Unraveling the parahormetic mechanism underlying the health-protecting effects of grapeseed procyanidins.

Proanthocyanidins (PACs), the predominant constituents within Grape Seed Extract (GSE), are intricate compounds composed of interconnected flavan-3-ol units. Renowned for their health-affirming properties, PACs offer a shield against a spectrum of inflammation associated diseases, such as diabetes, obesity, degenerations and possibly cancer. While monomeric and dimeric PACs undergo some absorption within the gastrointestinal tract, their larger oligomeric and polymeric counterparts are not bioavailable. However, higher molecular weight PACs engage with the colonic microbiota, fostering the production of bioavailable metabolites that undergo metabolic processes, culminating in the emergence of bioactive agents capable of modulating physiological processes. Within this investigation, a GSE enriched with polymeric PACs was employed to explore in detail their impact. Through comprehensive analysis, the present study unequivocally verified the gastrointestinal-mediated transformation of medium to high molecular weight polymeric PACs, thereby establishing the bioaccessibility of a principal catabolite termed 5-(3',4'-dihydroxyphenyl)-γ-valerolactone (VL). Notably, our findings, encompassing cell biology, chemistry and proteomics, converge to the proposal of the notion of the capacity of VL to activate, upon oxidation to the corresponding quinone, the nuclear factor E2-related factor 2 (Nrf2) pathway-an intricate process that incites cellular defenses and mitigates stress-induced responses, such as a challenge brought by TNFα. This mechanistic paradigm seamlessly aligns with the concept of para-hormesis, ultimately orchestrating the resilience to stress and the preservation of cellular redox equilibrium and homeostasis as benchmarks of health.

Brazilian FRED Registry: A Prospective Multicenter Study for Brain Aneurysm Treatment-The BRED Study.

BACKGROUND AND PURPOSE: The development of flow diverters has changed the endovascular approach to intracranial aneurysms. On the basis of good results, the indications for flow diverters have expanded to include aneurysms of different shapes, locations, and sizes. The objective of the study was to report on the performance of the Flow Re-Direction Endoluminal Device (FRED) in intracranial aneurysm treatment at early and medium-term follow-up. MATERIALS AND METHODS: This single-arm, multicentric, prospective, observational study assessed aneurysm treatment with the FRED. The primary outcome was complete aneurysm occlusion at 6 and 12 months, and the secondary outcome was to evaluate the safety of the FRED with respect to stroke and death rates. RESULTS: Between June 2016 and August 2018, a total of 100 consecutive patients with 131 aneurysms were treated in 107 procedures. Total occlusion rates were 91% and 95% at 6 and 12 months. There was 1 death, and the total final morbidity rate was 1.8%. The complication rate was 4.6%. CONCLUSIONS: As reported previously, the FRED has proved to be a safe and effective tool, with high occlusion rates. The design of the stent makes it more difficult to perform balloon angioplasty compared with similar devices. A branch arising from the aneurysm sac was found to be a predictor of nonocclusion at 12 months, though larger series are needed to estimate the magnitude of the association.

A novel boronated-porphyrin as a radio-sensitizing agent for boron neutron capture therapy of tumours: in vitro and in vivo studies.

A water-soluble [meso-tetra(4-nido-carboranylphenyl)porphyrin] (H(2)TCP) bearing 36 boron atoms was studied for its accumulation and its radio/photo-sensitization efficiency towards murine melanotic melanoma cells. The amount of H(2)TCP in the cells increased with the porphyrin dose in the incubation medium up to 100 microM with no significant dark toxicity. Fluorescence microscopy observations showed that the porphyrin was largely localized intracellularly. Based on these "in vitro" results our investigations were pursued using the B16F1 melanotic melanoma subcutaneously transplanted in C57BL6 mice as "in vivo" model. Phormacokinetic studies were performed by injection of H(2)TCP intratumorally (1 mg/kg) and intravenously (10 mg/kg). At 0.5h after i.t. administration or at 24 h after i.v. injection, the amounts of (10)B in the tumour were about 60 ppm and about 6 ppm, respectively. The distribution of H(2)TCP in the tumour after intravenous or intratumoural injection was also assessed by fluorescence microscopy analyses. Under these conditions, preliminary BNCT studies were carried out using a new thermal column called HYTOR (HYbrid Thermal spectrum sHifter TapirO Reactor) inserted in the fast nuclear reactor Tapiro at Enea Casaccia, Italy. The mice were exposed to HYTHOR radiation field for 20 min at a reactor power of 5 kW. In spite of different amounts of (10)B in the tumour at the irradiation time, a similar significant delay in tumour growth (5-6 days) was induced by neutron irradiation in intratoumorally and intravenously injected mice. The response of the melanotic melanoma to H(2)TCP-BNCT was compared with that obtained by irradiation after intraperitoneal injection of boron-phenylalanine.

Photosensitization with zinc (II) phthalocyanine as a switch in the decision between apoptosis and necrosis.

Photodynamic therapy (PDT) of tumors and other diseases is based on the uptake of a photosensitizing dye in target cells, which are damaged by reactive oxygen intermediates generated on irradiation with light in which the wavelengths match the dye absorption spectrum. PDT can induce cell death by necrosis and apoptosis both in vivo and in vitro, but the factors determining the contribution of either mechanism to the overall process are not completely defined. Our studies on the photosensitization of 4R transformed fibroblasts with the second-generation photosensitizer zinc (II) phthalocyanine (ZnPc) aim at determining the effect of important experimental parameters such as time of cell incubation (2 or 24 h) with ZnPc before irradiation and ZnPc concentration in the incubation medium on cell death. Furthermore, we propose possible correlations between the cell death mechanism and primary photo-damage sites; these are mainly determined by the intracellular localization of the photosensitizer. The mechanism of cell death was determined by both electron microscopy analysis of the morphological alterations induced by photosensitization and measurement of caspase 3 activation. The initial photodamage sites were determined by measuring the activities of several functions typical of mitochondria, lysosomes, Golgi apparatus, cytosol, and plasma membrane. The intracellular localization of ZnPc after 2- or 24-h incubation was determined by fluorescence microscopy. Necrosis, associated with early loss of plasma membrane integrity and complete depletion of intracellular ATP, represents the prevailing mode of death for 4R cells dark-incubated for 2 h with ZnPc and irradiated with light doses reducing viability by 99.9%. In contrast, irradiation performed 24 h after ZnPc incubation causes only partial inhibition of plasma membrane activities, and cell death occurs largely by apoptosis. ZnPc is mainly localized in the Golgi apparatus after 2- and 24-h incubation, and in all of the cases this compartment represents a primary target of photodamage. Only after prolonged incubation is mitochondrial localization of ZnPc clearly detected by fluorescence microscopy; this could be a determining factor for promotion of apoptosis. Our data demonstrate that it is possible to modulate the mechanism of cell death by appropriate protocols; this may be relevant for enhancing the therapeutic efficacy of PDT.

Quantitation and facilitated de novo sequencing of proteins by isotopic N-terminal labeling of peptides with a fragmentation-directing moiety.

We describe a method for comparative quantitation and de novo peptide sequencing of proteins separated either by standard chromatographic methods or by one- and two-dimensional polyacrylamide gel electrophoresis. The approach is based on the use of an isotopically labeled reagent to quantitate (by mass spectrometry) the ratio of peptides from digests of a protein being expressed under different conditions. The method allows quantitation of the changes occurring in spots or bands that contain more than one protein and has a greater dynamic range than most staining methods. Since the reagent carries a fixed positive charge under acidic conditions and labels only the N-terminal of peptides, the interpretation of tandem mass spectra to obtain sequence information is greatly simplified. The sequences can easily be extracted for homology searches instead of using indirect mass spectral-based searches and are independent of posttranslational modifications.

Ganglioside GM1 protection from apoptosis of rat heart fibroblasts.

Ceramide is involved as a mediator of apoptosis induced by a variety of signaling molecules or stressful events. Ceramide-derived sphingosine 1-phosphate behaves as an antiapoptotic agent. The ganglioside GM1 is known to protect neuronal cell lines from apoptosis induced by serum/growth factor withdrawal and its effect is mediated in part by the direct activation of the trkA NGF receptor [G. Ferrari et al. (1995) J. Biol. Chem. 270, 3074-3080]. We show that GM1, similarly to sphingosine 1-phosphate, protects rat heart fibroblasts from apoptosis induced by the protein kinase C inhibitor staurosporine and by C2-ceramide. Furthermore, we show that GM1 induces the synthesis of sphingosine 1-phosphate and that this effect is partially prevented by the sphingosine kinase inhibitor N,N-dimethylsphingosine. We conclude that the antiapoptotic action of GM1 is largely to be ascribed to an increased sphingosine kinase activity.

Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence.

The occurrence and the mode of opening of the mitochondrial permeability transition pore (MTP) were investigated directly in intact cells by monitoring the fluorescence of mitochondrial entrapped calcein. When MH1C1 cells and hepatocytes were loaded with calcein AM, calcein was also present within mitochondria, because (i) its mitochondrial signal was quenched by the addition of tetramethylrhodamine methyl ester and (ii) calcein-loaded mitochondria could be visualized after digitonin permeabilization. Under the latter condition, the addition of Ca2+ induced a prompt and massive release of the accumulated calcein, which was prevented by CsA, indicating that calcein release could, in principle, probe MTP opening in intact cells as well. To study this process, we developed a procedure by which the cytosolic calcein signal was quenched by Co2+. In hepatocytes and MH1C1 cells coloaded with Co2+ and calcein AM, treatment with MTP inducers caused a rapid, though limited, decrease in mitochondrial calcein fluorescence, which was significantly reduced by CsA. We also observed a constant and spontaneous decrease in mitochondrial calcein fluorescence, which was completely prevented by CsA. Thus MTP likely fluctuates rapidly between open and closed states in intact cells.

Effects of prostaglandins and nitric oxide on rat macrophage lipid metabolism in culture: implications for arterial wall-leukocyte interplay in atherosclerosis.

Macrophages/foam cells have a pivotal role in atherogenesis although little is known about the way lipid imbalance, a hallmark of atherosclerosis, leads to lipid accumulation in these cells. Modified low-density lipoproteins are associated with macrophage lipid dysfunction in atherosclerosis, but a possible role for altered lipogenesis leading to lipid accumulation remains to be elucidated. Since endothelium-derived nitric oxide (NO) and prostaglandins (PGs) are physiological autacoids whose production may be impaired in atherosclerosis, the effects of these mediators on de novo lipid synthesis in 24-h cultured rat peritoneal macrophages is investigated. In resident (unstimulated) cells, 1 microM PGE2 and the stable analog of PGI2 carbaprostacyclin (cPGI2, 1 microM) deviated the overall [1-14C]acetate from incorporation into cholesterol, free fatty acids and triacylglycerols favoring the formation of phospholipids. In inflammatory (thioglycollate-elicited) macrophages, these eicosanoids likewise reduced 14C-incorporations into all the lipid fractions tested. Also, cPGI2 and PGE2 reduced [4-14C]cholesterol uptake from inflammatory cells but did not interfere in 14C-cholesterol export. The PGE2-derivative PGA2 (10-20 microM) reduced 14C-incorporations into all the lipids in resident cells while it enhanced phospholipid synthesis by up to 129% at the expense of reduced incorporations into the other test lipids. The NO donor S-nitroso-N-acetylpenicillamine (SNAP, 1-10 microM), when added to macrophages in the presence of superoxide dismutase (SOD, to avoid the reaction of superoxide with NO), significantly reduced lipogenesis especially in inflammatory cells. These findings suggest that endothelium-derived NO and PGs may be associated with macrophage lipid accumulation by modulating lipogenesis and cholesterol uptake within these cells.

Imaging the mitochondrial permeability transition pore in intact cells.

The involvement of mitochondrial permeability transition pore (MTP) in cellular processes is generally investigated by indirect means, such as changes in mitochondrial membrane potential or pharmacological inhibition. However, such effects could not be related univocally to MTP. In addition, source of errors could be represented by the increased retention of membrane potential probes induced by cyclosporin A (CsA) and the interactions between fluorescent probes. We developed a direct technique for monitoring MTP. Cells were co-loaded with calcein-AM and CoCl2, resulting in the quenching of the cytosolic signal without affecting the mitochondrial fluorescence. MTP inducers caused a rapid decrease in mitochondrial calcein fluorescence which, however, was not completely prevented by CsA. Besides the large and rapid efflux of calcein induced by MTP agonists, we also observed a constant and spontaneous decrease of mitochondrial calcein which was completely prevented by CsA. Thus, MTP likely fluctuates between open and closed states in intact cells.

Determination of L-carnitine, acetyl-L-carnitine and propionyl-L-carnitine in human plasma by high-performance liquid chromatography after pre-column derivatization with 1-aminoanthracene.

A new sensitive high-performance liquid chromatographic procedure for the determination of L-carnitine (LC), acetyl-L-carnitine (ALC) and propionyl-L-carnitine (PLC) in human plasma has been developed. Precolumn derivatization with 1-aminoanthracene (1AA), performed in phosphate buffer in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) as catalyst, is involved. The fluorescent derivatives were isocratically separated on a reversed-phase column (C18). The eluate was monitored with a fluorimetric detector set at 248 nm (excitation wavelength) and 418 nm (emission wavelength). Because of the presence of endogenous carnitines, the validation was performed using dialyzed plasma. The identity of the derivatized compounds was assessed by mass spectrometry and the purity of the chromatographic peaks was confirmed by HPLC-tandem mass spectrometry. The limits of quantitation were 5 nmol/ml for LC, 1 nmol/ml for ALC and 0.25 nmol/ml for PLC. The recovery of the extraction procedure was in the range 82.6%-95.4% for all 3 compounds. Good linearity (R approximately 0.99) was observed within the calibration ranges studied: 5-160 nmol/ml for LC, 1-32 nmol/ml for ALC and 0.25-8 nmol/ml for PLC. Precision was in the range 0.3-16.8% and accuracy was always lower than 10.6%.

Mitochondrial alterations induced by aspirin in rat hepatocytes expressing mitochondrially targeted green fluorescent protein (mtGFP).

Mitochondria in primary living hepatocytes were visualized in cells transfected with a chimeric plasmid encoding for the green fluorescent protein (GFP) of Aequorea victoria engineered to be specifically targeted to mitochondria, as described recently (Rizutto et al. (1995) Curr. Biol. 5, 635-642). The identification of the fluorescent organelles as authentic mitochondria was confirmed by double labeling with rhodamine 123. Acetylsalicylate treatment of hepatocytes induced in mitochondria typical morphological alterations closely analogous to the swelling promoted by acetylsalicylate in isolated mitochondria. Cyclosporin A, which in isolated mitochondria prevents the changes induced by acetylsalicylate, had no protective action but induced per se specific alterations in the morphology of mitochondria. Moreover, exposure of hepatocytes to cyclosporin A followed by acetylsalicylate caused the same mitochondrial changes induced by each of the two compounds separately. The structural alterations caused by acetylsalicylate were constantly associated with a decrease in mitochondrial urea synthesis and cell viability.

Inhibition of macroautophagy and proteolysis in the isolated rat hepatocyte by a nontransportable derivative of the multiple antigen peptide Leu8-Lys4-Lys2-Lys-beta Ala.

The multiple antigen peptide derivative, Leu8-Lys4-Lys2-Lys-beta Ala (Leu8-MAP), was synthesized by attaching the carboxyl of leucine to the NH2 termini of a branched lysine core, termed MAP, creating a molecule of about 1900 Da with 8 leucine residues. On a molar basis (independent of the number of leucine substitutions), Leu8-MAP was as effective as leucine in suppressing macroautophagy and proteolysis; moreover, it exhibited the same apparent Km (about 0.1 mM). The effect was specific for leucine since Ile8-MAP was inactive. It is of interest, though, that Leu8-MAP did not elicit the multiphasic response typical of leucine but instead evoked the single site inhibition normally seen with leucine plus the co-regulator alanine. Some free leucine was produced from Leu8-MAP during hepatocyte incubations, but the amounts were insufficient to account for the inhibition. Although this degradation created species of Leu-MAP that had lost 1-3 residues of leucine, their inhibitory effectiveness was not diminished. Because the extracellular/intracellular distribution ratio of [3H]-Leu8-MAP was 100:1 or greater, the direct transport of Leu8-MAP across the plasma membrane into the cytosolic compartment can be excluded. Hence, cytosolic concentrations of Leu8-MAP will be at least 100-fold smaller than those of leucine under conditions of comparable proteolytic inhibition. For these and related reasons, effects attributable to the recognition of Leu8-MAP cannot be explained by signals generated within the cytosol. They could, however, be mediated from site(s) on the plasma membrane or within associated vesicles.

Leucine-specific binding of photoreactive Leu7-MAP to a high molecular weight protein on the plasma membrane of the isolated rat hepatocyte.

Leu8-MAP (Multiple Antigen Peptide) is an effective inhibitor of macroautophagy and proteolysis in the isolated rat hepatocyte, having an apparent Km (0.1 mM) equaling leucine. Since it is not transported into the cytosolic compartment, it very likely mediates its effect through a plasma membrane site. In an attempt to identify the site we photoreacted intact cells with a biologically active, iodinatable azide derivative of Leu7-MAP. A approximately 340,000 M(r) protein whose labeling was protected 83% with 20 mM Leu was found in plasma membrane fractions when electrophoresed in 7.5-20% gradient gels under nonreducing conditions; addition of 20 mM dithiothreitol generated smaller m.w. products, possibly subunits, of consistent size. No specific labeling was observed with photoreactive derivatives of Ile7-MAP or Val7-MAP.

De novo autophagic vacuole formation in hepatocytes permeabilized by Staphylococcus aureus alpha-toxin. Inhibition by nonhydrolyzable GTP analogs.

The role of GTP-binding proteins in autophagic vacuole formation was investigated in isolated rat hepatocytes permeabilized by alpha-toxin from Staphylococcus aureus, an agent which creates stable plasma membrane channels allowing exchange of small (< or = 1000 Da) molecules. Vacuole formation was monitored from the uptake of 125I-tyramine-cellobiitol (125ITC) into osmotically sensitive vacuoles isolated on colloidal silica density gradients. Separation was based on an established observation that autophagic vacuoles are retained in a heavy midgradient band when samples are layered, but are selectively shifted to dense fractions when they are previously dispersed in the gradient material. The vacuolar uptake of 125ITC was concentration-dependent and required exogenous ATP: 94% was directly mediated by sequestration; 6% was acquired by fluid-phase endocytosis as monitored by [carboxyl-14C]dextran-carboxyl. Although the amino acid control of proteolysis was lost, addition of the nonhydrolyzable GTP analog GTP gamma S (as well as GMP-PNP) decreased fractional rates of direct vacuolar 125ITC uptake and long-lived proteolysis by similar amounts (1.02-1.03% h-1), substantiating the notion that the effects were the direct result of autophagic inhibition. These and associated findings, supported by quantitative electron microscopy, indicate the presence of ongoing macro- and microautophagy in alpha-toxin-permeabilized cells and suggest that one or more GTP-binding proteins is required in macroautophagic vacuole formation.

Multiphasic control of proteolysis by leucine and alanine in the isolated rat hepatocyte.

Autophagically mediated proteolysis in the perfused rat liver is under complex multiphasic control by a small group of amino acids dominated by leucine. Because there have been no prior reports of such regulation in the isolated hepatocyte, our goal was to determine whether it is a manifestation of interactions between diverse cells in the intact liver or, alternatively, the expression of a unique control mechanism within a single population of cells. Hepatocytes were isolated from livers of ad libitum-fed rats and incubated with cycloheximide at low density (approximately 10(6) cells/ml) for the determination of valine release. As in perfusion experiments with synchronously fed rats, proteolytic responses to leucine in cells from fed rats were mediated through two inhibitory mechanisms that alternated randomly on a day-to-day basis. The first (L) represented a typical multiphasic dose-response with low- and high-concentration inhibition separated by a sharp zonal loss of inhibition that could be abolished by alanine. The second (H) mediated inhibition only at high concentrations. It disappeared after 24 h of starvation, leaving L as the prevailing mode. The findings indicate that both macroautophagy and the multiphasic mechanism for regulating it coexist in a single population of hepatocytes, making the cells suitable for studies aimed at defining the putative plasma membrane site of leucine recognition.

Control of hepatic proteolysis by leucine and isovaleryl-L-carnitine through a common locus. Evidence for a possible mechanism of recognition at the plasma membrane.

Deprivation-induced proteolysis in the perfused rat liver is controlled through the multiphasic action of 7 regulatory amino acids of which L-leucine plays the dominant role. Recently, isovaleryl-L-carnitine (IVC) was shown to mimic the leucine's effects, suggesting that the two molecules share structural features that are recognized at a common site(s). In this study we find that each evokes identical responses consisting of inhibitory effects at 0.08 and 0.8 mM, separated by a sharp zonal loss of inhibition at 0.15 mM. As monitored by density shifts of beta-hexosaminidase in colloidal silica gradients, macroautophagy is suppressed by both. Responses to Leu and IVC at 0.08 and 0.15 mM are stereospecific and require a reactive group at the alpha-carbon (or equivalent) and a high degree of branched chain specificity. In addition, 0.5 mM Ala coregulates with IVC and Leu by decreasing the zonal loss at 0.15 mM. The fact that the multiphasic responses can be duplicated with equimolar mixtures of Leu + IVC indicates that both react at the same site(s). IVC is readily taken up by a saturable process, but owing to its rapid hydrolysis in the cell, the ratio of internal to external IVC remains low over a 4-fold concentration range. These findings, together with a kinetic analysis of concerted responses to regulatory amino acids, suggest that the recognition sites are at a position in the cell, possibly at the plasma membrane, to react reversibly with plasma amino acids.

4-Amino-6-methylhept-2-enoic acid: a leucine analogue and potential probe for localizing sites of proteolytic control in the hepatocyte.

A recent analysis of leucine analogues has suggested that the carboxyl group is not required for mediating low concentration proteolytic inhibition in liver cells. In designing a probe to localize the regulatory site(s), we tested this hypothesis by synthesizing an analogue with a 2-carbon insert between the carboxyl and alpha-carbon. The Wittig product, a trans olefin, was fully active. Surprisingly, low concentration activity was lost when the double bond was eliminated by hydrogenation although some inhibitory effectiveness at high concentrations was evident. Since the double bond extends the carboxyl group away from the alpha-carbon, the results support the above hypothesis as well as the feasibility of adding functional groups to the carboxyl end of leucine.

Amino acid control of proteolysis in perfused livers of synchronously fed rats. Mechanism and specificity of alanine co-regulation.

The primary control of autophagically mediated proteolysis in perfused rat liver is carried out via two alternate mechanisms in response to specific regulatory amino acids. One (L) elicits direct inhibition at low and high plasma levels, but requires a co-regulatory amino acid to express inhibition at normal concentrations. The second (H) is ineffective at normal levels and below, but active at higher concentrations. Because regulation is subject to unpredictable variability with ad libitum feeding, we have utilized rats synchronously fed 4 h day-1 to stabilize responses. Proteolytic control is seen to evolve in stages: H appears 12 h after the start of feeding; by 18 h L emerges, alternating with H in a statistically predictable way; with omission of the 24-h feeding, H disappears and L remains constant through 42 h. In both 18- and 42-h rats, alanine, glutamate, and aspartate exhibit similar inhibitory activity when added singly to the regulatory group at normal plasma concentrations. However, since alanine, but not glutamate or aspartate, evokes proteolytic acceleration when it is deleted from a full plasma mixture, alanine appears to be the sole co-regulator. Alanine yields co-regulatory effects with normal plasma leucine (0.2 mM) in 18- and 42-h animals and interacts synergistically with 0.8 mM leucine in 42-h but not in 18-h rats where leucine alone inhibits strongly. Because the inactivation of alanine amino-transferase by aminooxyacetate (determined from the conversion of [14C]alanine to glucose) does not alter the co-regulatory and synergistic effects of alanine, regulation by alanine must be mediated from a site of recognition before transamination.