Regulation of TSC2 lysosome translocation and mitochondrial turnover by TSC2 acetylation status.

Sirtuin1 (SIRT1) activity decreases the tuberous sclerosis complex 2 (TSC2) lysine acetylation status, inhibiting the mechanistic target of rapamycin complex 1 (mTORC1) signalling and concomitantly, activating autophagy. This study analyzes the role of TSC2 acetylation levels in its translocation to the lysosome and the mitochondrial turnover in both mouse embryonic fibroblast (MEF) and in mouse insulinoma cells (MIN6) as a model of pancreatic ß cells. Resveratrol (RESV), an activator of SIRT1 activity, promotes TSC2 deacetylation and its translocation to the lysosome, inhibiting mTORC1 activity. An improvement in mitochondrial turnover was also observed in cells treated with RESV, associated with an increase in the fissioned mitochondria, positive autophagic and mitophagic fluxes and an enhancement of mitochondrial biogenesis. This study proves that TSC2 in its deacetylated form is essential for regulating mTORC1 signalling and the maintenance of the mitochondrial quality control, which is involved in the homeostasis of pancreatic beta cells and prevents from several metabolic disorders such as Type 2 Diabetes Mellitus.

Cell immortalization facilitates prelamin A clearance by increasing both cell proliferation and autophagic flux.

Hutchinson-Gilford Progeria Syndrome is an ultrarare disease which is characterized by an accelerated senescence phenotype with deleterious consequences to people suffering this pathology. The production of an abnormal protein derived from lamin A, called progerin, presents a farnesylated domain, which is not eliminated by the causal mutation of the disease, and accumulates in the interior of the nucleus, provoking a disruption of nuclear membrane, chromatin organization and an altered gene expression. The mutation in these patients occurs in a single nucleotide change, which creates a de novo splicing site, producing a shorter version of the protein. Apart from this mutation, an alteration in the metalloproteinase Zmpste24, involved in the maturation of lamin A, causing a similar alteration than in progeria. However, in this case, patients accumulate a protein, called prelamin A, which generates similar alterations in the nucleus than progerin. The reduction of prelamin A protein levels facilitates the recovery of the phenotype in different mice models of the disease, reducing the aging process. Different strategies have been studied for eliminating this toxic protein. Here, we report that immortalization of primary cells derived from the Zmpste24 KO mice, facilitates prelamin A degradation by different mechanisms, being essential, the enhancing proliferative capacity that the immortalized cells present. Then, these data suggest that using different treatments for increasing proliferative capacity of these cells, potentially could have a beneficial effect, facilitating prelamin A toxicity.

Insulin Resistance and Diabetes Mellitus in Alzheimer's Disease.

Type 2 diabetes mellitus is a progressive disease that is characterized by the appearance of insulin resistance. The term insulin resistance is very wide and could affect different proteins involved in insulin signaling, as well as other mechanisms. In this review, we have analyzed the main molecular mechanisms that could be involved in the connection between type 2 diabetes and neurodegeneration, in general, and more specifically with the appearance of Alzheimer's disease. We have studied, in more detail, the different processes involved, such as inflammation, endoplasmic reticulum stress, autophagy, and mitochondrial dysfunction.

Essential role of glucokinase in the protection of pancreatic ß cells to the glucose energetic status.

Energy sensing is indispensable to balance anabolic and catabolic processes for the maintenance of cell viability. Pancreatic ß cells are especially relevant because of their involvement in the coordination of insulin secretion when glucose concentration arises in the local milieu. In this work, we uncover the increased susceptibility of pancreatic ß cells to cell death in response to different energy stressors. Upon glucose decline, from 25 to 5 mM, caused by stimulation with either 2-deoxyglucose or metformin, only pancreatic ß cells showed an increase in cell death. Very interestingly, when we transfected either mouse insulinoma cell or human embryo kidney cells with a phospho-mutant form of B cell lymphoma 2 associated agonist of cell death at serine 155 (BAD S155D), an increase in the pro-survival factor B cell lymphoma 2 was detected in pancreatic ß cells and not in human embryonic kidney cells in the presence of the energetic stressors. This data suggests that the protective capacity of this mutant form is only present in cells that present glucokinase. In contrast, upon hyperactivation of mechanistic target of rapamycin complex 1 signaling by knocking-down tuberous sclerosis complex protein, we observed increased susceptibility to cell death in response to energy stress in both pancreatic and non-pancreatic ß cells. Therefore, mechanistic target of rapamycin complex 1 signaling presents a dual effect on cell viability. On the one hand, a chronic inhibition of mechanistic target of rapamycin complex 1 activity in response to the energy status is deleterious for pancreatic ß cells, being attenuated by the overexpression of B cell lymphoma 2 associated agonist of cell death S155D. On the other hand, mechanistic target of rapamycin complex 1 hyperactivity provokes a susceptibility to energetic stress-induced cell death. Taken together, these results may open potential implications for the use of glucokinase activators or mechanistic target of rapamycin complex 1 modulators for the maintenance of pancreatic ß cells for longer periods of time avoiding its loss in different pathologies such as type 2 diabetes mellitus.

Pancreatic ß cells overexpressing hIAPP impaired mitophagy and unbalanced mitochondrial dynamics.

Human islet amyloid polypeptide (hIAPP), or amylin, has the tendency to aggregate into insoluble amyloid fibrils, a typical feature of islets from type 2 diabetes individuals. Thus, we investigated comparatively the impact of hIAPP on key pathways involved in pancreatic beta survival. INS1E-hIAPP cells present a hyperactivation of MTORC1 and an inhibition of autophagy signaling, those cells showing an increase in cell size. Resveratrol, a MTORC1 inhibitor, can reverse TSC2 degradation that occurs in INS1E-hIAPP cells and diminished MTORC1 hyperactivation with concomitant autophagy stimulation. At the same time, a blockade in mitophagy was found in INS1E-hIAPP cells, as compared with control or INS1E-rIAPP cells. Consistently, human amylin overexpression generates a basal induction of nitrotyrosine levels and polyubiquitinated aggregates. Failure of the protein degradation machinery finally results in an accumulation of damaged and fissioned mitochondria, ROS production, and increased susceptibility to endoplasmic reticulum (ER)-stress-induced apoptosis. Overall, hIAPP overexpression in INS1E cells induced MTORC1 activation and mitophagy inhibition, favoring a pro-fission scenario of damaged mitochondria, these cells turn out to be more susceptible to the ER-stress-induced apoptosis and malfunction.

Nematicidal potential of hydrolates from the semi industrial vapor-pressure extraction of Spanish aromatic plants.

The nematicidal activity of hydrolate by-products from the semi industrial vapor-pressure essential oil extraction of selected aromatic plant species (commercial: Lavandula × intermedia Emeric ex Loisel. var. super, Thymus vulgaris L., T. zygis Loefl ex L. and experimentally pre-domesticated: L. luisieri (Rozeira) Rivas-Martínez) was investigated against the root-knot nematode Meloidogyne javanica by in vitro and in vivo bioassays. Liquid-liquid extraction of hydrolates yielded the corresponding aqueous and organic fractions which were biological and chemically studied. Hydrolates from L. × intermedia var. super, L. luisieri, T. vulgaris, and T. zygis showed strong in vitro nematicidal effects against M. javanica (J2 mortality and suppression of egg hatching). In the case of the Thymus species, the active components were found in the organic fraction, characterized by thymol as major component. Conversely, the nematicidal activity of L. × intermedia var. super and L. luisieri remained in the corresponding aqueous fractions. In vivo tests on tomato seedlings at sublethal doses of the hydrolates/organic fractions induced a significant reduction of nematode infectivity. In pot experiments, all hydrolates tested on tomato plants significantly affect the infection frequency and reproduction rate of the nematode population. This study demonstrates that L. × intermedia var. super, L. luisieri, T. vulgaris, and T. zygis hydrolates could be an exploitable source of potential waste protection products on root-knot nematodes.

Trypanocidal Effects of Essential Oils from Selected Medicinal Plants. Synergy among the Main Components.

Fourteen essential oils (EOs) from selected live germplasm of medicinal plants have been tested for their antitrypanosomal and cytotoxic activity. These plants have been domesticated and maintained under experimental cultivation. Their EOs were tested on epimastigote forms of Trypanosoma cruzi strain Y and human lung fibroblasts LC5 cell line, along with the major components of the active oils, both separately and in binary combinations. Mentha rotundifolia, Thymus zygis, T. vulgaris and Hyssopus officinalis were the most active EOs against T. cruzi. Among the main components of these EOs (1-8-cineole, thymol, p-cymene, piperitenone oxide, ß-pinene, γ-terpinene, carvacrol and linalool), the most active against the parasite and less toxic to human cells was thymol. In general, the activity of the main components did not exceed that of their origin EO, and the study of the activity of these compounds in combination indicates the existence of antagonistic and synergistic effects depending on the concentration tested.

Phytotoxic and Nematicidal Components of Lavandula luisieri.

Several preparations were obtained from the aerial parts of predomesticated Lavandula luisieri, including the essential oil and ethanolic, hexane, and ethyl acetate extractives. Additionally, pilot plant vapor pressure extraction was carried out at a pressure range of 0.5-1.0 bar to give a vapor pressure oil and an aqueous residue. A chemical study of the hexane extract led to the isolation of six necrodane derivatives (1, 2, and 4-7), with four of these (1, 2, 5, and 7) being new, as well as camphor, a cadinane sesquiterpene (9), tormentic acid, and ursolic acid. The EtOAc and EtOH extracts contained a mixture of phenolic compounds with rosmarinic acid being the major component. Workup of the aqueous residue resulted in the isolation of the necrodane 3 and (1R*,2S*,4R*)-p-menth-5-ene-1,2,8-triol (8), both new natural compounds. The structures of the new compounds were established based on their spectroscopic data. The phytotoxic and nematicidal activities of these compounds were evaluated.

Selective nematocidal effects of essential oils from two cultivated Artemisia absinthium populations.

Essential oils (EOs) obtained from two crops and populations of thujone-free cultivated Artemisia absinthium were tested against two nematode models, the mammalian parasite Trichinella spiralis, and the plant parasitic root knot nematode Meloidogyne javanica. The EOs were characterized by the presence of (Z)-epoxyocimene and chrysanthenol as major components and showed time and population dependent quantitative and qualitative variations in composition. The EOs showed a strong ex vivo activity against the L1 larvae of the nematode Trichinella spiralis with a reduction of infectivity between 72 and 100% at a dose range of 0.5-1 mg/ml in absence of cytotoxicity against mammalian cells. Moreover, the in vivo activity of the EO against T. spiralis showed a 66% reduction of intestinal adults. However, these oils were not effective against M. javanica.

Trypanocidal, trichomonacidal and cytotoxic components of cultivatedArtemisia absinthium Linnaeus (Asteraceae) essential oil

Artemisia absinthium is an aromatic and medicinal plant of ethnopharmacological interest and it has been widely studied. The use ofA. absinthiumbased on the collection of wild populations can result in variable compositions of the extracts and essential oils (EOs). The aim of this paper is the identification of the active components of the vapour pressure (VP) EO from a selected and cultivated A. absinthiumSpanish population (T2-11) against two parasitic protozoa with different metabolic pathways: Trypanosoma cruzi andTrichomonas vaginalis. VP showed activity on both parasites at the highest concentrations. The chromatographic fractionation of the VP T2-11 resulted in nine fractions (VLC1-9). The chemical composition of the fractions and the antiparasitic effects of fractions and their main compounds suggest that the activity of the VP is related with the presence oftrans-caryophyllene and dihydrochamazulene (main components of fractions VLC1 and VLC2 respectively). Additionally, the cytotoxicity of VP and fractions has been tested on several tumour and no tumour human cell lines. Fractions VLC1 and VLC2 were not cytotoxic against the nontumoural cell line HS5, suggesting selective antiparasitic activity for these two fractions. The VP and fractions inhibited the growth of human tumour cell lines in a dose-dependent manner.

Trypanocidal, trichomonacidal and cytotoxic components of cultivated Artemisia absinthium Linnaeus (Asteraceae) essential oil.

Artemisia absinthium is an aromatic and medicinal plant of ethnopharmacological interest and it has been widely studied. The use ofA. absinthium based on the collection of wild populations can result in variable compositions of the extracts and essential oils (EOs). The aim of this paper is the identification of the active components of the vapour pressure (VP) EO from a selected and cultivated A. absinthium Spanish population (T2-11) against two parasitic protozoa with different metabolic pathways: Trypanosoma cruzi and Trichomonas vaginalis. VP showed activity on both parasites at the highest concentrations. The chromatographic fractionation of the VP T2-11 resulted in nine fractions (VLC1-9). The chemical composition of the fractions and the antiparasitic effects of fractions and their main compounds suggest that the activity of the VP is related with the presence of trans-caryophyllene and dihydrochamazulene (main components of fractions VLC1 and VLC2 respectively). Additionally, the cytotoxicity of VP and fractions has been tested on several tumour and no tumour human cell lines. Fractions VLC1 and VLC2 were not cytotoxic against the nontumoural cell line HS5, suggesting selective antiparasitic activity for these two fractions. The VP and fractions inhibited the growth of human tumour cell lines in a dose-dependent manner.

Composition and antioxidant activity of Thymus vulgaris volatiles: comparison between supercritical fluid extraction and hydrodistillation.

Supercritical fluid extraction (SFE) of the volatile oil from Thymus vulgaris L. aerial flowering parts was performed under different conditions of pressure, temperature, mean particle size and CO(2) flow rate and the correspondent yield and composition were compared with those of the essential oil isolated by hydrodistillation (HD). Both the oils were analyzed by GC and GC-MS and 52 components were identified. The main volatile components obtained were p-cymene (10.0-42.6% for SFE and 28.9-34.8% for HD), gamma-terpinene (0.8-6.9% for SFE and 5.1-7.0% for HD), linalool (2.3-5.3% for SFE and 2.8-3.1% for HD), thymol (19.5-40.8% for SFE and 35.4-41.6% for HD), and carvacrol (1.4-3.1% for SFE and 2.6-3.1% for HD). The main difference was found to be the relative percentage of thymoquinone (not found in the essential oil) and carvacryl methyl ether (1.0-1.2% for HD versus t-0.4 for SFE) which can explain the higher antioxidant activity, assessed by Rancimat test, of the SFE volatiles when compared with HD. Thymoquinone is considered a strong antioxidant compound.

Supercritical fluid extraction of the volatile oil from Santolina chamaecyparissus.

Supercritical fluid extraction (SFE) of the volatile oil from Santolina chamaecyparissus L. flower heads was performed under different conditions of pressure, temperature, mean particle size and CO(2) flow rate. This oil was compared with the essential oil isolated by hydrodistillation (HD). The SFE volatile and essential oils were analysed by GC and GC-MS. The range of the main volatile components obtained with HD and SFE were, respectively: 1,8-cineole (25-30% and 7-48%), camphor (7-9% and 8-14%), borneol (7-8% and 2-11%), terpinen-4-ol (6-7% and 1-4%), terpinolene (1-4% and 1-7%) and isobornyl acetate (1-2% and 1-11%). The chemical composition of the extracts was greatly influenced by the conditions of pressure and temperature used. In fact, it was possible to enrich the sesquiterpene fraction by increasing the pressure from 8 to 9 MPa, while changing the temperature from 50 to 40 degrees C at 9 MPa enriched the volatiles in n-alkanes [corrected].

Enrichment of the thymoquinone content in volatile oil from Satureja montana using supercritical fluid extraction.

Supercritical fluid extraction (SFE) of the volatile oil from Satureja montana L. was performed under different conditions of pressure (90 and 100 bar), temperature (40 and 50 degrees C), mean particle sizes (0.4, 0.6 and 0.8 mm) and CO(2) flow rate (0.8, 1.1 and 1.3 kg/h) to understand the influence of these parameters on the composition and yield of this oil. The results were compared with those obtained for the essential oil isolated by hydrodistillation (HD). The volatile and the essential oil were analysed by GC and GC-MS. The main compounds are carvacrol (52.2-62.0% for HD vs. 41.7-64.5% for SFE), thymol (8.6-11.0% for HD vs. 6.0-11.3% for SFE), p-cymene (6.9-12.8% for HD vs. 6.0-17.8% for SFE), gamma-terpinene (6.4-9.4% for HD vs. 2.3-6.0% for SFE) and beta-bisabolene (2.0-2.7% for HD vs. 2.2-3.5% for SFE). The major difference between SFE and HD was the relative amount of thymoquinone, an oxygenated monoterpene with important biological activities, which can be ten-fold higher in volatile oil (1.6-3.0 for SFE vs. 0.2% for HD). The morphology of the glandular trichomes of S. montana and the effect of the grinding process on them was also evaluated by SEM.

Bioguided isolation and identification of the nonvolatile antioxidant compounds from fennel (Foeniculum vulgare Mill.) waste.

A bioguided isolation of an aqueous extract of fennel waste led to the isolation of 12 major phenolic compounds. Liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry (LC/UV/APCI-MS) combined with spectroscopic methods (NMR) was used for compound identification. Radical scavenging activity was tested using three methods: DPPH*, superoxide nitro-blue tetrazolium hypoxanthine/xanthine oxidase, and *OH/luminol chemiluminescence. In addition to products described in the literature, eight antioxidant compounds were isolated and identified for the first time in fennel: 3-caffeoylquinic acid, 4-caffeoylquinic acid, 1,5-O-dicaffeoylquinic acid, rosmarinic acid, eriodictyol-7-O-rutinoside, quercetin-3-O-galactoside, kaempferol-3-O-rutinoside, and kaempferol-3-O-glucoside. The structures of eriodictyol-7-O-rutinoside and quercetin-3-O-glucuronide were completely elucidated by two-dimensional NMR experiments. The isolated compounds exhibited a strong antiradical scavenging activity, which may contribute to the interpretation of the pharmacological effects of fennel.

Comparison between the radical scavenging activity and antioxidant activity of six distilled and nondistilled mediterranean herbs and aromatic plants.

Thirty-six different extracts of six herbs and aromatic plants (fennel, common melilot, milfoil, lavandin cv. Super, spike lavender, and tarragon) were evaluated for their radical scavenging activity by the DPPH*, NBT/hypoxanthine superoxide, and *OH/luminol chemiluminescence methods, and for their antioxidant activity by the beta-carotene blenching test. The total phenolic content was also determined by the Folin-Ciocalteu method. The plant material included cultivated plants and their wastes after being distilled for essential oils. Both remarkably high phenolic content and radical scavenging activities were found for the ethyl acetate and dichloromethane fractions among the different plant extracts. In general, the distilled plant material was found to exhibit a higher phenolic content as well as antioxidant and radical scavenging activities than the nondistilled material. Ethyl acetate and dichloromethane extracts, and even some crude extract, of both distilled and nondistilled plants exhibited activities comparable to those of commercial extracts/compounds, thus making it possible to consider some of them as a potential source of antioxidants of natural origin.