Cell-free Model Research Articles

The application of in vitro cell-free conversion systems to human prion diseases

A key event in the pathogenesis of prion diseases is the conversion of the normal cellular isoform of the prion protein into the disease-associated isoform, but the mechanisms operating in this critical event are not yet fully understood. A number of novel approaches have recently been developed to study factors influencing this process. One of these, the protein misfolding cyclical amplification (PMCA) technique, has been used to explore defined factors influencing the conversion of cellular prion protein in a cell-free model system. Although initially developed in animal models, this technique has been increasingly applied to human prion diseases. Recent studies have focused on the role of different isoforms of the disease-associated human prion protein and the effects of the naturally occurring polymorphism at codon 129 in the human prion protein gene on the conversion process, improving our understanding of the interaction between host and agent factors that influence the wide range of phenotypes in human prion diseases. This technique also allows a greatly enhanced sensitivity of detection of disease-associated prion protein in human tissues and fluids, which is potentially applicable to disease screening, particularly for variant Creutzfeldt-Jakob disease. The PMCA technique can also be used to model human susceptibility to a range of prions of non-human origin, which is likely to prove of considerable future interest as more novel and potentially pathogenic prion diseases are identified in animal species that form part of the human food chain.

Cytoprotection by almond skin extracts or catechins of hepatocyte cytotoxicity induced by hydroperoxide (oxidative stress model) versus glyoxal or methylglyoxal (carbonylation model)

Oxidative and carbonyl stress are detrimental in the pathogenesis of diabetic complications, as well as in other chronic diseases. However, this process may be decreased by dietary bioactive compounds. Almond skin is an abundant source of bioactive compounds and antioxidants, including polyphenolic flavonoids, which may contribute to the decrease in oxidative and carbonyl stress. In this study, four Almond Skin Extracts (ASEI, ASEII, ASEIII, and ASEIV) were prepared by different methods and evaluated for their antioxidant activity. The order of the polyphenol content (total μM gallic acid equivalents) of the four extracts was found to be, in decreasing order of effectiveness: ASEI > ASEIII > ASEIV > ASEII. The order of Ferric-reducing antioxidant power (FRAP, μM FeSO 4/g) value, in decreasing order was ASEI (216) > ASEIII (176) > ASEIV (89) > ASEII (85). The order of ASE effectiveness for decreasing protein carbonyation induced by the copper Fenton reaction was ASEI > ASEIV > ASEII > ASEIII. The order of antioxidant effectiveness for inhibiting tertiary-butyl hydroperoxide (TBH) induced microsomal lipid peroxidation was ASEI > ASEIV > ASEII, ASEIII. Also, the order of ASE effectiveness for inhibiting TBH induced hepatocyte cell death was: ASEIII, ASEIV > ASEI, ASEII. Catechin also protected hepatocytes from TBH induced hepatocyte, lipid peroxidation and cytotoxicity. In a cell free model, equimolar concentrations of catechin or epicatechin rescued serum albumin from protein carbonylation induced by methylglyoxal (MGO). Catechin, epicatechin and ASEI all decreased gloxal induced hepatocyte cell death and reactive oxygen species (ROS) formation in GSH-depleted hepatocytes. Catechin and epicatechin protected against GO or MGO induced hepatocyte cell death, protein carbonylation and ROS formation. Catechin was more effective than epicatechin. Our results suggest that (a) bioactive almond skin constituents in the non-lipophilic polyphenol extract were the most effective at protecting hepatocytes against hydroperoxide induced hepatocyte oxidative stress and in protecting against dicarbonyl induced cytotoxicity; (b) catechins, the major polyphenol in the extract, were also effective at preventing GO or MGO cytotoxicity likely by trapping GO and MGO and/or rescuing hepatocytes from protein carbonylation.

Mitochondrial control of caspase-dependent and -independent cell death

Mitochondria control whether a cell lives or dies. The role mitochondria play in deciding the fate of a cell was first identified in the mid-1990s, because mitochondria-enriched fractions were found to be necessary for activation of death proteases, the caspases, in a cell-free model of apoptotic cell death. Mitochondrial involvement in apoptosis was subsequently shown to be regulated by Bcl-2, a protein that was known to contribute to cancer in specific circumstances. The important role of mitochondria in promoting caspase activation has therefore been a major focus of apoptosis research; however, it is also clear that mitochondria contribute to cell death by caspase-independent mechanisms. In this review, we will highlight recent findings and discuss the mechanism underlying the mitochondrial control of apoptosis and caspase-independent cell death.

Only a Fraction of Quantal Content is Released During Exocytosis as Revealed by Electrochemical Cytometry of Secretory Vesicles

The primary method for neuronal communication involves the release of chemical messengers that are packaged intracellularly in vesicles. Although experiments measuring release at single cells have classically been thought to assess the entire content of vesicles, there is evidence in the literature that suggests that the total transmitter stored in vesicles is not expelled during exocytosis. In this work, we introduce a novel technology using a microfluidic-based platform to electrochemically probe individual PC12 cell vesicles isolated from the cell environment. We measure the total vesicular content using methodology that circumvents the biophysical processes of the cell associated with exocytosis. Direct comparisons of amperometric data from release experiments at single PC12 cells versus our cell-free model reveal that on average vesicles release only 40% of their total transmitter load. The data support the intriguing hypothesis that the average vesicle does not open all the way during the normal exocytosis process, resulting in incomplete distention of the vesicular contents. In addition, we have shown that vesicular catecholamine levels can be altered with pharmacological manipulation and variances observed from these treatments can be resolved at the single vesicle level in a high-throughput manner, a process that we have termed electrochemical cytometry. Upon establishing that release in exocytotic processes proceeds in an incomplete manner, electrochemical data quantified from both single cell release experiments and electrochemical cytometry of vesicles were related to vesicular volume from electron microscopy measurements to investigate the location of intravesicular catecholamine stores retained post-fusion.

Effects of Antiepileptic Agents on Homotypic Fusion of Synaptic Vesicles

We studied the effects of three antiepileptic drugs (AEDs) in a cell-free model system containing isolated synaptic vesicles (SVs) and cytosolic proteins, which allowed us to reproduce one of the stages of complex exocytosis. Ethosuximide, sodium valproate, and gabapentin intensified calcium- and Mg2+-ATP-induced fusion of SVs; the effect was indicative of the ability of these agents to influence the processes of simple and/or complex exocytosis in synaptic connections in the CNS structures. Antiepileptic drugs did not change the intensity of calcium-dependent fusion of liposomes and SVs treated by proteases. Therefore, the effect of AEDs can be realized via their interaction with proteins of SVs. After decrease in the level of cholesterol in the membranes of SVs using treatment by methyl-β- cyclodextrin, the ability of AEDs to activate fusion of SVs remained unchanged. Therefore, the studied AEDs act via proteins localized beyond the borders of cholesterol-enriched microdomains of the membrane. Drugs that induce convulsions (corazole and picrotoxin) did not change the characteristics of fusion of SVs under the in vitro action of AEDs. This is indicative of the absence of molecular targets for the above chemoconvulsants in the SV membranes, as compared with those in the plasma membranes of nerve terminals. According to our experiments, just proteins of SVs are functional targets for ethosuximide, sodium valproate, and gabapentin providing their anticonvulsant actions. The proposed model, which allows one to reproduce the membrane fusion, can be successfully used for the testing of drugs influencing a presynaptic link of synaptic contacts in the CNS.

Further Examination of Antiradical Properties of Crocus sativus Stigmas Extract Rich in Crocins

Data for antiradical properties of saffron extract and its bioactive constituents (crocins, crocetin) are limited and poorly discussed in comparison with those of extracts containing potent scavengers. Further examination was sought using the Folin-Ciocalteu (F-C) reagent and various free radical species produced in cell-free or cell model systems. Oregano and turmeric methanol extracts, rich in well established scavengers, and also crocetin, rosmarinic acid, and curcumin, representing the major types of constituents in the three studied extracts, were used as "reference". On the same weight basis, saffron extract activity was found to be rather negligible in all cell-free systems with regard to that found for reference ones. On the contrary, in the human monocyte system, saffron extracts or free crocetin were found to reduce ROS production as effectively as the phenolic antioxidants. Our findings point out that saffron extracts exhibit a remarkable intracellular antioxidant activity that cannot be revealed using assays repeatedly applied to the evaluation of phenolic-type antioxidants.

Aminopeptidase A contributes to the N‐terminal truncation of amyloid β‐peptide

Several lines of data previously indicated that N-terminally truncated forms of amyloid-beta (Abeta) peptides are likely the earliest and more abundant species immunohistochemically detectable in Alzheimer's disease-affected brains. It is noteworthy that the free N-terminal residue of full-length Abeta (fl-Abeta) is an aspartyl residue, suggesting that Abeta could be susceptible to exopeptidasic attack by aminopeptidase A (APA)-like proteases. In this context, we have examined whether APA could target Abeta peptides in both cell-free and cellular models. We first show that the general aminopeptidase inhibitor amastatin as well as two distinct aminopeptidase A inhibitors EC33 and pl302 both significantly increase the recovery of genuine fl-Abeta peptides generated by cells over-expressing Swedish-mutated beta amyloid precursor protein (APP) while the aminopeptidase N blocker pl250 did not modify fl-Abeta recovery. In agreement with this observation, we establish that over-expressed APA drastically reduces, in a calcium dependent manner, fl-Abeta but not APP IntraCellular Domain in a cell-free model of Abeta production. In agreement with the above data, we show that recombinant APA degrades fl-Abeta in a pl302-sensitive manner. Interestingly, we also show that EC33 and pl302 lower staurosporine-stimulated activation of caspase-3 in wild-type fibroblasts but not in betaAPP/beta-amyloid precursor protein-like protein 2 (APLP2) double knockout fibroblasts, suggesting that protecting endogenous fl-Abeta physiological production triggers neuroprotective phenotype. By contrast, EC33 does not modify staurosporine-induced caspase-3 activation in wild-type and Swedish-mutated betaAPP-HEK293 expressing cells that display exacerbated production of Abeta. Overall, our data establish that APA contributes to the N-terminal truncation of Abeta and suggest that this cleavage is likely abrogating a protective function associated with physiological but not supraphysiological levels of genuine fl-Abeta peptides.

Docking and fusion of synaptic vesicles in cell-free model system of exocytosis

The present study involves the testing and characterization of synaptic vesicle (SV) docking and fusion as the steps of exocytosis using two different approaches in vitro. The interaction of SVs was determined by the changing of particles size in suspensions by the method of dynamic light scattering (DLS). Fluorescence assay is represented for studying the mechanism of SV membrane fusion. The sizes of membrane particles were shown to increase in the medium containing cytoplasmic proteins of synaptosomes. Therefore, the cytosolic proteins are suggested to promote the SVs into close proximity where they may become stably bound or docked. The specific effect of synaptosomal cytosolic proteins on the interaction of SVs in the cell-free system was demonstrated. The incubation of SVs with liver cytosol proteins or in the bovine serum albumin solution did not lead to the enlargement of the particles size. The fusion reaction of the SVs membranes occurred within the micromolar range of Ca 2+ concentrations. Our studies have shown that in vitro process of exocytosis can be divided into Ca 2+-independent step, termed docking and followed by fusion step that is triggered by Ca 2+. The role of cytosolic proteins of synaptosomes in docking and fusion of SVs in cell-free system was further confirmed.

Bixin and lycopene modulation of free radical generation induced by cisplatin–DNA interaction

This work evaluated the modulation of reactive oxygen species (ROS) produced by the cisplatin–human DNA interaction in a cell-free experimental model by the carotenoids bixin and lycopene, extracted from natural dietary sources and purified through luminol- and Cypridina luciferin methoxy-analogue (MCLA)-enhanced chemiluminescence assays. The results showed that the ROS generation by DNA–cisplatin interaction was inhibited by both lycopene and bixin in a concentration-dependent manner. At a concentration of 100 μM, lycopene and bixin inhibited superoxide anion ( O 2 - ) generation at 90% and 82%, respectively, and the total ROS generation at 44% and 42%, respectively. The formation of significant amounts of isomers or degradation products of both carotenoids was not observed after ROS scavenging, as evaluated by high-performance liquid chromatography. Taken together, these results suggest that carotenoids can be helpful to modulate the oxidative stress found in cancer therapy with cisplatin.

Comparative Induction of 28S Ribosomal RNA Cleavage by Ricin and the Trichothecenes Deoxynivalenol and T-2 Toxin in the Macrophage

Ribosome-inactivating proteins (RIPs) and sesquiterpenoid trichothecene mycotoxins are known to bind to eukaryotic ribosomes, inhibit translation and activate mitogen-activated protein kinases. Here we compared the capacities of the RIP ricin to promote 28S ribosomal RNA (rRNA) cleavage with that of the trichothecenes, deoxynivalenol (DON), and T-2 toxin (T-2). In a cell-free model, exposure to ricin at 300 ng/ml for 30 min depurinated yeast 28S rRNA, however, neither DON (< or = 4 microg/ml) nor T-2 (< or = 2 microg/ml) exhibited this N-glycosidase activity. Incubation of RAW 264.7 macrophages with ricin (20-320 ng/ml), DON (250-5000 ng/ml), or T-2 (2-80 ng/ml) for 6 h, however, generated 28S rRNA-specific products consistent with cleavage sites near the 3' terminal end of murine 28S rRNA. Oligonucleotide extension analysis of treated RAW 264.7 cells revealed that ricin evoked 28S rRNA damage at one site in the alpha-sarcin/ricin (S/R)-loop (A4256) and two other sites (A3560 and A4045) in the peptidyl transferase center. Although DON or T-2 did not damage the S/R loop, these trichothecenes did promote cleavage at A3560 and A4045. In addition, incubation of the cells with ricin (> or = 20 ng/ml), DON (> or = 250 ng/ml), or T-2 (> or = 10 ng/ml) induced RNase activity as well as RNase L mRNA and protein expression. These data suggest that only ricin directly damaged 28S rRNA under cell-free conditions but that ricin, DON, and T-2 promoted intracellular 28S rRNA cleavage, potentially by facilitating the action of endogenous RNases and/or by upregulating RNase expression.

Leydig cell re-generation and expression of cell signaling molecules in the germ cell-free testis

Leydig cells in the rat testis can be specifically ablated with ethane dimethane sulfonate (EDS) and will subsequently re-generate. In this study, we have characterized Leydig cell re-generation and expression of selected cell-signaling molecules in a germ cell-free model of EDS action. This model offers the advantage that re-generation occurs on a stable background without confounding changes from the regressing and repopulating germ cell population. Adult rats were treated with busulfan to remove the germ cell population and Leydig cells were then ablated with EDS. Testicular testosterone levels declined markedly within 24 h of EDS treatment and started to recover after 8 days. After EDS treatment there were marked declines in levels of Leydig cell-specific mRNA transcripts coding for steroidogenic enzymes cytochrome P450 11a1 (Cyp11a1), cytochrome P450 17a1 (Cyp17a1), 3beta-hydroxysteroid dehydrogenase type 1 (Hsd3b1), 17beta-hydroxysteroid dehydrogenase type 3 (Hsd17b3) and the LH receptor. Levels of all transcripts recovered within 20 days of EDS treatment apart from Hsd17b3, which remained undetectable up to 20 days. Immunohistochemical localization of CYP11A1 during the phase of early Leydig cell re-generation showed that the Leydig cell precursors are spindle-shaped peritubular cells. Studies on factors which may be involved in Leydig cell re-generation showed there were significant but transient increases in platelet-derived growth factor A (Pdgfa), leukemia inhibitory factor (Lif), and neurofilament heavy polypeptide (Nefh) after EDS, while desert hedgehog (Dhh) levels declined sharply but recovered by 3 days. This study shows that the Leydig cell precursors are peritubular cells and that expression of Pdgfa and Lif is increased at the start of the re-generation process when precursor proliferation is likely to be taking place.

Statistical approach in alginate membrane formulation for cell encapsulation in a GMP-based cell factory

A cell encapsulation technology in alginate has been developed with the aim of obtaining cell controlled release or three-dimensional cultures. The aim of this work is to verify the predictability of alginate capsules for large-scale production by Good Manufacturing Practice (GMP) standardized procedures in a cell factory. A cell-free capsule model was performed following the GMP guidelines: an opaque agent suspension in a bivalent cation solution (Ca 2+, Ba 2+, Sr 2+) was dropped in a sodium alginate solution, obtaining capsules presenting a liquid core surrounded by a gel alginate membrane. The concentration of the ion, and the treatment with protamine, can considerably vary the characteristics of the capsules (weight, whole diameter, core diameter, gel capsule thickness, capsule strength). It is therefore possible to optimize the performance of the capsules, relating the molecular structure and size of the polymeric membrane to the desired functional properties. Technological resources are available for large-scale cell encapsulation intended for advanced therapies (gene therapy, somatic cell therapy and tissue engineering) in a cell factory, following GMP guidelines.

High-throughput screening for reactive oxygen species scavengers targeting mitochondria

A high-throughput screening (HTS) method for reactive oxygen species (ROS) scavengers was established to explore compounds protecting mitochondria. Freshly prepared rat liver mitochondria and the ROS fluorescent probe, 2',7'-dichlorofluorescin diacetate (DCFH-DA), were coincubated for 90 min to establish the assay in 384-well plates. The fluorescence of DCF values was monitored with a microplate reader. The Z' factor value for HTS was 0.81. A sample library containing 75,230 compounds was screened, and 10 compounds with high ROS scavenging activity were identified, among which the compound, 5061, with the strongest ROS scavenging activity was reevaluated. 5061 showed potent free radical scavenging activity both in cell-free models and cell models, which were in good accordance with the HTS assay. The established fluorescent HTS assay was simple, sensitive, stable, highly reproducible and robust and suitable for the HTS assay for ROS scavengers targeting mitochondria.

Cellular Oligomerization of α-Synuclein Is Determined by the Interaction of Oxidized Catechols with a C-terminal Sequence

The mechanisms that govern the formation of alpha-synuclein (alpha-syn) aggregates are not well understood but are considered a central event in the pathogenesis of Parkinson's disease (PD). A critically important modulator of alpha-syn aggregation in vitro is dopamine and other catechols, which can prevent the formation of alpha-syn aggregates in cell-free and cellular model systems. Despite the profound importance of this interaction for the pathogenesis of PD, the processes by which catechols alter alpha-syn aggregation are unclear. Molecular and biochemical approaches were employed to evaluate the mechanism of catechol-alpha-syn interactions and the effect on inclusion formation. The data show that the intracellular inhibition of alpha-syn aggregation requires the oxidation of catechols and the specific noncovalent interaction of the oxidized catechols with residues (125)YEMPS(129) in the C-terminal region of the protein. Cell-free studies using novel near infrared fluorescence methodology for the detection of covalent protein-ortho-quinone adducts showed that although covalent modification of alpha-syn occurs, this does not affect alpha-syn fibril formation. In addition, oxidized catechols are unable to prevent both thermal and acid-induced protein aggregation as well as fibrils formed from a protein that lacks a YEMPS amino acid sequence, suggesting a specific effect for alpha-syn. These results suggest that inappropriate C-terminal cleavage of alpha-syn, which is known to occur in vivo in PD brain or a decline of intracellular catechol levels might affect disease progression, resulting in accelerated alpha-syn inclusion formation and dopaminergic neurodegeneration.

Mechanisms of the in vivo inhibition of calcification of bioprosthetic porcine aortic valve cusps and aortic wall with triglycidylamine/mercapto bisphosphonate

Heart valve replacements fabricated from glutaraldehyde (Glut)-crosslinked heterograft materials, porcine aortic valves or bovine pericardium, have been widely used in cardiac surgery to treat heart valve disease. However, these bioprosthetic heart valves often fail in long-term clinical implants due to pathologic calcification of the bioprosthetic leaflets, and for stentless porcine aortic valve bioprostheses, bioprosthetic aortic wall calcification also typically occurs. Previous use of the epoxide-based crosslinker, triglycidyl amine (TGA), on cardiac bioprosthetic valve materials demonstrated superior biocompatibility, mechanics, and calcification resistance for porcine aortic valve cusps (but not porcine aortic wall) and bovine pericardium, vs. Glut-prepared controls. However, TGA preparation did not completely prevent long-term calcification of cusps or pericardium. Herein we report further mechanistic investigations of an added therapeutic component to this system, 2-mercaptoethylidene-1,1-bisphosphonic acid (MABP), a custom synthesized thiol bisphosphonate, which has previously been shown in a preliminary report to prevent bioprosthetic heterograft biomaterial calcification when used in combination with initial TGA crosslinking for 7 days. In the present studies, we have further investigated the effectiveness of MABP in experiments that examined: (1) The use of MABP after optimal TGA crosslinking, in order to avoid any competitive interference of MABP-reactions with TGA during crosslinking; (2) Furthermore, recognizing the importance of alkaline phosphatase (ALP) in the formation of dystrophic calcific nodules, we have investigated the hypothesis that the mechanism by which MABP primarily functions is through the reduction of ALP activity. Results from cell-free model systems, cell culture studies, and rat subcutaneous implants, show that materials functionalized with MABP after TGA crosslinking have reduced ALP activity, and in vivo have no significant calcification in long-term implant studies. It is concluded that bioprosthetic heart valves prepared in this fashion are compelling alternatives for Glut-prepared bioprostheses.

Comparison of Extracellular and Intracellular Potency of Botulinum Neurotoxins

Levels of botulinum neurotoxin (BoNT) proteolytic activity were compared using a cell-free assay and living neurons to measure extracellular and intracellular enzymatic activity. Within the cell-free reaction model, BoNT serotypes A and E (BoNT/A and BoNT/E, respectively) were reversibly inhibited by chelating Zn2+ with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN). BoNT/E required relatively long incubation with TPEN to achieve total inhibition, whereas BoNT/A was inhibited immediately upon mixing. When naïve Zn2+-containing BoNTs were applied to cultured neurons, the cellular action of each BoNT was rapidly inhibited by subsequent addition of TPEN, which is membrane permeable. Excess Zn2+ added to the culture medium several hours after poisoning fully restored intracellular toxin activity. Unlike TPEN, EDTA irreversibly inhibited both BoNT/A and -E within the cell-free in vitro reaction. Excess Zn2+ did not reactivate the EDTA-treated toxins. However, application of EDTA-treated BoNT/A or -E to cultured neurons demonstrated normal toxin action in terms of both blocking neurotransmission and SNAP-25 proteolysis. Different concentrations of EDTA produced toxin preparations with incrementally reduced in vitro proteolytic activities, which, when applied to living neurons showed undiminished cellular potency. This suggests that EDTA renders the BoNT proteolytic domain conformationally inactive when tested with the cell-free reaction, but this change is corrected during entry into neurons. The effect of EDTA is unrelated to Zn2+ because TPEN could be applied to living cells before or after poisoning to produce rapid and reversible inhibition of both BoNTs. Therefore, bound Zn2+ is not required for toxin entry into neurons, and removal of Zn2+ from cytosolic BoNTs does not irreversibly alter toxin structure or function. We conclude that EDTA directly alters both BoNTs in a manner that is independent of Zn2+.

A recombinant transductor–effector system: In vitro study of G inhibitory protein (G-alpha-i1) direct activators

Mutations and altered functionality of the inhibitory subfamily of G proteins (Gi) are involved in pathological states. Compounds able to activate Gi in a receptor-independent manner would be useful to treat these pathological conditions. Aimed to study Gi direct activation we have reconstituted a recombinant transductor–effector complex cloning both the mammalian Gα i1 subunit and adenylate cyclase (AC). The myristoylation of Gα, fundamental for interaction with AC, was obtained in the procaryotic expression host Escherichia coli transformed with a single plasmid containing both the coding sequences for human Gα i1 and Saccharomyces cerevisiae myristoyl transferase. AC-V isoform was obtained by the expression of its cytosolic domains. A recent synthesized molecule, named BC5, was tested to evaluate its pharmacological profile in a Gi/AC cell-free complex model. In this functional transductor–effector system BC5 was able to activate Gi signalling, moreover providing a new tool to give a better insight into G-protein receptor-independent modulation.

Engineered Hybrid Dimers: Tracking the Activation Pathway of Caspase-7

Caspase-7 is an obligate dimer of catalytic domains, with generation of activity requiring limited proteolysis within a region that separates the large and small chains of each domain. Using hybrid dimers we distinguish the relative contribution of each domain to catalysis by the whole molecule. We demonstrate that the zymogen arises from direct dimerization and not domain swapping. In contrast to previous conclusions, we show that only one of the catalytic domains must be proteolyzed to enable activation. The processed domain of this singly cleaved zymogen has the same catalytic activity as a domain of fully active caspase-7. A transient intermediate of singly cleaved dimeric caspase-7 can be found in a cell-free model of apoptosis induction. However, we see no evidence for an analogous intermediate of the related executioner caspase-3. Our study demonstrates the efficiency by which the executioner caspases are activated in vivo.

Direct and indirect effects of single walled carbon nanotubes on RAW 264.7 macrophages: Role of iron

Single-walled carbon nanotubes (SWCNT), nano-cylinders with an extremely small diameter (1–2 nm) and high aspect ratio, have unique physico-chemical, electronic and mechanical properties and may exhibit unusual interactions with cells and tissues, thus necessitating studies of their toxicity and health effects. Manufactured SWCNT usually contain significant amounts of iron that may act as a catalyst of oxidative stress. Because macrophages are the primary responders to different particles that initiate and propagate inflammatory reactions and oxidative stress, we utilized two types of SWCNT: (1) iron-rich (non-purified) SWCNT (26 wt.% of iron) and (2) iron-stripped (purified) SWCNT (0.23 wt.% of iron) to study their interactions with RAW 264.7 macrophages. Ultrasonication resulted in predominantly well-dispersed and separated SWCNT strands as evidenced by scanning electron microscopy. Neither purified nor non-purified SWCNT were able to generate intracellular production of superoxide radicals or nitric oxide in RAW 264.7 macrophages as documented by flow-cytometry and fluorescence microscopy. SWCNT with different iron content displayed different redox activity in a cell-free model system as revealed by EPR-detectable formation of ascorbate radicals resulting from ascorbate oxidation. In the presence of zymosan-stimulated RAW 264.7 macrophages, non-purified iron-rich SWCNT were more effective in generating hydroxyl radicals (documented by EPR spin-trapping with 5,5-dimethyl-1-pyrroline- N-oxide, DMPO) than purified SWCNT. Similarly, EPR spin-trapping experiments in the presence of zymosan-stimulated RAW 264.7 macrophages showed that non-purified SWCNT more effectively converted superoxide radicals generated by xanthine oxidase/xanthine into hydroxyl radicals as compared to purified SWCNT. Iron-rich SWCNT caused significant loss of intracellular low molecular weight thiols (GSH) and accumulation of lipid hydroperoxides in both zymosan-and PMA-stimulated RAW 264.7 macrophages. Catalase was able to partially protect macrophages against SWCNT induced elevation of biomarkers of oxidative stress (enhancement of lipid peroxidation and GSH depletion). Thus, the presence of iron in SWCNT may be important in determining redox-dependent responses of macrophages.

Antioxidant activity of polyphenols from solid olive residues of c.v. Coratina

The antioxidant profile of extracts from solid olive residue (SOR) of c.v. Coratina, a cultivar widely diffused in the south of Italy, using both cell-free and cell-based experimental models, was investigated. A total hydroalcoholic extract (polyphenols content 19.7%) and a purified extract (Oleaselect™) (polyphenols content 35.1%) were tested for their ability to quench the stable free radical DPPH, the peroxyl radicals (ORAC assay), by monitoring the loss in fluorescence of R-phycoerythrin induced by the peroxyl radical generator AAPH and their ability to inhibit the cumene hydroperoxide-induced lysis of rat red blood cells (RBC). The total hydroalcoholic extract showed IC 50 26.96 ± 1.53 μg/ml in the DPPH assay, that 10 μg/ml were equivalent to 2.11 ± 0.12 μg/ml Trolox (ORAC assay) and IC 50 1.7 ± 0.20 μg/ml in the RBC hemolysis. The Oleaselect™ extract was 4 to 5 folds more active than the hydroalcoholic extract in all the experimental models, with IC 50 values of 7.36 ± 0.38 μg/ml in the DPPH test and of 0.38 ± 0.03 μg/ml in RBC; the antioxidant activity in the ORAC assay was slightly greater than that of Trolox (10 μg/ml equivalent to 11.45 ± 0.40 μg/ml). The scavenging effect of the extract in the ORAC assay was compared to that of verbascoside (the main polyphenol component) and of caffeic acid (the basic constituent of verbascoside): the results indicate that caffeic acid (10 μg/ml equivalent to 35.70 ± 2.95 μg/ml Trolox) is more potent than verbascoside (10 μg/ml equivalent to 15.42 ± 1.21 μg/ml Trolox) in entrapping peroxyl radicals. Finally the antioxidant activity of the Oleaselect™ extract was confirmed in human umbilical endothelial cells (EC) exposed to the site-specific peroxyl radical inducer AAPH, where a massive lipid peroxidation process (marker the fluorescence probe BODIPY) takes place, paralleled by a marked loss of cell viability (calcein assay). The purified extract (1–20 μg/ml) pre-incubated with EC for 1 h dose-dependently inhibited both the lipid-peroxidation damage and cell death. Taking into account the total polyphenol content, these results clearly indicate a greater antioxidant activity for the purified extract, due to a cooperative antioxidant interaction among its polyphenol constituents.