholine, as well as the involvement of oxidative stress in this process. The activity of AChE was increased when retinal cells were incubated with Abeta(25-35) (25 microM, 24 h) and antioxidants such as alpha-tocopherol acetate and nitric oxide synthase (NOS) inhibitors were capable of preventing this effect. Despite Abeta(25-35) did not affect cell membrane integrity, the redox capacity of cells decreased. The incubation with this amyloidogenic peptide led to an increment of reactive oxygen species formation (20%), of lipid peroxidation (65%), and basal intracellular calcium levels (40%). The data obtained show that the enhancement of AChE activity induced by Abeta(25-35) is mediated by oxidative stress, and that vitamin E and NOS inhibitors, by preventing the compromise of the enzyme activity, can have an important role in the maintenance of acetylcholine synaptic levels, thus preventing or improving cognitive and memory functions of AD patients.

Metabolic profiling of human lung cancer tissue by 1H high resolution magic angle spinning (HRMAS) NMR spectroscopy.

This work aims at characterizing the metabolic profile of human lung cancer, to gain new insights into tumor metabolism and to identify possible biomarkers with potential diagnostic value in the future. Paired samples of tumor and noninvolved adjacent tissues from 12 lung tumors have been directly analyzed by (1)H HRMAS NMR (500/600 MHz) enabling, for the first time to our knowledge, the identification of over 50 compounds. The effect of temperature on tissue stability during acquisition time has also been investigated, demonstrating that analysis should be performed within less than two hours at low temperature (277 K), to minimize glycerophosphocholine (GPC) and phosphocholine (PC) conversion to choline and reduce variations in some amino acids. The application of Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) to the standard 1D (1)H spectra resulted in good separation between tumor and control samples, showing that inherently different metabolic signatures characterize the two tissue types. On the basis of spectral integration measurements, lactate, PC, and GPC were found to be elevated in tumors, while glucose, myo-inositol, inosine/adenosine, and acetate were reduced. These results show the valuable potential of HRMAS NMR-metabonomics for investigating the metabolic phenotype of lung cancer.

Analytical Approaches toward Successful Human Cell Metabolome Studies by NMR Spectroscopy

The aim of this work was to investigate the effects of cell handling and storage on cell integrity and (1)H high resolution magic angle spinning (HRMAS) NMR spectra. Three different cell types have been considered (lung tumoral, amniocytes, and MG-63 osteosarcoma cells) in order for sample-dependent effects to be identified. Cell integrity of fresh cells and cells frozen in cryopreservative solution was approximately 70-80%, with the former showing higher membrane degradation, probably enzymatic, as indicated by increased phosphocholine (PC) and/or glycerophosphocholine (GPC). Unprotected freezing (either gradual or snap-freezing) was found to lyse cells completely, similar to mechanical cell lysis. Besides enhanced metabolites visibility, lysed cells showed a different lipid profile compared to intact cells, with increased choline, PC, and GPC and decreased phosphatidylcholine (PTC). Cell lysis has, therefore, a significant effect on cell lipid composition, making handling reproducibility an important issue in lipid analysis. Sample spinning was found to disrupt 5-25% of cells, depending on cell type, and HRMAS was shown to be preferable to solution-state NMR of suspensions or supernatant, giving enhanced information on lipids and comparable resolution for smaller metabolites. Relaxation- and diffusion-edited NMR experiments gave limited information on intact cells, compared to lysed cells. The (1)H HRMAS spectra of the three cell types are compared and discussed.

Galantamine protects against oxidative stress induced by amyloid-beta peptide in cortical neurons

Galantamine is currently used in the treatment of patients with mild‐to‐moderate Alzheimer’s disease (AD). Although its action is mostly directed at the regulation of cholinergic transmission, galantamine can also afford neuroprotection against amyloid‐beta peptide (Aβ), which is involved in AD pathogenesis. In this study, we used cultured rat cortical neurons treated with two forms of Aβ1–40, fresh and previously aged (enriched in fibrils). First, we confirmed that galantamine prevented neurodegeneration induced by both peptide forms in a concentration‐dependent manner. Moreover, we observed that when neurons were co‐incubated with fresh Aβ1–40 plus galantamine, the amount of amyloid aggregates was reduced. As oxidative conditions influence Aβ aggregation, we investigated whether galantamine prevents oxidative stress induced by this peptide. The data show that either fresh or aged Aβ1–40 significantly increased the amount of reactive oxygen species and lipoperoxidation, these effects being prevented by galantamine. In Aβ1–40‐treated neurons, the depletion of reduced glutathione (GSH) seems to be related to the decrease in glutathione peroxidase and glutathione reductase activities. These alterations in the GSH antioxidant system were prevented by galantamine. Overall, these results constitute the first evidence that galantamine can prevent the neuronal oxidative damage induced by Aβ, providing an in vitro basis for the beneficial actions of galantamine in the AD neurodegenerative process.

Genomic characterization of three urinary bladder cancer cell lines: understanding genomic types of urinary bladder cancer.

Several genomic regions are frequently altered and associated with the type, stage and progression of urinary bladder cancer (UBC). We present the characterization of 5637, T24 and HT1376 UBC cell lines by karyotyping, fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH) and multiplex ligation-dependent probe amplification (MLPA) analysis. Some cytogenetic anomalies present in UBC were found in the three cell lines, such as chromosome 20 aneuploidy and the loss of 9p21. Some gene loci losses (e.g. CDKN2A) and gains (e.g. HRAS, BCL2L1 and PTPN1) were coincident across all cell lines. Although some significant heterogeneity and complexity were detected between them, their genomic profiles exhibited a similar pattern to UBC. We suggest that 5637 and HT1376 represent the E2F3/RB1 pathway due to amplification of 6p22.3, concomitant with loss of one copy of RB1 and mutation of the remaining copy. The HT1376 presented a 10q deletion involving PTEN region and no alteration of PIK3CA region which, in combination with the inactivation of TP53, bears more invasive and metastatic properties than 5637. The T24 belongs to the alternative pathway of FGFR3/CCND1 by presenting mutated HRAS and over-represented CCND1. These cell lines cover the more frequent subtypes of UBC and are reliable models that can be used, as a group, in preclinical studies.

Genetic gains and losses in oral squamous cell carcinoma: impact on clinical management

PurposeThe identification of genetic markers associated with oral cancer is considered essential to improve the diagnosis, prognosis, early tumor and relapse detection and, ultimately, to delineate individualized therapeutic approaches. Here, we aimed at identifying such markers.MethodsMultiplex Ligation-dependent Probe Amplification (MLPA) analyses encompassing 133 cancer-related genes were performed on a panel of primary oral tumor samples and its corresponding resection margins (macroscopically tumor-free tissue) allowing, in both types of tissue, the detection of a wide arrange of copy number imbalances on various human chromosomes.ResultsWe found that in tumor tissue, from the 133 cancer-related genes included in this study, those that most frequently exhibited copy number gains were located on chromosomal arms 3q, 6p, 8q, 11q, 16p, 16q, 17p, 17q and 19q, whereas those most frequently exhibiting copy number losses were located on chromosomal arms 2q, 3p, 4q, 5q, 8p, 9p, 11q and 18q. Several imbalances were highlighted, i.e., losses of ERBB4, CTNNB1, NFKB1, IL2, IL12B, TUSC3, CDKN2A, CASP1, and gains of MME, BCL6, VEGF, PTK2, PTP4A3, RNF139, CCND1, FGF3, CTTN, MVP, CDH1, BRCA1, CDKN2D, BAX, as well as exon 4 of TP53. Comparisons between tumor and matched macroscopically tumor-free tissues allowed us to build a logistic regression model to predict the tissue type (benign versus malignant). In this model, the TUSC3 gene showed statistical significance, indicating that loss of this gene may serve as a good indicator of malignancy.ConclusionsOur results point towards relevance of the above mentioned cancer-related genes as putative genetic markers for oral cancer. For practical clinical purposes, these genetic markers should be validated in additional studies.

Potential markers of cisplatin treatment response unveiled by NMR metabolomics of human lung cells.

In this work, (1)H high resolution magic angle spinning (HRMAS) nuclear magnetic resonance (NMR) spectroscopy was used to characterize the variations in the metabolome (small metabolites and mobile lipids) of A549 human lung cells in response to exposure to the alkylating drug cisplatin. Multivariate analysis and signal integration of spectral data were carried out to unveil exposure-induced effects and follow their time course. Parallel and strongly correlated increases in lipids (particularly unsaturated triglycerides) and nucleotide sugars (particularly uridine diphosphate N-acetylglucosamine) were found in cisplatin-treated cells, highlighting these compounds as potential biomarkers of treatment response. Other significant changes upon drug exposure comprised an increase in sorbitol and decreases in niacinamide and several amino acids (glutamine, alanine, lysine, methionine, citrulline, phenylalanine and tyrosine). These results show that in vitro NMR metabolomics is a powerful tool for detecting variations in a range of intracellular compounds upon drug exposure, thus offering the possibility of identifying candidate metabolite markers for in vivo monitoring of tumor responsiveness to treatment.

Genetic imbalances detected by multiplex ligation-dependent probe amplification in a cohort of patients with oral squamous cell carcinoma—the first step towards clinical personalized medicine

Oral tumors are a growing health problem worldwide; thus, it is mandatory to establish genetic markers in order to improve diagnosis and early detection of tumors, control relapses and, ultimately, delineate individualized therapies. This study was the first to evaluate and discuss the clinical applicability of a multiplex ligation-dependent probe amplification (MLPA) probe panel directed to head and neck cancer. Thirty primary oral squamous cell tumors were analyzed using the P428 MLPA probe panel. We detected genetic imbalances in 26 patients and observed a consistent pattern of distribution of genetic alterations in terms of losses and gains for some chromosomes, particularly for chromosomes 3, 8, and 11. Regarding the latter, some specific genes were highlighted due to frequent losses of genetic material—RARB, FHIT, CSMD1, GATA4, and MTUS1—and others due to gains—MCCC1, MYC, WISP1, PTK2, CCND1, FGF4, FADD, and CTTN. We also verified that the gains of MYC and WISP1 genes seem to suggest higher propensity of tumors localized in the floor of the mouth. This study proved the value of this MLPA probe panel for a first-tier analysis of oral tumors. The probemix was developed to include target regions that have been already shown to be of diagnostic/prognostic relevance for oral tumors. Furthermore, this study emphasized several of those specific genetic targets, suggesting its importance to oral tumor development, to predict patients’ outcomes, and also to guide the development of novel molecular therapies.

Prion protein aggregation and neurotoxicity in cortical neurons.

Abstract:  Prion diseases are degenerative disorders of the central nervous system characterized by cerebral protein aggregation and deposition. A cellular glycoprotein, PrPC is converted in an altered isoform, PrPSc, that accumulates in the brain, and is believed to be responsible for the neuronal loss observed in prion diseases. The synthetic peptide PrP106‐126 shares many characteristics with PrPSc and is largely used to explore the toxic mechanisms underlying prion diseases. In this article we analyzed the neurotoxic effects of PrP106‐126 in primary rat brain cortical neurons, correlating these results with the presence of amyloid plaques in cultures. Incubation of cells with PrP106‐126, 25 μM, for 2 days did not significantly decrease neuronal viability, although we have observed an increase of basal intracellular calcium levels, reactive oxygen species (ROS) formation, and lipid peroxidation. The presence of congophylic and thioflavin S‐amyloid‐positive plaques in cortical cultures was only observed after a 5‐day‐treatment period, correlating with a significant decrease of neuronal viability, as assessed by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) leakage. The data obtained support the idea that PrP106‐126 aggregates in vitro and that the aggregation state is important for its neurotoxicity but also suggest that this synthetic peptide, even when is not aggregated invitro, can compromise cell homeostasis.

X-chromosome terminal deletion in a female with premature ovarian failure: Haploinsufficiency of X-linked genes as a possible explanation

BackgroundPremature ovarian failure (POF) has repeatedly been associated to X-chromosome deletions. FMR1 gene premutation alleles carrier women have an increased risk for POF. We intent to determine the cause of POF in a 29 year old female, evaluating both of these situations.MethodsConcomitant analysis of FMR1 gene CGG repeat number and karyotype revealed an X-chromosome terminal deletion. Fluorescence in situ further characterized the breakpoint. A methylation assay for FMR1 gene allowed to determine its methylation status, and hence, the methylation status of the normal X-chromosome.ResultsWe report a POF patient with a 46,X,del(X)(q26) karyotype and with skewed X-chromosome inactivation of the structural abnormal X-chromosome.ConclusionsDespite the hemizygosity of FMR1 gene, the patient does not present Fragile X syndrome features, since the normal X-chromosome is not subject to methylation. The described deletion supports the hypothesis that haploinsufficiency of X-linked genes can be on the basis of POF, and special attention should be paid to X-linked genes in region Xq28 since they escape inactivation and might have a role in this disorder. A full clinical and cytogenetic characterization of all POF cases is important to highlight a pattern and help to understand which genes are crucial for normal ovarian development.