Amandio Vieira

Control of EGF Receptor Signaling by Clathrin-Mediated Endocytosis

Epidermal growth factor receptor (EGFR) signaling was analyzed in mammalian cells conditionally defective for receptor-mediated endocytosis. EGF-dependent cell proliferation was enhanced in endocytosis-defective cells. However, early EGF-dependent signaling events were not uniformly up-regulated. A subset of signal transducers required the normal endocytic trafficking of EGFR for full activation. Thus, endocytic trafficking of activated EGFR plays a critical role not only in attenuating EGFR signaling but also in establishing and controlling specific signaling pathways.

Flavonoid intake and disability-adjusted life years due to Alzheimer’s and related dementias: a population-based study involving twenty-three developed countries

OBJECTIVE Dietary flavonoids and their metabolites may have neuroprotective effects against age-associated neurodegenerative disorders such as Alzheimers and related dementias (dementia). There is a lack of population studies, however, on correlations between flavonoid intake and dementia. The main objective of the present study was to analyse such a relationship at a large-scale population level. DESIGN Based on global data (FAO, WHO), databases were generated for: (i) flavonoid content of foods; (ii) per capita national dietary intakes of flavonoids and other dietary factors; and (iii) disability-adjusted life years - a measure of burden and death - due to dementia. Five major flavonoid subclasses were examined. To minimize influences due to accuracy and reliability of the disease source data, twenty-three developed countries were selected after statistical evaluation. RESULTS Flavonols and combined flavonoids (all five combined) intakes were the only two parameters with significant (P < 0.05) negative dementia correlations. Multiple linear regression models confirmed this relationship, and excluded confounding from some other dietary and non-dietary factors. Similar analyses with non-dementia, neurological/psychiatric diseases did not yield significant correlations. CONCLUSIONS At a global level, and in the context of different genetic backgrounds, our results suggest that higher consumption of dietary flavonoids, especially flavonols, is associated with lower population rates of dementia in these countries.

An assessment of dietary flavonoid intake in the UK and Ireland.

Accurate estimates of flavonoid intake are important for public health studies and potential policies related to these phytochemicals. As an alternative to studies involving population samples and individual food consumption surveys, the international FAO Food Balance Sheets (FBS) were used in the current study to estimate flavonoid consumption among the populations of the UK and Republic of Ireland. A supplemented USDA database was prepared for flavonoid analyses of the foods reported in the FBS. Twenty-three flavonoids from five groups (anthocyanidins, flavonols, flavanols, flavanones, and flavones) were analyzed. Estimated per-capita daily flavonoid intake (all five groups) was 182 mg and 177 mg for the UK and Ireland, respectively. In both cases, anthocyanidins and flavanols accounted for about 65% of total consumption. Combined intake of flavones, flavanones, and flavonols was 60 mg/day in the UK and 69 mg/day in Ireland. These flavonoid intake values are compared with those previously reported for the UK and other countries. Overall, these novel results contribute to establishing accurate reference points for national flavonoid intakes.

Comparative antioxidant activities and synergism of resveratrol and oxyresveratrol

Resveratrol (1) and oxyresveratrol (2) are phytoalexins with antioxidant activities (AAs) and proposed effects against several pathological processes. The main objective of this study was to provide a novel, comparative assessment of their AAs, and to test for potential synergism in their combined activities, or in combination with another phytochemical antioxidant, curcumin (3). The phytochemicals were tested at 10 µM total concentrations in a heme-based assay that involved, as the final step, quantification of tetramethyl-phenylene-diamine oxidation. Significant AAs were observed for both 1 and 2, 27–33% inhibition of oxidation (p < 0.05 relative to non-phytochemical control). The combination of 1 and 2 in the same assay (5 µM each) suggested a moderate synergistic effect of about 10% (41% inhibition of oxidation by 1/2 under the same conditions as for 1 and 2 separately). Combinations of 1/3 and 2/3 were also synergistic, but 1/3 had a two-fold greater AA (p < 0.05) than 2/3 (or 1/2). Our results indicate that (i) 1 and 2 are effective antioxidants in the assay, (ii) in combination, their AAs can synergise, and (iii) in relation to 2, 1 has a much greater synergistic potential with 3. The latter suggests different synergy mechanisms of the curcuminoid with each of the two stilbene phytoalexins.

Oxidative stress disrupts internalization and endocytic trafficking of transferrin in a human malignant keratinocyte line.

Oxidative stress is involved in epidermal cell pathology. One potential mechanism for this toxicity that has previously not been explored in epidermal cells involves modulation of endocytic trafficking and the implications that such modulation can have for altered cell function. The effects of oxidative stress on endocytic trafficking are not well understood, particularly relating to how general or cell-type specific such effects may be. With induction of oxidative stress by hydrogen peroxide, for example, both impaired and enhanced cell-surface binding and endocytic trafficking have been reported for transferrin (Tf), a circulatory iron-carrier protein. The objective of the current study was to characterize the effect of oxidative stress on internalization and endocytic trafficking of Tf in an epidermoid cell line (A431). Evidence is presented for a significant dose-dependent impairment of cellular Tf internalization after treatment with hydrogen peroxide over a wide range of concentrations from 0.06 to 5.8 mM. Scatchard analysis of binding revealed that peroxide treatments resulted in a large decrease, more than fourfold, in the number of cell-surace Tf-binding sites (Bmax) but little change in the dissociation constant (Kd). With respect to endocytic trafficking of Tf, evidence is presented that transport of internalized transferrin back out of the cell (i.e., Tf recycling) is significantly impaired as a result of oxidative stress at all the peroxide concentrations tested. The oxidative stress-dependent changes in endocytic trafficking in these malignant human keratinocytes are compared with those reported for other cell types.

DNA methylation, riboswitches, and transcription factor activity: fundamental mechanisms of gene–nutrient interactions involving vitamins

Nutrient–gene interactions occur with a variety of nutrients including some minerals, vitamins, polyunsaturated fatty acids and other lipids. Fundamental molecular mechanisms that underlie many of the effects of nutrients on gene expression are presented herein. Two of the mechanisms described influence gene transcription: DNA methylation and transcription factor activation. Another mechanism, riboswitching, can regulate gene expression at different levels, for example, at the mRNA translation level. The first two mechanisms are widely distributed across animal phyla. Riboswitches are documented primarily in more primitive organisms, but may prove to be of wider relevance. Riboswitches are known for several vitamins; those involving thiamine are presented here. The role of folates and retinoids in DNA methylation and transcriptional factor (nuclear retinoid receptor) activities, respectively, is presented in the context of cell proliferation and differentiation, and related physiological or pathological effects during embryogenesis and cancer.

Transthyretin aggregates induce production of reactive nitrogen species.

Background and Objective: Misfolded and aggregated transthyretins (agTTR) contribute to neurodegenerative amyloid diseases such as familial amyloid polyneuropathy and senile systemic amyloidosis. The neurotoxicity mechanisms of agTTR, however, are not well understood. In the current study, the possible contribution of reactive nitrogen species (RNS) to such mechanisms was investigated by examining agTTR-mediated changes in cellular RNS levels. Methods and Results: The production of RNS was assessed through nitrate and nitrite assays in two human cell lines after exposure to agTTR (2.4 µm pre-aggregation concentration). In both epidermoid (A431) and schwannoma (sNF94.3) cell lines, agTTR induced significant increases in RNS (p < 0.05 relative to the same concentration of normal TTR, or no-TTR controls). Redox modulators such as apocynin (1-(4-hydroxy-3-methoxy-phenyl)ethanone) and l-NMMA (NG-monomethyl-l-arginine) were tested for their effects on RNS production. These modulators decreased RNS production in both cell lines; although the effects of l-NMMA were statistically significant only in the schwannoma cells. Moreover, cells treated with agTTR exhibited decreases in metabolic activity relative to TTR- or non-TTR-treated cells (p < 0.05) as assessed by reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Conclusion: The results provide novel evidence for involvement of RNS in pro-oxidative effects of agTTR in two different human cell lines, and show that agTTR can induce more generalized changes in cellular metabolic activity.

Comparative 17β-estradiol response and lipoprotein interactions of an avian apolipoprotein

Apolipoprotein D (apo D), a member of the lipocalin protein family, has been identified and cloned in several mammalian species; its physiological functions, however, remain poorly understood. As with other lipocalins, apo D can bind small hydrophobic ligands. Lipids and hormones, such as cholesterol, arachidonic acid, and progesterone can bind to apo D; but the physiological significance of these interactions is not clear. We previously reported the existence of an avian (Gallus domesticus) apo D-like protein, and indicated a possible role for it in reproduction. This report provides a further comparative characterization of this avian protein. Evidence is presented that the putative avian apo D, like some (e.g., human) but not other (e.g., rat) mammalian apo Ds, preferentially associates with high density lipoproteins (HDL) in the circulation. These results confirm the apolipoprotein nature of the avian apo D-like protein, and indicate that it has conserved the HDL-interaction property of some mammalian apo Ds. The response of circulatory levels of the avian protein to 17beta-estradiol treatment is also examined. Large estrogen-dependent increases are known to occur in the circulatory levels of some avian apolipoproteins, such as apo B and vitellogenins, that represent major yolk precursors and nutrient sources for the embryo. Although the avian apo D-like protein is also a known yolk precursor, the minor estrogen-dependent increase observed for this apolipoprotein (less than 7% that of apo B) distinguishes it from the major yolk-precursor apolipoproteins. The response of the avian apo D-like protein to 17beta-estradiol is more like that of other yolk precursor proteins that transport regulatory molecules such as vitamin A and thyroid hormones.

Pro-oxidative effects of aggregated transthyretin in human Schwannoma cells.

Neurotoxicity mechanisms of amyloidogenic polypeptides such as transthyretin (TTR) are not well understood. Misfolded and aggregated TTRs (agTTR) lead to age-related diseases such as senile systemic amyloidosis and familial amyloid polyneuropathy (FAP). Among other clinical manifestations in TTR amyloidic disease, peripheral nerve tissue, including Schwann cell, degeneration has been observed. In this study, we examined potential toxic effects of agTTR in human Schwannoma cells (sNF94.3 peripheral nerve sheath line). Cells were treated with agTTR (2.4μM pre-aggregation concentration) or, as controls, normal, soluble TTR (2.4μM) or no-TTR treatment, and then analyzed for different pro-oxidant and anti-oxidant markers: hydrogen peroxide (H2O2), catalase (CAT), glutathione (GSH), and more generalized cellular antioxidant capacity. In the latter case, cytosolic fractions were prepared after agTTR (or control) treatments and analyzed in oxidation assays. Relative to treatment with normal soluble TTR, cells treated with agTTR increase their release of H2O2. Residual CAT activity is decreased after agTTR treatment. The Schwannoma cells also exhibit significantly lower levels of GSH after agTTR treatment (p<0.05, relative to controls). More generally, cytosols from agTTR-treated cells exhibited a lower capacity to prevent oxidation relative to those from control cells (TTR-treated, or non-TTR-treated). These results suggest that agTTR (a) stimulates production of reactive oxygen species, (b) leads to lower levels of endogenous antioxidants, and (c) decreases overall cellular antioxidant capacity, in Schwannoma cells.

Comparative Phenolic Contents and Antioxidant Activities of Myristica and Pimenta Extracts

Antioxidant activities of two commercial spices, Myristica fragrans (HOUTT, nutmeg) and Pimenta dioica (L., allspice), were analyzed for the first time in a hemin-enhanced oxidation (HET) assay. Hemin is a potentially cytotoxic factor that can act as an efficient prooxidant. Relative activities of the two spices were compared in HET and other assays. When standardized for total phenolic content (mg gallic acid equivalents, GAE), ethanolic and aqueous Myristica extracts had 4- and 10-fold greater activity relative to Pimenta (p < 0.05). Myristica also had a 3-fold greater aqueous:ethanolic activity ratio compared to Pimenta. Our results (a) provide novel evidence for potent Myristica antioxidants, especially water- soluble ones, in HET assays, and (b) provide a basis for further testing of Myristica phytochemicals in heme-related pathological models. Most other reported protocols also indicate a higher Myristica potency relative to Pimenta, with standardized relative antioxidant activity variations up to 27-fold. Such variation, as well as the distinct reactive species implicated in some pathologies, emphasize the importance of comparing multiple assays to evaluate antioxidant activities.