Role of intracellular signaling pathways and their inhibitors in the treatment of inflammation.

Inflammation is not only a defense mechanism of the innate immune system against invaders, but it is also involved in the pathogenesis of many diseases such as atherosclerosis, thrombosis, diabetes, epilepsy, and many neurodegenerative disorders. The World Health Organization (WHO) reports worldwide estimates of people (9.6% in males and 18.0% in females) aged over 60 years, suffering from symptomatic osteoarthritis, and around 339 million suffering from asthma. Other chronic inflammatory diseases, such as ulcerative colitis and Crohn's disease are also highly prevalent. The existing anti-inflammatory agents, both non-steroidal and steroidal, are highly effective; however, their prolonged use is marred by the severity of associated side effects. A holistic approach to ensure patient compliance requires understanding the pathophysiology of inflammation and exploring new targets for drug development. In this regard, various intracellular cell signaling pathways and their signaling molecules have been identified to be associated with inflammation. Therefore, chemical inhibitors of these pathways may be potential candidates for novel anti-inflammatory drug approaches. This review focuses on the anti-inflammatory effect of these inhibitors (for JAK/STAT, MAPK, and mTOR pathways) describing their mechanism of action through literature search, current patents, and molecules under clinical trials.

Molecular mechanisms of brain water transport.

Our brains consist of 80% water, which is continuously shifted between different compartments and cell types during physiological and pathophysiological processes. Disturbances in brain water homeostasis occur with pathologies such as brain oedema and hydrocephalus, in which fluid accumulation leads to elevated intracranial pressure. Targeted pharmacological treatments do not exist for these conditions owing to our incomplete understanding of the molecular mechanisms governing brain water transport. Historically, the transmembrane movement of brain water was assumed to occur as passive movement of water along the osmotic gradient, greatly accelerated by water channels termed aquaporins. Although aquaporins govern the majority of fluid handling in the kidney, they do not suffice to explain the overall brain water movement: either they are not present in the membranes across which water flows or they appear not to be required for the observed flow of water. Notably, brain fluid can be secreted against an osmotic gradient, suggesting that conventional osmotic water flow may not describe all transmembrane fluid transport in the brain. The cotransport of water is an unconventional molecular mechanism that is introduced in this Review as a missing link to bridge the gap in our understanding of cellular and barrier brain water transport.

Dehydroeffusol Pprevents Amyloid ß1-42-mediated Hippocampal Neurodegeneration via Reducing Intracellular Zn2+ Toxicity.

Dehydroeffusol, a phenanthrene isolated from Juncus effusus, is a Chinese medicine. To explore an efficacy of dehydroeffusol administration for prevention and cure of Alzheimer's disease, here we examined the effect of dehydroeffusol on amyloid ß1-42 (Aß1-42)-mediated hippocampal neurodegeneration. Dehydroeffusol (15 mg/kg body weight) was orally administered to mice once a day for 6 days and then human Aß1-42 was injected intracerebroventricularly followed by oral administration for 12 days. Neurodegeneration in the dentate granule cell layer, which was determined 2 weeks after Aß1-42 injection, was rescued by dehydroeffusol administration. Aß staining (uptake) was not reduced in the dentate granule cell layer by pre-administration of dehydroeffusol for 6 days, while increase in intracellular Zn2+ induced with Aß1-42 was reduced, suggesting that pre-administration of dehydroeffusol prior to Aß1-42 injection is effective for Aß1-42-mediated neurodegeneration that was linked with intracellular Zn2+ toxicity. As a matter of fact, pre-administration of dehydroeffusol rescued Aß1-42-mediated neurodegeneration. Interestingly, pre-administration of dehydroeffusol increased synthesis of metallothioneins, intracellular Zn2+-binding proteins, in the dentate granule cell layer, which can capture Zn2+ from Zn-Aß1-42 complexes. The present study indicates that pre-administration of dehydroeffusol protects Aß1-42-mediated neurodegeneration in the hippocampus by reducing intracellular Zn2+ toxicity, which is linked with induced synthesis of metallothioneins. Dehydroeffusol, a novel inducer of metallothioneins, may protect Aß1-42-induced pathogenesis in Alzheimer's disease.

Epigallocatechin-3-gallate mobilizes intracellular Ca2+ in prostate cancer cells through combined Ca2+ entry and Ca2+-induced Ca2+ release.

AIMS: To elucidate the mechanism by which (-)-epigallocatechin-3-gallate (EGCG) mediates intracellular Ca2+ increase in androgen-independent prostate cancer (PCa) cells. MAIN METHODS: Following exposure to different doses of EGCG, viability of DU145 and PC3 PCa cells was evaluated by MTT assay and the intracellular Ca2+ dynamics by the fluorescent Ca2+ chelator Fura-2. The expression of different channels was investigated by qPCR analysis and sulfhydryl bonds by Ellman's assay. KEY FINDINGS: EGCG inhibited DU145 and PC3 proliferation with IC50 = 46 and 56 µM, respectively, and induced dose-dependent peaks of internal Ca2+ that were dependent on extracellular Ca2+. The expression of TRPC4 and TRPC6 channels was revealed by qPCR in PC3 cells, but lack of effect by modulators and blockers ruled out an exclusive role for these, as well as for voltage-dependent T-type Ca2+ channels. Application of dithiothreitol and catalase and sulfhydryl (SH) measurements showed that EGCG-induced Ca2+ rise depends on SH oxidation, while the effect of EGTA, dantrolene, and the PLC inhibitor U73122 suggested that EGCG-induced Ca2+ influx acts as a trigger for Ca2+-induced Ca2+ release, involving both ryanodine and IP3 receptors. Different from EGCG, ATP caused a rapid Ca2+ increase, which was independent of external Ca2+, but sensitive to U73122. SIGNIFICANCE: EGCG induces an internal Ca2+ increase in PCa cells by a multi-step mechanism. As dysregulation of cytosolic Ca2+ is directly linked to apoptosis in PCa cells, these data confirm the possibility of using EGCG as a synergistic adjuvant in combined therapies for recalcitrant malignancies like androgen-independent PCa.

Saikosaponin d causes apoptotic death of cultured neocortical neurons by increasing membrane permeability and elevating intracellular Ca2+ concentration.

Saikosaponins (SSs) are a class of naturally occurring oleanane-type triterpenoid saponins found in Radix bupleuri that has been widely used in traditional Chinese medicine. As the main active principals of Radix bupleuri, SSs have been shown to suppress mouse motor activity, impair learning and memory, and decrease hippocampal neurogenesis. In the present study, we investigated the effect of five SSs (SSa, SSb1, SSb2, SSc, and SSd) on neuronal viability and the underlying mechanisms in cultured murine neocortical neurons. We demonstrate that SSa, SSb1 and SSd produce concentration-dependent apoptotic neuronal death and induce robust increase in intracellular Ca2+ concentration ([Ca2+]i) at low micromolar concentrations with a rank order of SSd > SSa > SSb1, whereas SSb2 and SSc have no detectable effect on both neuronal survival and [Ca2+]i. Mechanistically, SSd-induced elevation in [Ca2+]i is the primary result of enhanced extracellular Ca2+ influx, which likely triggers Ca2+-induced Ca2+ release through ryanodine receptor activation, but not SERCA inhibition. SSd-induced Ca2+ entry occurs through a non-selective mechanism since blockers of major neuronal Ca2+ entry pathways, including L-type Ca2+ channel, NMDA receptor, AMPA receptor, Na+-Ca2+ exchanger, and TRPV1, all failed to attenuate the Ca2+ response to SSd. Further studies demonstrate that SSd increases calcein efflux and induces an inward current in neocortical neurons. Together, these data demonstrate that SSd elevates [Ca2+]i due to its ability to increase membrane permeability, likely by forming pores in the surface of membrane, which leads to massive Ca2+ influx and apoptotic neuronal death in neocortical neurons.

Folic Acid Impairs the Uptake of 5-Methyltetrahydrofolate in Human Umbilical Vascular Endothelial Cells.

BACKGROUND: Adequate folate status supports endothelial structure and function. Folic acid (FA), an oxidized synthetic folate, which is present in the plasma of patients consuming fortified food or FA supplements, may impair cellular uptake of physiological, reduced folates. We studied the effect of FA on uptake of the dominant circulatory folate, 5-methyltetrahydrofolate (5MTHF) in endothelial cells. METHODS AND RESULTS: For short-term effects of FA, primary human umbilical vein endothelial cells (HUVECs) were maintained in growth medium containing 200 nM 5MTHF and preincubated with 20 nM FA 10 minutes before the 5MTHF uptake assessment. For long-term effects, HUVECs were cultured for 3 passages in growth medium containing either 200 nM 5MTHF, or a combination of 100 nM 5MTHF and 100 nM FA. 5MTHF uptake was assessed after exposing cells to 200 nM [C5]-5MTHF, after which intracellular [C5]-5MTHF was quantified using liquid chromatography/tandem mass spectrometry. Acute FA exposure caused a 57% reduction in 5MTHF uptake compared with control conditions (51 ± 12 vs. 22 ± 7 fmol·min·mg protein; P = 0.01). Long-term exposure to FA reduced 5MTHF uptake by 41% (51 ± 12 vs. 30 ± 11 fmol·min·mg protein; P = 0.05) and reduced total cellular 5MTHF levels by 47 ± 21% in HUVEC (P = 0.02). CONCLUSION: Unmetabolized FA, which appears in the plasma after consumption of fortified food or FA supplements, may impair uptake of 5MTHF, the dominant bioactive form of folate, in HUVEC.

Protective effects of Cassia tora leaves in experimental cataract by modulating intracellular communication, membrane co-transporters, energy metabolism and the ubiquitin-proteasome pathway.

CONTEXT: Cataract is the clouding of eye lens which causes impairment in vision and accounts for the leading factor of global blindness. Functional food-based prevention of cataract finds application in vision research because of its availability and easy access to all classes of the society. Cassia tora Linn. (Caesalpinaceae) is an edible plant mentioned in the traditional systems of medicine for whole body health, especially to the eyes. OBJECTIVE: The present study evaluates the potential of ethyl acetate fraction of Cassia tora leaves (ECT) on experimental cataract. MATERIALS AND METHODS: Cataract was induced by a single subcutaneous injection of sodium selenite (4 µg/g body weight) on 10th day. ECT was supplemented orally from 8th day up to 12th day at a concentration of 5 µg/g body weight and marker parameters were evaluated after 30 days. RESULTS: The production of MPO and the activation of calpain were reduced 52.17% and 36.67% by ECT in lens tissue, respectively. It modulated the energy status by significantly increasing the activity of CCO 1 (55.56%) and ATP production (41.88%). ECT maintained the ionic balance in the lens by reducing the level of sodium (50%) and increasing the level of potassium (42.5%). It also reduced cell junction modifications and preserved a functional ubiquitin-proteasome pathway. DISCUSSION AND CONCLUSION: The results reinforce the growing attention on wild plant food resources for preventive protection against cataract. The data suggest the value of Cassia tora leaves as a functional food for ameliorating cataract pathology.

In vitro neuroprotective potential of lichen metabolite fumarprotocetraric acid via intracellular redox modulation.

The lichen-forming fungi Cetraria islandica has been largely used in folk medicines, and it has recently showed promising in vitro antioxidant effects in glial-like cells. Current work aimed at investigating the neuroprotective potential of its major isolated secondary metabolite: the depsidone fumarprotocetraric acid (FUM). H2O2 was used herein to induce oxidative stress (OS)-mediated cytotoxicity in two models of neurons and astrocytes cells (SH-SY5Y and U373-MG cell lines). We found that a pre-treatment with FUM significantly enhanced cell viability compared to H2O2-treated cells, and we selected the optimal concentrations in each model (1 and 25µg/ml, respectively) for assessing its cytoprotective mechanisms. FUM, which exerted effective peroxyl radical scavenging effect in the chemical oxygen radical antioxidant capacity (ORAC) assay, alleviated the alterations in OS markers provoked by H2O2. It attenuated intracellular ROS formation, lipid peroxidation and GSH depletion. At mitochondrial level, FUM prevented from the dissipation of mitochondrial membrane potential and the increase in mitochondrial calcium, implying a protective role against oxidative damage in mitochondrial membrane. Similarly, FUM pre-treatment diminished H2O2-induced apoptosis, as evidenced by the reduction in caspase-3 activity and expression; inmunoblot analysis also revealed a decrease in Bax and an increase in Bcl-2 proteins levels. Furthermore, FUM up-regulated the expression of the antioxidant enzymes catalase, superoxide dismutase-1, and hemeoxigenase-1. These findings and the activation of Nrf2 binding activity in nuclear extracts suggest a plausible involvement of Nrf2 signaling pathway in the cytoprotection by FUM. In conclusion, FUM emerges as a potential drug candidate in the therapy of OS-related diseases, such as the neurodegenerative disorders.

Antimicrobial Activity of Ferulic Acid Against Cronobacter sakazakii and Possible Mechanism of Action.

Cronobacter sakazakii is an opportunistic pathogen transmitted by food that affects mainly newborns, infants, and immune-compromised adults. In this study, the antibacterial activity of ferulic acid was tested against C. sakazakii strains. Minimum inhibitory concentration of ferulic acid against C. sakazakii strains was determined using the agar dilution method. Changes in intracellular pH, membrane potential and intracellular ATP concentration were measured to elucidate the possible antibacterial mechanism. Moreover, SYTO 9 nucleic acid staining was used to assess the effect of ferulic acid on bacterial membrane integrity. Cell morphology changes were observed under a field emission scanning electron microscope. The minimum inhibitory concentrations of ferulic acid against C. sakazakii strains ranged from 2.5 to 5.0 mg/mL. Addition of ferulic acid exerted an immediate and sustained inhibition of C. sakazakii proliferation. Ferulic acid affected the membrane integrity of C. sakazakii, as evidenced by intracellular ATP concentration decrease. Moreover, reduction of intracellular pH and cell membrane hyperpolarization were detected in C. sakazakii after exposure to ferulic acid. Reduction of green fluorescence indicated the injury of cell membrane. Electronic microscopy confirmed that cell membrane of C. sakazakii was damaged by ferulic acid. Our results demonstrate that ferulic acid has moderate antimicrobial activity against C. sakazakii. It exerts its antimicrobial action partly through causing cell membrane dysfunction and changes in cellular morphology. Considering its antimicrobial properties, together with its well-known nutritional functions, ferulic acid has potential to be developed as a supplement in infant formula or other foods to control C. sakazakii.

Resveratrol induces intracellular Ca(2+) rise via T-type Ca(2+) channels in a mesothelioma cell line.

AIMS: Intracellular calcium (Ca(2+)) is known to play an important role in cancer development and growth. Resveratrol (Res) is a stilbene polyphenol occurring in several plant species and known for various possible beneficial effects, including its ability to inhibit proliferation and to induce apoptosis in cancer cells. This study was designed to determine whether Res affects Ca(2+) signaling in cancer cells. MAIN METHODS: We used the REN human mesothelioma cell line, as an in vitro cancer cell model, and the non-malignant human mesothelial MeT5A cell line, as normal cell model. Cytosolic Ca(2+) concentration was measured by the fluorescent indicator Fura-2. Immunofluorescence, Western blot, and siRNA technique were employed to assess the involvement of T-type Ca(2+) channels. Cell viability was determined by the calcein assay. KEY FINDINGS: REN cells transiently exposed to 1-10µM Res showed increasing peaks of Ca(2+) that were absent in Ca(2+)-free medium and were reduced by non-selective (Ni(2+)), and highly selective (NNC 55-0396) T-type Ca(2+) channels antagonist, and by siRNA knockout of Cav3.2T-type Ca(2+) channel gene. Dose-dependent curve of Res-induced Ca(2+) peaks showed a rightward shift in normal MeT-5A mesothelial cells (EC50=4.9µM) with respect to REN cells (EC50=2.7µM). Moreover, incubation with 3 and 10µM Res for 7days resulted in cell growth inhibition for REN, but not for MeT-5A cells. SIGNIFICANCE: Res induces Ca(2+) influx, possibly mediated through T-type Ca(2+) channels, with significant selectivity towards mesothelioma cells, suggesting a possible use as an adjuvant to chemotherapy drugs for mesothelioma clinical treatment.

Cardioprotective Effect of Ranolazine in the Process of Ischemia-reperfusion in Adult Rat Cardiomyocytes.

INTRODUCTION AND OBJECTIVES: Ranolazine is used as a complementary treatment for angina in symptomatic patients who are inadequately controlled with first-line antianginal therapies. Ranolazine inhibits sodium voltage-dependent channels, suggesting their possible involvement in the reperfusion process by preventing the sodium and calcium overload that occurs during ischemia. In this study, we characterized the effect of ranolazine on calcium homeostasis in isolated adult cardiac myocytes from rats subjected to a simulated ischemia and reperfusion protocol. METHODS: The effects of ranolazine on changes in intracellular calcium concentration were evaluated at different times using field electrostimulation. The study of intracellular calcium was performed using microfluorimetry with the fluorescent indicator, Fura-2, and by confocal microscopy with the indicator, Fluo-3. RESULTS: We found that cardiomyocytes subjected to ischemia-reperfusion showed an increase in the diastolic calcium concentration and a decrease in the amplitude of intracellular calcium transients. The application of ranolazine during ischemia significantly improved intracellular calcium handling, preventing intracellular calcium overload, decreasing the diastolic calcium concentration, increasing the sarcoplasmic reticulum calcium load, and preserving the amplitude of the intracellular calcium transient, which was reflected by successful recovery in the process of excitation-contraction coupling during reperfusion. However, these effects of ranolazine did not occur when it was applied during reperfusion or when applied in both ischemia and reperfusion. CONCLUSIONS: Ranolazine shows beneficial effects in cardiomyocytes exposed to ischemia/reperfusion but only when applied during ischemia. This effect is achieved through its improvement of calcium handling during ischemia.

Metabotropic glutamate receptor 5 couples cellular prion protein to intracellular signalling in Alzheimer's disease.

Alzheimer's disease-related phenotypes in mice can be rescued by blockade of either cellular prion protein or metabotropic glutamate receptor 5. We sought genetic and biochemical evidence that these proteins function cooperatively as an obligate complex in the brain. We show that cellular prion protein associates via transmembrane metabotropic glutamate receptor 5 with the intracellular protein mediators Homer1b/c, calcium/calmodulin-dependent protein kinase II, and the Alzheimer's disease risk gene product protein tyrosine kinase 2 beta. Coupling of cellular prion protein to these intracellular proteins is modified by soluble amyloid-ß oligomers, by mouse brain Alzheimer's disease transgenes or by human Alzheimer's disease pathology. Amyloid-ß oligomer-triggered phosphorylation of intracellular protein mediators and impairment of synaptic plasticity in vitro requires Prnp-Grm5 genetic interaction, being absent in transheterozygous loss-of-function, but present in either single heterozygote. Importantly, genetic coupling between Prnp and Grm5 is also responsible for signalling, for survival and for synapse loss in Alzheimer's disease transgenic model mice. Thus, the interaction between metabotropic glutamate receptor 5 and cellular prion protein has a central role in Alzheimer's disease pathogenesis, and the complex is a potential target for disease-modifying intervention.

Nutritional Strategies to Modulate Intracellular and Extracellular Buffering Capacity During High-Intensity Exercise.

Intramuscular acidosis is a contributing factor to fatigue during high-intensity exercise. Many nutritional strategies aiming to increase intra- and extracellular buffering capacity have been investigated. Among these, supplementation of beta-alanine (~3-6.4 g/day for 4 weeks or longer), the rate-limiting factor to the intramuscular synthesis of carnosine (i.e. an intracellular buffer), has been shown to result in positive effects on exercise performance in which acidosis is a contributing factor to fatigue. Furthermore, sodium bicarbonate, sodium citrate and sodium/calcium lactate supplementation have been employed in an attempt to increase the extracellular buffering capacity. Although all attempts have increased blood bicarbonate concentrations, evidence indicates that sodium bicarbonate (0.3 g/kg body mass) is the most effective in improving high-intensity exercise performance. The evidence supporting the ergogenic effects of sodium citrate and lactate remain weak. These nutritional strategies are not without side effects, as gastrointestinal distress is often associated with the effective doses of sodium bicarbonate, sodium citrate and calcium lactate. Similarly, paresthesia (i.e. tingling sensation of the skin) is currently the only known side effect associated with beta-alanine supplementation, and it is caused by the acute elevation in plasma beta-alanine concentration after a single dose of beta-alanine. Finally, the co-supplementation of beta-alanine and sodium bicarbonate may result in additive ergogenic gains during high-intensity exercise, although studies are required to investigate this combination in a wide range of sports.

Inhibitory effect of plant phenolics on fMLP-induced intracellular calcium rise and chemiluminescence of human polymorphonuclear leukocytes and their chemotactic activity in vitro.

CONTEXT: Polymorphonuclear leukocytes (PMNs) produce oxidants, contributing to systemic oxidative stress. Diets rich in plant polyphenols seem to decrease the risk of oxidative stress-induced disorders including cardiovascular disease. OBJECTIVE: The objective of this study was to examine the in vitro effect of each of the 14 polyphenols on PMNs chemotaxis, intracellular calcium response, oxidants production. MATERIALS AND METHODS: Blood samples and PMNs suspensions were obtained from 60 healthy non-smoking donors and incubated with a selected polyphenol (0.5-10 µM) or a control solvent. We assessed resting and fMLP-dependent changes of intracellular calcium concentration ([Ca(2+)]i) in PMNs with the Fura-2AM method and measured fMLP-induced luminol enhanced whole blood chemiluminescence (fMLP-LBCL). Polyphenol chemoattractant activity for PMNs was tested with Boyden chambers. RESULTS: Polyphenols had no effect on resting [Ca(2+)]i. Unaffected by other compounds, fMLP-dependent increase of [Ca(2+)]i was inhibited by quercetin and catechol (5 µM) by 32 ± 14 and 12 ± 10% (p < 0.04), respectively. Seven of the 14 tested substances (5 µM) influenced fMLP-LBCL by decreasing it. Catechol, quercetin, and gallic acid acted most potently reducing fMLP-LBCL by 49 ± 5, 42 ± 15, and 28 ± 18% (p < 0.05), respectively. 3,4-Dihydroxyhydrocinnamic, 3,4-dihydroxyphenylacetic, 4-hydroxybenzoic acid, and catechin (5 µM) revealed distinct (p < 0.02) chemoattractant activity with a chemotactic index of 1.9 ± 0.8, 1.8 ± 0.7, 1.6 ± 0.6, 1.4 ± 0.2, respectively. CONCLUSION AND DISCUSSION: Catechol, quercetin, and gallic acid at concentrations commensurate in human plasma strongly suppressed the oxidative response of PMNs. Regarding quercetin and catechol, this could result from an inhibition of [Ca(2+)]i response.

3,5,4'-Trihydroxy-6,7,3'-trimethoxyflavone protects astrocytes against oxidative stress via interference with cell signaling and by reducing the levels of intracellular reactive oxygen species.

Oxidative stress is tightly involved in various neurodegenerative diseases such as Parkinson's and Alzheimer's diseases, and conditions such as ischemia. Astrocytes, the most abundant glial cells in the brain, protect neurons from reactive oxygen species (ROS) and provide them with trophic support. Therefore, any damage to astrocytes will affect neuronal survival. In a previous study we have demonstrated that an extract prepared from the plant Achillea fragrantissima (Af) prevented the oxidative stress-induced death of astrocytes and attenuated the intracellular accumulation of ROS in astrocytes under oxidative stress. In the present study, using activity guided fractionation, we have purified from this plant the active compound, determined to be a flavonoid named 3,5,4'-trihydroxy-6,7,3'-trimethoxyflavone (TTF). The effects of TTF in any biological system have not been studied previously, and this is the first study to characterize the anti-oxidant and protective effects of this compound in the context of neurodegenerative diseases. Using primary cultures of astrocytes we have found that TTF prevented the hydrogen peroxide (H2O2)-induced death of astrocytes, and attenuated the intracellular accumulation of ROS following treatment of these cells with H2O2 or the peroxyl radicals generating molecule 2,2'-Azobis(amidinopropane) (ABAP). TTF also interfered with cell signaling events and inhibited the phosphorylation of the signaling proteins stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), extracellular signal regulated kinase (ERK 1/2) and mitogen activated protein kinase kinase (MEK1) and the phosphorylation of the transcription factor cyclic AMP response element-binding protein (CREB). The mechanism of the protective effect of TTF against H2O2-cytotoxicity could not be attributed to a direct H2O2 scavenging but rather to the scavenging of free radicals as was shown in cell free systems. Thus, TTF might be a therapeutic candidate for the prevention/treatment of neurodegenerative diseases where oxidative stress is part of the pathophysiology.

Intracellular Aß pathology and early cognitive impairments in a transgenic rat overexpressing human amyloid precursor protein: a multidimensional study.

Numerous studies have implicated the abnormal accumulation of intraneuronal amyloid-ß (Aß) as an important contributor to Alzheimer's disease (AD) pathology, capable of triggering neuroinflammation, tau hyperphosphorylation and cognitive deficits. However, the occurrence and pathological relevance of intracellular Aß remain a matter of controversial debate. In this study, we have used a multidimensional approach including high-magnification and super-resolution microscopy, cerebro-spinal fluid (CSF) mass spectrometry analysis and ELISA to investigate the Aß pathology and its associated cognitive impairments, in a novel transgenic rat model overexpressing human APP. Our microscopy studies with quantitative co-localization analysis revealed the presence of intraneuronal Aß in transgenic rats, with an immunological signal that was clearly distinguished from that of the amyloid precursor protein (APP) and its C-terminal fragments (CTFs). The early intraneuronal pathology was accompanied by a significant elevation of soluble Aß42 peptides that paralleled the presence and progression of early cognitive deficits, several months prior to amyloid plaque deposition. Aß38, Aß39, Aß40 and Aß42 peptides were detected in the rat CSF by MALDI-MS analysis even at the plaque-free stages; suggesting that a combination of intracellular and soluble extracellular Aß may be responsible for impairing cognition at early time points. Taken together, our results demonstrate that the intraneuronal development of AD-like amyloid pathology includes a mixture of molecular species (Aß, APP and CTFs) of which a considerable component is Aß; and that the early presence of these species within neurons has deleterious effects in the CNS, even before the development of full-blown AD-like pathology.

Omega-3 fatty acid supplementation changes intracellular phospholipase A2 activity and membrane fatty acid profiles in individuals at ultra-high risk for psychosis.

The identification of an ultra-high risk (UHR) profile for psychosis and a greater understanding of its prodrome have led to increasing interest in early intervention to delay or prevent the onset of psychotic illness. In a randomized placebo-controlled trial, we have identified long-chain ω-3 (ω-3) polyunsaturated fatty acid (PUFA) supplementation as potentially useful, as it reduced the rate of transition to psychosis by 22.6% 1 year after baseline in a cohort of 81 young people at UHR of transition to psychosis. However, the mechanisms whereby the ω-3 PUFAs might be neuroprotective are incompletely understood. Here, we report on the effects of ω-3 PUFA supplementation on intracellular phospholipase A2 (inPLA(2)) activity, the main enzymes regulating phospholipid metabolism, as well as on peripheral membrane lipid profiles in the individuals who participated in this randomized placebo-controlled trial. Patients were studied cross-sectionally (n=80) and longitudinally (n=65) before and after a 12-week intervention with 1.2 g per day ω-3 PUFAs or placebo, followed by a 40-week observation period to establish the rates of transition to psychosis. We investigated inPLA(2) and erythrocyte membrane FAs in the treatment groups (ω-3 PUFAs vs placebo) and the outcome groups (psychotic vs non-psychotic). The levels of membrane ω-3 and ω-6 PUFAs and inPLA(2) were significantly related. Some of the significant associations (that is, long-chain ω-6 PUFAs, arachidonic acid) with inPLA(2) activity were in opposite directions in individuals who did (a positive correlation) and who did not (a negative correlation) transition to psychosis. Supplementation with ω-3 PUFA resulted in a significant decrease in inPLA(2) activity. We conclude that ω-3 PUFA supplementation may act by normalizing inPLA(2) activity and δ-6-desaturase-mediated metabolism of ω-3 and ω-6 PUFAs, suggesting their role in neuroprogression of psychosis.

Elevated [Ca2+]i levels occur with decreased calpain activity in aged fibroblasts and their reversal by energy-rich compounds: new paradigm for Alzheimer's disease prevention.

Elevated intracellular Ca2+ levels in the aging brain are widely thought to hyperactivate Ca2+ signaling and Ca2+-dependent enzymes, leading to neuronal death through an excitatory mechanism in Alzheimer's disease (AD). This "Ca2+ overload" hypothesis has been questioned by our theoretical analyses. To better understand the relationship between the "level" and functionality of Ca2+ in aging, in this study we simultaneously measured intracellular Ca2+ transients and calpain activity in cultured human fibroblasts. We found that Ca2+ transitions elicited by bradykinin were indeed overstayed or elevated in levels in old cells but, remarkably, calpain activity was decreased compared to young cells. Also, treating young cells with the energy inhibitor rotenone or with H2O2 recapitulated the Ca2+ overstay and calpain inactivation found in old cells. More importantly, treating old cells with high-energy compounds such as phosphoenol pyruvate or phosphocreatine, which boosted cellular ATP content, reduced the Ca2+ overstay and re-activated calpain. Moreover, Ca2+ levels and calpain activity were dramatically raised in the dying cells killed by detergent. Finally, Ca2+ oscillations induced by low dose of bradykinin in old cells exhibited lower spike frequency, but higher overall levels. Collectively, these results suggest that (a) Ca2+ overload in old cells arises from an inefficient Ca2+ handling system compromised by age-related energy depletion and oxidative stress; and (b) despite elevated levels, the functionality of Ca2+ signaling has diminished in old cells. Thus, the study reinforces the concept that tonic promotion of bioenergetics and Ca2+ signaling function is a reasonable and new paradigm to protect the aging brain cells to prevent AD.

Zinc and zinc transporters in prostate carcinogenesis.

The healthy human prostate accumulates the highest level of zinc of any soft tissue in the body. This unique property is retained in BPH, but is lost in prostatic malignancy, which implicates changes in zinc and its transporters in carcinogenesis. Indeed, zinc concentrations diminish early in the course of prostate carcinogenesis, preceding histopathological changes, and continue to decline during progression toward castration-resistant disease. Numerous studies suggest that increased zinc intake might protect against progression of prostatic malignancy. In spite of increased dietary intake, zinc accumulation might be limited by the diminished expression of zinc uptake transporters, resulting in decreased intratumoural zinc levels. This finding can explain the conflicting results of various epidemiological studies evaluating the role of zinc supplementation on primary and secondary prostate cancer prevention. Overall, more research into the mechanisms of zinc homeostasis are needed to fully understand its impact on prostate carcinogenesis. Only then can the potential of zinc and zinc transport proteins be harnessed in the diagnosis and treatment of men with prostate cancer.

The inhibitory activity of atractylenolide Ш, a sesquiterpenoid, on IgE-mediated mast cell activation and passive cutaneous anaphylaxis (PCA).

ETHNOPHARMACOLOGICAL RELEVANCE: AT Ш, a sesquiterpenoid, is the major component of Atractylodes japonica Koidz that has been used as a traditional oriental medicine. AIM OF THE STUDY: We investigated the anti-allergic activity of AT Ш and its mechanism of action. MATERIALS AND METHODS: The released amount of ß-hexosaminidase in mast cells, a key parameter of degranulation, was measured. Anti-allergic potential of AT Ш was evaluated using passive cutaneous anaphylaxis in vivo. The anti-allergic mechanism of AT Ш was investigated by immunoblotting analysis, RT-PCR and measurement of [Ca(2+)]i in mast cells. RESULTS: AT Ш significantly inhibited IgE/Ag-mediated degranulation with an IC(50) value (36 ± 4 µM) in RBL-2H3 cells without affecting cell viability. It also suppressed IgE/Ag-mediated passive cutaneous anaphylaxis (PCA) response with an ED(50) value (65 ± 41 mg/kg) in vivo. AT Ш suppressed the production of interleukin (IL-4) and tumor necrosis factor (TNF)-alpha mRNAs more potent than the Src-family kinase inhibitor PP2 in RBL-2H3 cells at all concentrations. In order to elucidate the anti-allergic mechanisms of AT Ш in mast cells, we examined the activated levels of signaling molecules. AT Ш inhibited the phosphorylation of Lyn, Fyn, Syk, LAT, PLCγ, Gab2, Akt, p38, and JNK kinases expression. IgE/Ag-mediated [Ca(2+)]i elevation was significantly inhibited by AT Ш. CONCLUSIONS: Our study suggests that AT Ш might be used as a therapeutic agent for allergic diseases.