Phenolic Compounds from Tropea Red Onion as Dietary Agents for Protection against Heavy Metals Toxicity.

The present study aims to highlight the cell protective effect of Tropea red onion (TRO) hydroalcoholic extract and some of its components against "non-essential" heavy metals. For this purpose, the cytoprotective roles of cyanidin, cyanidin-3-O-glucoside and quercetin against Cd, Hg and Pb and of TRO extract against Hg and Pb have been investigated, and data are reported here. To the best of our knowledge, this is the first detailed evaluation of the protective effect against cell damage induced by "non-essential" heavy metals through the simultaneous administration of cyanidin, cyanidin-3-O-glucoside and quercetin with CdCl2, HgCl2 or PbCl2 and the TRO extract against HgCl2 and PbCl2. Present data are also compared with our previous results from the TRO extract against Cd. The antioxidant capacity of the extract was also determined by the ferric reducing antioxidant power (FRAP) and the bovine brain peroxidation assay. Both of the assays indicated a good antioxidant capacity of the extract. Cell viability and the impact on necrotic cell death were examined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test and lactate dehydrogenase (LDH) release assay. After 24 h of exposure, Caco-2 cell viability decreased by approximately 50% at 0.25 µM for Cd, Hg and Pb and, after 72 h, the ranking order of "non-essential" heavy metal toxicity on cell viability was PbCl2 > CdCl2 > HgCl2. Cell viability was assessed by treating the cells with the biomolecules at doses of 25, 50 and 100 µg/mL for 24 and 72 h. The same analysis was carried out on Caco-2 cells treated with combinations of TRO extract, cyanidin, cyanidin-3-O-glucoside, or quercetin and "non-essential" heavy metals. Treatments with the bioactive metabolites did not significantly improve cell viability. The identical treatment of Caco-2 cells produced instead LDH release, suggesting a decrease in cell viability. Consistently with the finding that TRO extract showed a good antioxidant activity, we suggest that its higher cytotoxicity, compared to that of the individual assayed phytochemicals, may be derived by the combined antioxidant and chelating properties of all the molecules present in the extract. Therefore, from all the acquired experimental evidence, it appears that the TRO extract may be a better promising protective agent against the toxic effect of Cd, Hg and Pb compared to its bioactive metabolites.

Evaluation of the Potential Protective Effect of Ellagic Acid against Heavy Metal (Cadmium, Mercury, and Lead) Toxicity in SH-SY5Y Neuroblastoma Cells.

Ellagic acid (EA), a polyphenolic constituent of plant origin, has been thoroughly investigated for its hypothesised pharmacological properties among which antioxidant and neuroprotective activities are included. The present study was designed to explore whether EA could attenuate heavy metal (cadmium, mercury, and lead)-induced neurotoxicity in SH-SY5Y cells, which were utilized as a model system for brain cells. MTT and LDH assays were performed to examine the viability of the SH-SY5Y cells after exposure to Cd, Hg, and Pb (either individually or in combination with EA) as well as the effects of necrotic cell death, respectively. Furthermore, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), a cell-based assay, was performed to determine whether EA could protect SH-SY5Y from heavy metal-induced oxidative stress. Results allowed us to assess the capability of EA to enhance the number of viable SH-SY5Y cells after exposure to heavy metal toxicity. Pre-treatment with EA showed a considerable, concentration-dependent, cytoprotective effect, particularly against Cd2+-induced toxicity. This effect was confirmed through the reduction of LDH release after the simultaneous cell treatment with Cd2+ and EA compared with Cd2+-treated cells. Furthermore, a significant, concentration-dependent decrease in reactive oxygen species (ROS) production, induced by H2O2 or heavy metals, was observed in the same model. Overall, the obtained results provide further insight into the protective role of EA against heavy metal-induced neurotoxicity and oxidative stress, thus indicating the potential beneficial effects of the consumption of EA-rich foods. However, to confirm its effects, well-designed human randomized controlled trials are needed to fill the existing gap between experimental and clinical research.

Potential Protective Effects of Spirulina (Spirulina platensis) against In Vitro Toxicity Induced by Heavy Metals (Cadmium, Mercury, and Lead) on SH-SY5Y Neuroblastoma Cells.

Spirulina, a filamentous microalga, is used all over the world as a nutraceutical dietary supplement. Recent studies have focused on examining its chelating activity and antioxidant properties, especially as a candidate for protection against neurotoxicity caused by heavy metals. The MTT test and LDH assay were used to examine the viability of the SH-SY5Y cells for 24, 48, and 72 h, to Cd, Hg, and Pb, individually or in combination with Spirulina, and the effects of necrotic cell death. In comparison to the control group, the viability of SH-SY5Y cells decreased after 24 h of exposure, with Cd being more toxic than Hg and Pb being less lethal. The effects of heavy metal toxicity on cell survival were ranked in order after 72 h under identical experimental circumstances as follows: Hg, Pb, and Cd. The viability of the cells was then tested after being exposed to Spirulina at doses of 5 at 50 (%v/v) for 24, 48, and 72 h, respectively. SH-SY5Y cells that had been treated with mixtures of heavy metals and Spirulina underwent the same assay. Cell viability is considerably increased by using Spirulina treatments at the prescribed periods and doses. Instead, the same procedure, when applied to SH-SY5Y cells, caused the release of LDH, which is consistent with the reduction in cell viability. We demonstrated for the first time, considering all the available data, that Spirulina 5, 25, and 50 (%v/v) enhanced the number of viable SH-SY5Y cells utilized as a model system for brain cells. Overall, the data from the present study provide a first insight into the promising positive role of Spirulina against the potentially toxic effects of metals.

The Effect of Calcium Ions on hIAPP Channel Activity: Possible Implications in T2DM.

The calcium ion (Ca2+) has been linked to type 2 diabetes mellitus (T2DM), although the role of Ca2+ in this disorder is the subject of intense investigation. Serum Ca2+ dyshomeostasis is associated with the development of insulin resistance, reduced insulin sensitivity, and impaired glucose tolerance. However, the molecular mechanisms involving Ca2+ ions in pancreatic ß-cell loss and subsequently in T2DM remain poorly understood. Implicated in the decline in ß-cell functions are aggregates of human islet amyloid polypeptide (hIAPP), a small peptide secreted by ß-cells that shows a strong tendency to self-aggregate into ß-sheet-rich aggregates that evolve toward the formation of amyloid deposits and mature fibrils. The soluble oligomers of hIAPP can permeabilize the cell membrane by interacting with bilayer lipids. Our study aimed to evaluate the effect of Ca2+ on the ability of the peptide to incorporate and form ion channels in zwitterionic planar lipid membranes (PLMs) composed of palmitoyl-oleoyl-phosphatidylcholine (POPC) and on the aggregation process of hIAPP molecules in solution. Our results may help to clarify the link between Ca2+ ions, hIAPP peptide, and consequently the pathophysiology of T2DM.

Interaction of Quercetin, Cyanidin, and Their O-Glucosides with Planar Lipid Models: Implications for Their Biological Effects.

Flavonoids are specialized metabolites produced by plants, as free aglycones or as glycosylated derivatives, which are particularly endowed with a variety of beneficial health properties. The antioxidant, anti-inflammatory, antimicrobial, anticancer, antifungal, antiviral, anti-Alzheimer's, anti-obesity, antidiabetic, and antihypertensive effects of flavonoids are now known. These bioactive phytochemicals have been shown to act on different molecular targets in cells including the plasma membrane. Due to their polyhydroxylated structure, lipophilicity, and planar conformation, they can either bind at the bilayer interface or interact with the hydrophobic fatty acid tails of the membrane. The interaction of quercetin, cyanidin, and their O-glucosides with planar lipid membranes (PLMs) similar in composition to those of the intestine was monitored using an electrophysiological approach. The obtained results show that the tested flavonoids interact with PLM and form conductive units. The modality of interaction with the lipids of the bilayer and the alteration of the biophysical parameters of PLMs induced by the tested substances provided information on their location in the membrane, helping to elucidate the mechanism of action which underlies some pharmacological properties of flavonoids. To our knowledge, the interaction of quercetin, cyanidin, and their O-glucosides with PLM surrogates of the intestinal membrane has never been previously monitored.

Antioxidant activity and protective effect of the outer scales hydroalcoholic extract of Allium cepa L. var. Tropea on toxicity damage induced by Cadmium in Caco-2 cells.

The characterization of bioactive metabolites and the protective effects against Cadmium (Cd) of the hydroalcoholic extract from Allium cepa var. Tropea (Tropea red onion) in human Caco-2 colon adenocarcinoma cells and in vitro antioxidant effects were investigated. Tropea red onion extract showed high levels of bioactive compounds and a strong activity as radical scavenger and inhibitor of lipid peroxidation. The most abundant specialized metabolites were quercetin derivatives. In addition, the extract inhibited NO release in a dose-dependent manner. Exposure of Cd treated human Caco-2 cells to the Tropea red onion extract resulted in a higher cytoprotection with a significant falloff of cells damage produced by Cd. The effect of Tropea red onion evaluated with the MTT assay and LDH test, was time-dependent and more evident after 24h of treatment. Caco-2 cells treated with the Tropea red onion extract and CdCl2 at 24h showed a significant cytoprotection at concentrations equal to 50 µg/mL + 25 µM and 100 µg/mL + 25 µM while at 72h at a concentration of 25 µg/mL + 25 µM. We observed with MTT and LDH assays that treatment of cells with Tropea red onion extract at 24h was able to significantly prevent Cd-induced cytotoxicity alone.

Evidence of the different effect of mercury and cadmium on the hIAPP aggregation process.

hIAPP is a hormone consisting of 37 aminoacids that shows a strong tendency to self-assemble into ß-sheet-rich aggregates, which evolve to form insoluble aggregates that seem to be associated with ß-cell degeneration in Type 2 Diabetes Mellitus. Numerous factors, intrinsic and extrinsic to the peptide molecule, appear to influence the hIAPP aggregation process. Different metal ions are able to interact with the hIAPP molecule, modulating its secondary structure and subsequently the peptide's capacity to aggregate. In this study, the effect of Hg2+ and Cd2+ on the hIAPP aggregation process was evaluated using direct and indirect methods. The kinetics and morphology of amyloid aggregate formation were respectively evaluated with Thioflavin T assays and electron microscopy, while the ability of the peptide to incorporate into POPC PLMs and form ion channels was monitored by single-channel current measurements. Hg2+ and Cd2+ each seem to modulate the peptide's ability to aggregate in a different way, suggesting a different mechanism of hIAPP toxicity.

Levels of Mercury, Methylmercury and Selenium in Fish: Insights into Children Food Safety.

Total mercury (THg), methylmercury (MeHg), and selenium (Se) concentrations were measured in various commercially important fish species. The benefit-risk binomial associated with these chemicals was assessed in children through the probability of exceeding the provisional tolerable weekly intakes (PTWIs) of the contaminants and the Se recommended dietary allowance (RDA). The Se:Hg molar ratios, selenium health benefit values (HBVSe), and monthly consumption rate limits (CRmm) for each species were also calculated. THg and Se were analyzed by atomic absorption spectrophotometer (Shimadzu, Milan, Italy), while MeHg was determined by Trace Ultra gas chromatograph connected with a PolarisQ MS (Thermo Fisher Scientific, Waltham, MA, USA). None of the analyzed fish had Hg levels above the European Community regulatory limits, while most large predators had MeHg levels over the threshold concentration set by US EPA. The estimated weekly intakes of THg and MeHg exceeded in many cases the PTWIs and the Se estimated daily intakes were provided from 0.71% to 2.75% of the RDA. Se:Hg molar ratios above 1 and positive HBVSe index suggested that Se in fish could be enough to alleviate the potential toxic effect of Hg. However, high-risk groups as children should consume fish in moderation because a large consumption pattern, especially of swordfish and tunas, might be of concern for health.

Study of Resveratrol's Interaction with Planar Lipid Models: Insights into Its Location in Lipid Bilayers.

Resveratrol, a polyphenolic molecule found in edible fruits and vegetables, shows a wide range of beneficial effects on human health, including anti-microbial, anti-inflammatory, anti-cancer, and anti-aging properties. Due to its poor water solubility and high liposome-water partition coefficient, the biomembrane seems to be the main target of resveratrol, although the mode of interaction with membrane lipids and its location within the cell membrane are still unclear. In this study, using electrophysiological measurements, we study the interaction of resveratrol with planar lipid membranes (PLMs) of different composition. We found that resveratrol incorporates into palmitoyl-oleoyl-phosphatidylcholine (POPC) and POPC:Ch PLMs and forms conductive units unlike those found in dioleoyl-phosphatidylserine (DOPS):dioleoyl-phosphatidylethanolamine (DOPE) PLMs. The variation of the biophysical parameters of PLMs in the presence of resveratrol provides information on its location within a lipid double layer, thus contributing to an understanding of its mechanism of action.

Trace Metals in Pork Meat Products Marketed in Italy: Occurrence and Health Risk Characterization.

This study provides valuable information on the levels of various trace metals (Pb, Cd, Hg, Zn, Cu, Cr) in meat products (baked ham, raw ham, mortadella, cured sausage, würstel, salami) from South Italy and calculates potential health risk toxicity associated with their consumption for the total population and for children. In the samples studied metal concentrations are within the permissible legal limits (Cd: 0.01-0.03 µg g-1 w.w., Hg: 0.01-0.02 µg g-1 w.w., Zn: 5.71-7.32 µg g-1 w.w., Cu: 1.08-1.21 µg g-1 w.w., Cr: 0.15-0.23 µg g-1 w.w.), except for Pb (Pb: 0.22-0.38 µg g-1 w.w.). The estimated intake values are within the provisional tolerable daily intake limits for toxic metals and recommended daily intake values for essential metals in both tested groups. The noncarcinogenic risk values of the individual metals indicate that there is no health risk, but their combined effects might constitute a potential risk for children. Furthermore, the cumulative cancer risk of all samples studied exceeds the recommended threshold risk limit (> 10-4) in both total population and children, indicating a risk of potential health problems for consumers especially for children, who are more vulnerable to toxic metal exposure.

Evidence of cadmium and mercury involvement in the Aß42 aggregation process.

Aß42 is a small peptide formed from 42 aminoacids that presents a great propensity to aggregate until it forms fibrils. Aß42 aggregation and fibrillation are very complex processes whose molecular mechanisms seem to depend on characteristics intrinsic to the peptide molecule, as well as extrinsic factors. Peptide concentration, mean pH and several substances, including metal ions, are principal extrinsic factors for the oligomerization process. Different metals affect the aggregation of the Aß42 molecule, and their toxicity favours the misfolding and aggregation of the peptide. In this study, we evaluate the effect of different concentrations of Cd2+ and Hg2+ on the Aß42 peptide in solution by different methods. The toxicity of Aß42 was evaluated with the MTT assay, while the aggregation process was monitored by single-channel measurements, electrophoresis and western blot. Cd2+ and Hg2+ seem to favour the formation of high-molecular-weight aggregates, to decrease ion channel turnover inside the membrane and to significantly increase Aß42 toxicity.

Concentration-dependent effects of mercury and lead on Aß42: possible implications for Alzheimer's disease.

Mercury (Hg) and lead (Pb) are known to be toxic non-radioactive elements, with well-described neurotoxicology. Much evidence supports the implication of metals as potential risk cofactors in Alzheimer's disease (AD). Although the action mechanism of the two metals remains unclear, Hg and Pb toxicity in AD could depend on their ability to favour misfolding and aggregation of amyloid beta proteins (Aßs) that seem to have toxic properties, particularly in their aggregated state. In our study, we evaluated the effect of Hg and Pb both on the Aß42 ion channel incorporated in a planar lipid membrane made up of phosphatidylcholine containing 30% cholesterol and on the secondary structure of Aß42 in an aqueous environment. The effects of Hg and Pb on the Aß42 peptide were observed for its channel incorporated into a membrane as well as for the peptide in solution. A decreasing Aß42 channel frequency and the formation of large and amorphous aggregates in solution that are prone to precipitate were both dependent on metal concentration. These experimental data suggest that Hg and Pb interact directly with Aßs, strengthening the hypothesis that the two metals may be a risk factor in AD.

Effect of calcium ions on human calcitonin. Possible implications for bone resorption by osteoclasts.

Calcium ions (Ca(2+)) are indispensable for life and are involved in important physiological actions, which makes maintaining a constant level of blood Ca(2+) essential. Ca(2+) is mainly stored in bones which serve as a reservoir and its homeostasis is modulated by various hormones. Human calcitonin (hCt) is a small peptide hormone that exerts its physiological effect on Ca(2+) metabolism by means of osteoclast-mediated bone resorption inhibition. Most of these actions are mediated through peptide/receptor interaction that acts via a second messenger. However, in vitro studies have shown that hCt can interact with membrane lipids to form ion channels in membrane models. This ability is due to the peptide's secondary structure and aggregation state, that can be modulated by different molecules. In our study, we evaluated the effect of Ca(2+), at different concentrations, both on the hCt ion channel incorporated into a planar lipid membrane made up of phosphatidylcholine containing 15% phosphatidylglycerol and on the secondary structure of hCt in an aqueous environment. Ca(2+) is able to interact with the hCt peptide by acting on the channel incorporated into the membrane as well as on the peptide in solution, both by increasing hCt channel frequency and in solution promoting α-helix formation, that counteracts the fibrillating process. These experimental observations, suggesting that hCt senses Ca(2+) concentration variations, strengthen the hypothesis that channel formation represents an extra source of Ca(2+) entry into osteoclasts in addition to the well-known interaction of the monomer with the specific receptor.

Heavy metals toxicity: effect of cadmium ions on amyloid beta protein 1-42. Possible implications for Alzheimer's disease.

Cadmium (Cd) is an environmental contaminant, highly toxic to humans. This biologically non-essential element accumulates in the body, especially in the kidney, liver, lung and brain and can induce several toxic effects, depending on the concentration and the exposure time. Cd has been linked to Alzheimer's disease (AD) as a probable risk factor, as it shows higher concentrations in brain tissues of AD patients than in healthy people, its implication in the formation of neurofibrillary tangles and in the aggregation process of amyloid beta peptides (AßPs). AßPs seem to have toxic properties, particularly in their aggregated state; insoluble AßP forms, such as small and large aggregates, protofibrils and fibrils, appear to be implicated in the pathogenesis of AD. In our study, we have evaluated the effect of Cd, at different concentrations, both on the AßP1-42 ion channel incorporated in a planar lipid membrane made up of phosphatidylcholine containing 30 % cholesterol and on the secondary structure of AßP1-42 in aqueous environment. Cadmium is able to interact with the AßP1-42 peptide by acting on the channel incorporated into the membrane as well as on the peptide in solution, both decreasing AßP1-42 channel frequency and in solution forming large and amorphous aggregates prone to precipitate. These experimental observations suggesting a toxic role for Cd strengthen the hypothesis that Cd may interact directly with AßPs and may be a risk factor in AD.

AßP1-42 incorporation and channel formation in planar lipid membranes: the role of cholesterol and its oxidation products.

Amyloid beta peptide (AßP) is a natural peptide, normally released into the cerebrospinal fluid (CSF), that plays a key role in Alzheimer's disease. The conversion of the peptide from a native soluble form to a non-native and often insoluble form, such as small and large aggregates, protofibrils and fibrils of AßP appears to be implicated in the pathogenesis of AD. Although the molecular mechanisms of AßP neurotoxicity are not fully understood, a large body of data suggests that the primary target of amyloid peptides is the cell membrane of neurons, that may modulate the structural and functional conversion of AßP into assemblies involved in pathological processes. In our study, we provide a systematic investigation of AßP1-42's ability to incorporate and form channel-like events in membranes of different lipid composition and focus on cholesterol and its oxidation products. We propose that cholesterol and its oxidation products can be considered neuroprotective factors because a) by favouring AßP1-42 insertion into membranes, the fibrillation/clearance balance shifts toward clearance; b) by shifting channel selectivity toward anions, the membrane potential is moved far from the threshold of membrane excitability, thus decreasing the influx of calcium into the cell.

Choline modulation of the aß p1-40 channel reconstituted into a model lipid membrane.

Nicotinic acetylcholine receptors (AChRs), implicated in memory and learning, in subjects affected by Alzheimer's disease result altered. Stimulation of α7-nAChRs inhibits amyloid plaques and increases ACh release. ß-amyloid peptide (AßP) forms ion channels in the cell and model phospholipid membranes that are retained responsible in Alzheimer disease. We tested if choline, precursor of ACh, could affect the AßP1-40 channels in oxidized cholesterol (OxCh) and in palmitoyl-oleoyl-phosphatidylcholine (POPC):Ch lipid bilayers. Choline concentrations of 5 × 10(-11) M-1.5 × 10(-8) M added to the cis- or trans-side of membrane quickly increased AßP1-40 ion channel frequency (events/min) and ion conductance in OxCh membranes, but not in POPC:Ch membranes. Circular Dichroism (CD) spectroscopy shows that after 24 and 48 hours of incubation with AßP1-40, choline stabilizes the random coil conformation of the peptide, making it less prone to fibrillate. These actions seem to be specific in that ACh is ineffective either in solution or on AßP1-40 channel incorporated into PLMs.

Kissper, a kiwi fruit peptide with channel-like activity: structural and functional features.

Kissper is a 39-residue peptide isolated from kiwi fruit (Actinidia deliciosa). Its primary structure, elucidated by direct protein sequencing, is identical to the N-terminal region of kiwellin, a recently reported kiwi fruit allergenic protein, suggesting that kissper derives from the in vivo processing of kiwellin. The peptide does not show high sequence identity with any other polypeptide of known function. However, it displays a pattern of cysteines similar, but not identical, to those observed in some plant and animal proteins, including toxins involved in defence mechanisms. A number of these proteins are also active on mammalian cells. Functional characterization of kissper showed pH-dependent and voltage-gated pore-forming activity, together with anion selectivity and channeling in model synthetic PLMs, made up of POPC and of DOPS:DOPE:POPC. A 2DNMR analysis indicates that in aqueous solution kissper has only short regions of regular secondary structure, without any evident similarity with other bioactive peptides. Comparative analysis of the structural and functional features suggests that kissper is a member of a new class of pore-forming peptides with potential effects on human health.

Frog intestinal sac as an in vitro method for the assessment of intestinal permeability in humans: Application to carrier transported drugs.

The aim of this study was to investigate the presence of pharmaceutically relevant drug transporters in frog intestine which has been proposed as model for intestinal permeability screening assays of passively absorbed drugs in humans [Trapani, G., Franco, M., Trapani, A., Lopedota, A., Latrofa, A., Gallucci, E., Micelli, S., Liso, G., 2004. Frog intestinal sac: a new in vitro method for the assessment of intestinal permeability. J. Pharm. Sci. 93, 2909-2919]. The expression of transporters in frog intestine was supported by the following observations: (i) the involvement of purine nucleobase transport system was deduced by inhibition of acyclovir transport in the presence of adenine; (ii) baclofen or l-dopa transport was inhibited by the digitalis steroid ouabain and it may be related to the Na(+) electrochemical potential difference, presumably involving amino acid transporters; (iii) the presence of proton-dependent peptide transporters was argued evaluating the effect of the pH change (from pH 5.9 to pH 7.4) on the transport of glutathione; (iv) the possible expression in the frog intestine of an efflux system distinct from P-glycoprotein (Pgp) in the benzylpenicillin transport was deduced using a glucose enriched frog Ringer with or without the known Pgp inhibitor verapamil; (v) the contribution of Pgp-mediated efflux system in determining the frog intestinal absorption of drugs was supported by the specific inhibition of cimetidine or nadolol transport in the presence of verapamil. These results indicate that pharmaceutically relevant drug transporters should be also expressed in frog intestine. In this work, an attempt was also made to compare the measured P(app) values in the frog intestinal model for the aforementioned series of actively/effluxed transported drugs in humans to the corresponding literature values for the fraction absorbed. The P(app) values used in these comparisons were obtained at high concentrations of drugs at which probably saturation of the carrier occurs. Interestingly, it was found that drugs that are completely absorbed had P(app) values >3 x 10(-6)cm/s, while drugs absorbed <90% had P(app) values lower than 1 x 10(-6)cm/s. In these cases, indeed, a borderline region characterized by the apparent permeability coefficient P(app) value between 1 x 10(-6) and 3 x 10(-6)cm/s should be considered for which the prediction of the absorbed fraction after oral administration in humans become more uncertain by the frog intestinal sac system.

Acetyl-[Asn30,Tyr32]-calcitonin fragment 8-32 forms channels in phospholipid planar lipid membranes.

The N-terminally truncated derivative of salmon calcitonin (sCt) (acetyl-[Asn(30),Tyr(32)]-calcitonin fragment 8-32) (AC 187) lacks hormonal activity and is a potent and selective antagonist of the hormone and amylin receptor. It was investigated for its capability to interact and form channels in palmitoleoylphosphatidylcholine:dioleoylphosphatidylglycerol planar lipid membranes. Interestingly, AC 187 exhibits channel activity, whose parameters, i.e., central conductance (Lambda (c)), occurrence (number of channels/min), voltage-dependence and lifetime, are similar to those found for sCt although, in the same experimental conditions, it takes longer to incorporate into the membrane than sCt. This channel activity can be modulated by changing either the holding potential or the pH of the medium, or by adding picomolar concentrations of SDS. One evident difference between the two peptides is that sCt is unselective (1.03) while AC 187 displays a cationic selectivity (P (K) (+)/P (Cl) (-) = 2.7) at pH 7, increasing to 3.87 when the pH drops to 3.8. The present findings indicate that the 1-7 disulfide bridge is sufficient but not necessary for membrane interaction, in accordance with the observation reported on the interaction with membrane receptors. Furthermore, the remarkable pH dependence of the cationic channel could be taken into consideration for full biotechnological study.

Water permeability of rat liver mitochondria: A biophysical study.

The movement of water accompanying solutes between the cytoplasm and the mitochondrial spaces is central for mitochondrial volume homeostasis, an important function for mitochondrial activities and for preventing the deleterious effects of excess matrix swelling or contraction. While the discovery of aquaporin water channels in the inner mitochondrial membrane provided valuable insights into the basis of mitochondrial plasticity, questions regarding the identity of mitochondrial water permeability and its regulatory mechanism remain open. Here, we use a stopped flow light scattering approach to define the water permeability and Arrhenius activation energy of the rat liver whole intact mitochondrion and its membrane subcompartments. The water permeabilities of whole brain and testis mitochondria as well as liposome models of the lipid bilayer composing the liver inner mitochondrial membrane are also characterized. Besides finding remarkably high water permeabilities for both mitochondria and their membrane subcompartments, the existence of additional pathways of water movement other than aquaporins are suggested.