Fluorescent Analysis of the Cell-Selective Alzheimer's Disease Aβ Peptide Surface Membrane Binding: Influence of Membrane Components

We performed a fluorescent analysis of the binding of Aβ to the surface membrane of different types of cells lines such as PC12, GT1-7, and ex vivo neurons. Analyses were performed on sorted cells with membrane bound Aβ Competitive binding between Aβ phosphatidyl serine- (PtdSer-) specific binder annexin V and an anti-PtdSer antibody provided compelling data confirming the involvement of PtdSer as one of the surface membrane signal molecules for Aβ. We found that populations of cells that exhibited high surface membrane binding affinity for Aβ also show higher membrane cholesterol levels compared to cells that did not bind Aβ. This direct relationship was upheld in cholesterol-enriched or cholesterol-depleted cell membranes. We conclude that the initial process for the cell-selective binding by Aβ, to later conversion of elemental Aβ units into larger structures such as fibrils or to the potentially toxic ion channel aggregates, is highly influenced by the membrane content of PtdSer and cholesterol in the cell surface membrane

Aβ ion channels. Prospects for treating Alzheimer's disease with Aβ channel blockers

AbstractThe main pathological features in the Alzheimer’s brain are progressive depositions of amyloid protein plaques among nerve cells, and neurofibrillary tangles within the nerve cells. The major components of plaques are Aβ peptides. Numerous reports have provided evidence that Aβ peptides are cytotoxic and may play a role in the pathogenesis of AD. An increasing number of research reports support the concept that the Aβ–membrane interaction event may be followed by the insertion of Aβ into the membrane in a structural configuration which forms an ion channel. This review summarizes experimental procedures which have been designed to test the hypothesis that the interaction of Aβ with a variety of membranes, both artificial and natural, results in the subsequent formation of Aβ ion channels We describe experiments, by ourselves and others, that support the view that Aβ is cytotoxic largely due to the action of Aβ channels in the cell membrane. The interaction of Aβ with the surface of the cell membrane may results in the activation of a chain of processes that, when large enough, become cytotoxic and induce cell death by apoptosis. Remarkably, the blockage of Aβ ion channels at the surface of the cell absolutely prevents the activation of these processes at different intracellular levels, thereby preserving the life of the cells. As a prospect for therapy for Alzheimer’s disease, our findings at cellular level may be testable on AD animal models to elucidate the potential role and the magnitude of the contribution of the Aβ channels for induction of the disease

Avaliação do impacto dos cruzamentos Bos taurus x Bos indicus e tecnologias post mortem na qualidade gustativa de lombos de novilhos terminados a pasto

This experiment aimed to evaluate the effects of Brahman crossbreeding and postmortem technologies (electrical stimulation and vacuum aging) on eating quality of loins from pasture-finished bulls. Fifty yearling bulls representing five Brahman-influenced types (n = 10 each): Brahman (BRAH), F1-Angus (F1ANG), F1-Chianina (F1CHI), F1-Romosinuano (F1ROM), and F1-Simmental (F1SIM) were supplemented on pasture until reaching a desirable conformation at a suitable live weight of ca. 480 kg. All carcasses were classified as “Bullocks” according to U.S. standards. Carcass’s right sides were subjected to high-voltage electrical stimulation (ES) while the left sides were not stimulated (NOES). Longissimus lumborum (LL) steaks from ES and NOES carcasses were allotted either to the vacuum aging control treatment for 2 d (NOAGING) or 10 d (AGING).  LL steaks were evaluated for Warner-Bratzler shear force (WBSF) and sensory traits by trained panelists. No differences in WBSF, juiciness, or flavor ratings were detected among breed types (P > 0.05). Sensory ratings for tenderness-related traits varied little with breed type (P < 0.05). Steaks from F1ANG received higher ratings for muscle fiber tenderness, overall tenderness, and amount of connective tissue, and differed (P < 0.05) from those of F1ROM and F1SIM which received the lowest ratings. Bullock loins were more responsive to ES+AGING in WBSF reduction and desirable tenderness ratings than other postmortem treatments (P < 0.05) by reaching a greater proportion (72%) of “tender” (WBSF < 40.1 N) steaks than AGING (48%), ES (36%), and NOES-NOAGING (24%) samples (P < 0.01). Tenderness of bullock loin steaks is marginally improved by crossbreeding; therefore, the application of ES+AGING is necessary to ensure a higher proportion of tenderloin steaks.Se evaluaron los efectos de cruzamientos interraciales y tecnologías postmortem sobre la calidad gustativa de lomos de toros terminados a pasto.  Cincuenta toros añosos representando cinco tipos raciales (n = 10 cada uno): Brahman puro (BRAH), F1-Angus (F1ANG), F1-Chianina (F1CHI), F1-Romosinuano (F1ROM) y F1-Simmental (F1SIM) se suplementaron a pastoreo hasta alcanzar una conformación satisfactoria a un peso vivo de ca. 480 kg. Todas las canales clasificaron como “Bullocks” (Toretes) por la norma estadounidense. Los lados derechos de cada canal se sometieron a estimulación eléctrica de alto voltaje (ES) mientras que los lados izquierdos no fueron estimulados (NOES).  Bistés de longissimus lumborum ES y NOES se asignaron a tratamientos de maduración al vacío por 2 d (NOMADURADOS) ó 10 d (MADURADOS). Los bistés se evaluaron para fuerza de corte Warner-Bratzler (FCWB) y rasgos sensoriales calificados por panelistas capacitados. No se detectaron diferencias en valores FCWB, o calificaciones para jugosidad o intensidad de sabor entre tipos raciales (P > 0.05).  Los rasgos relacionados con terneza variaron poco con el tipo racial (P < 0.05). Los bistecs de F1ANG recibieron calificaciones más altas para terneza de fibra muscular, terneza general y cantidad de tejido conectivo, y difirieron (P < 0.05) de los F1ROM y F1SIM, que recibieron las calificaciones más bajas. Los bistés ES+MADURADOS tuvieron menor FCWB y mayores calificaciones para rasgos asociados con la terneza que los ES ó los MADURADOS (P < 0.05) alcanzando una mayor proporción (72%) de "bistés tiernos" (FCWB < 40.1 N) que la de MADURADOS (48%), ES (36%) y NOES-NOMADURADOS (24%) (P < 0.01). La mejora en terneza de lomos de toretes Brahman mediante cruzamiento con razas taurinas es marginal; por lo tanto, se requiere aplicar el tratamiento combinado de ES y maduración al vacío para asegurar una proporción mayor de bistés tiernos.Foram avaliados os efeitos de cruzamentos interraciais e tecnologias postmortem na qualidade do sabor de lombos de touros terminados a pasto. Cinquenta touros envelhecidos representando cinco tipos de raça (n = 10 cada): Brahman puro (BRAH), F1-Angus (F1ANG), F1-Chianina (F1CHI), F1-Romosinuano (F1ROM) e F1-Simmental (F1SIM) foram suplementados. pastado com uma conformação satisfatória com um peso vivo de ca. 480kg Toda as carcaças classificadas como “Boi” (Toretes) pela norma americana. Os lados direitos de cada canal foram submetidos à estimulação elétrica de alta voltagem (ES), enquanto os lados esquerdos não foram estimulados (NOES). Bifes ES e NOES longissimus lumborum foram submetidos a tratamentos de maturação a vácuo por 2 d (NOMATURED) ou 10 d (MATURED). Os bifes foram avaliados quanto à resistência ao cisalhamento Warner-Bratzler (FCWB) e características sensoriais avaliadas por provadores treinados. Não foram detectadas diferenças nos valores de FCWB, ou classificações de suculência ou intensidade de sabor entre os tipos de raça (P > 0,05). As características relacionadas à ternura variaram pouco com o tipo de raça (P < 0,05). Os bifes F1ANG receberam pontuações mais altas para maciez da fibra muscular, maciez geral e quantidade de tecido conjuntivo, e diferiram (P <0,05) do F1ROM e F1SIM, que receberam as pontuações mais baixas. Os bifes ES + MADUROS apresentaram menor FCWB e maiores classificações para características associadas à maciez do que os bifes ES ou MATURADOS (P < 0,05), atingindo uma proporção maior (72%) de "bifes macios" (FCWB < 40,1 N) do que os bifes MATURADOS (48 %), ES (36%) e NOES-NOMATURADO (24%) (P < 0,01). A melhora na maciez dos lombos de touros Brahman pelo cruzamento com raças tauromáquicas é marginal; portanto, o tratamento combinado de ES e envelhecimento a vácuo é necessário para garantir uma maior proporção de bifes macios

Endocrine and Paracrine Regulation of Endometrial Angiogenesis

The human endometrium is a complex tissue comprised of different cell types, including epithelial, stromal, inflammatory, perivascular, and blood vessel cells. The hormonal receptivity and distribution of these cell populations change during the menstrual cycle. Cyclical endometrial growth is dependent on its ability to regenerate a vascular capillary network, which grows in parallel with the proliferation and differentiation of the endometrial lining. Natural hormonal effects on the endometrium and endocrine manipulation of this tissue, in response to the use of exogenous steroid therapies, can affect endometrial capillary proliferation and function, leading to clinical abnormalities of uterine bleeding. We propose that the regulation of endometrial angiogenesis is mediated indirectly via complex interactions among cell types. Our laboratory has focused on a prototypical member of the angiogenic proteins, vascular endothelial growth factor (VEGF)-A. In this paper we present data demonstrating that VEGF-A expression in normal endometrial epithelial and stromal cells and in Ishikawa adenocarcinoma cells is increased by an ovarian steroid, estradiol. Infiltrating immune cells, particularly polymorphonuclear granulocytes, also are sources of VEGF-A. In inflammatory conditions involving the endometrium (e.g., endometriosis), a proinflammatory cytokine, IL-1Β, can mediate neoangiogenesis by inducing VEGF-A gene transcription. Thus, endometrial vascularization is effected by both endocrine and paracrine pathways.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73496/1/j.1749-6632.2001.tb03795.x.pd

Seladin-1 expression is regulated by promoter methylation in adrenal cancer

<p>Abstract</p> <p>Background</p> <p>Seladin-1 overexpression exerts a protective mechanism against apoptosis. Seladin-1 mRNA is variably expressed in normal human tissues. Adrenal glands show the highest levels of seladin-1 expression, which are significantly reduced in adrenal carcinomas (ACC). Since up to now seladin-1 mutations were not described, we investigated whether promoter methylation could account for the down-regulation of seladin-1 expression in ACC.</p> <p>Methods</p> <p>A methylation sensitive site was identified in the seladin-1 gene. We treated DNA extracted from two ACC cell lines (H295R and SW13) with the demethylating agent 5-Aza-2-deoxycytidine (5-Aza). Furthermore, to evaluate the presence of an epigenetic regulation also 'in vivo', seladin-1 methylation and its mRNA expression were measured in 9 ACC and in 5 normal adrenal glands.</p> <p>Results</p> <p>The treatment of cell lines with 5-Aza induced a significant increase of seladin-1 mRNA expression in H295R (fold increase, F.I. = 1.8; p = 0.02) and SW13 (F.I. = 2.9; p = 0.03). In ACC, methylation density of seladin-1 promoter was higher (2682 ± 686) than in normal adrenal glands (362 ± 97; p = 0.02). Seladin-1 mRNA expression in ACC (1452 ± 196) was significantly lower than in normal adrenal glands (3614 ± 949; p = 0.01).</p> <p>Conclusion</p> <p>On this basis, methylation could be involved in the altered pattern of seladin-1 gene expression in ACC.</p

Protein Folding and Misfolding on Surfaces

Protein folding, misfolding and aggregation, as well as the way misfolded and aggregated proteins affects cell viability are emerging as key themes in molecular and structural biology and in molecular medicine. Recent advances in the knowledge of the biophysical basis of protein folding have led to propose the energy landscape theory which provides a consistent framework to better understand how a protein folds rapidly and efficiently to the compact, biologically active structure. The increased knowledge on protein folding has highlighted its strict relation to protein misfolding and aggregation, either process being in close competition with the other, both relying on the same physicochemical basis. The theory has also provided information to better understand the structural and environmental factors affecting protein folding resulting in protein misfolding and aggregation into ordered or disordered polymeric assemblies. Among these, particular importance is given to the effects of surfaces. The latter, in some cases make possible rapid and efficient protein folding but most often recruit proteins/peptides increasing their local concentration thus favouring misfolding and accelerating the rate of nucleation. It is also emerging that surfaces can modify the path of protein misfolding and aggregation generating oligomers and polymers structurally different from those arising in the bulk solution and endowed with different physical properties and cytotoxicities

Mitochondrial Ca2+ Overload Underlies Aβ Oligomers Neurotoxicity Providing an Unexpected Mechanism of Neuroprotection by NSAIDs

Dysregulation of intracellular Ca2+ homeostasis may underlie amyloid β peptide (Aβ) toxicity in Alzheimer's Disease (AD) but the mechanism is unknown. In search for this mechanism we found that Aβ1–42 oligomers, the assembly state correlating best with cognitive decline in AD, but not Aβ fibrils, induce a massive entry of Ca2+ in neurons and promote mitochondrial Ca2+ overload as shown by bioluminescence imaging of targeted aequorin in individual neurons. Aβ oligomers induce also mitochondrial permeability transition, cytochrome c release, apoptosis and cell death. Mitochondrial depolarization prevents mitochondrial Ca2+ overload, cytochrome c release and cell death. In addition, we found that a series of non-steroidal anti-inflammatory drugs (NSAIDs) including salicylate, sulindac sulfide, indomethacin, ibuprofen and R-flurbiprofen depolarize mitochondria and inhibit mitochondrial Ca2+ overload, cytochrome c release and cell death induced by Aβ oligomers. Our results indicate that i) mitochondrial Ca2+ overload underlies the neurotoxicity induced by Aβ oligomers and ii) inhibition of mitochondrial Ca2+ overload provides a novel mechanism of neuroprotection by NSAIDs against Aβ oligomers and AD