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Insulin resistance (IR) and its health consequences (diabetes, hypertension, cardiovascular disease, obesity etc.) affect between 25 and 35% of Westernized populations. Decreased fatty acid (FA) oxidation in skeletal muscle is implicated in obesity-related IR. Carnitine-acylcarnitine translocase (CACT) transports long-chain FAs both into mitochondria (as carnitine esters for energy-generating processes) and out of mitochondria. To determine whether CACT activity correlates with decreased FA oxidation we measured CACT concentrations in cellular and mitochondrial extracts from the skeletal muscle of 19 obese IR individuals and of 19 lean controls. We also evaluated carnitine transport in skeletal muscle mitochondria in both groups. Mitochondrial CACT was decreased at translational and transductional level, and carnitine-carnitine and acylcarnitine-carnitine exchange rates were significantly lower in IR subjects. Aberrant acylcarnitine flux into mitochondria was not correlated with decreased activity of other components of the mitochondrial carnitine system (i.e., carnitine palmitoyl transferase-I and II). Our data suggest that by restraining entry of FA-coenzyme A into mitochondria, low CACT levels increase cytosolic FA levels and their incorporation into glycerolipids. The low level of CACT in IR muscle may contribute to the elevated muscle concentrations of triglycerides, diacylglycerol, and FA-coenzyme A characteristic of IR muscle.

Three caffeoylquinic acids, isolated from the Peruvian plants Tessaria integrifolia and Mikania cordifolia that are used medicinally as anti-inflammatory agents, were tested for their activities on monocyte migration and superoxide anion production. 3,5-Di-O-caffeoylquinic and 4,5-di-O-caffeoylquinic acids exhibited an appreciable anti-inflammatory activity in vitro, while the tricaffeoyl derivative was inactive.

Carnitine, a name derived from the Latin carnis (flesh), was isolated from meat extracts in 19051 and early its chemical formula (C7H15NO3) was proposed. Its structure, a trimethylbetaine of γ-amino-β-hydroxybutyric acid, was correctly identified and published about twenty years later.2 Initially, some circumstances led to consider carnitine as a vitamin. By about 1945, all of the important vitamins of the B group had been identified, but the interest in the discovery of still missing B-vitamins, their lack being possibly correlated with anemia, was tremendous. In those years Fraenkel and coworkers observed that the mealworm Tenebrio molitor required for normal growth and survival, in addition to at least eight of the known B-vitamins, also folic acid and a new factor contained in brewers yeast or in liver extract, which they tentatively named vitamin-BT (T for Tenebrio).3 The unfavorable properties of this factor (it was hygroscopic and extremely water soluble, thus, hard to crystallize) made its isolation difficult but, finally, the missing vitamin-BT was identified as carnitine.4 The widespread distribution of carnitine was established in microorganisms, lower animals, and in all organs of mammals, and in plants too.5

This work was supported by The Scientific and Technological Research Council of Turkey (TUBITAK) (Project Number: 117S216) to S.O. Senotherapeutics are new drugs that can modulate senescence phenomena within tissues and reduce the onset of age-related pathologies. Senotherapeutics are divided into senolytics and senomorphics. The senolytics selectively kill senescent cells, while the senomorphics delay or block the onset of senescence. Metformin has been used to treat diabetes for several decades. Recently, it has been proposed that metformin may have anti-aging properties as it prevents DNA damage and inflammation. We evaluated the senomorphic effect of 6 weeks of therapeutic metformin treatment on the biology of human adipose mesenchymal stromal cells (MSCs). The study was combined with a proteome analysis of changes occurring in MSCs' intracellular and secretome protein composition in order to identify molecular pathways associated with the observed biological phenomena. The metformin reduced the replicative senescence and cell death phenomena associated with prolonged in vitro cultivation. The continuous metformin supplementation delayed and/or reduced the impairment of MSC functions as evidenced by the presence of three specific pathways in metformin-treated samples: 1) the alpha-adrenergic signaling, which contributes to regulation of MSCs physiological secretory activity, 2) the signaling pathway associated with MSCs detoxification activity, and 3) the aspartate degradation pathway for optimal energy production. The senomorphic function of metformin seemed related to its reactive oxygen species (ROS) scavenging activity. In metformin-treated samples, the CEBPA, TP53 and USF1 transcription factors appeared to be involved in the regulation of several factors (SOD1, SOD2, CAT, GLRX, GSTP1) blocking ROS. Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) 117S216

Several investigations on senescence and its causative role in aging have underscored the importance of developing senotherapeutics, a field focused on killing senescent cells and/or preventing their accumulation within tissues. Using polyphenols in counteracting senescence may facilitate the development of senotherapeutics given their presence in the human diet, their confirmed tolerability and absence of severe side effects, and their role in preventing senescence and inducing the death of senescent cells. Against that background, we evaluated the effect of piceatannol, a natural polyphenol, on the senescence of mesenchymal stromal cells (MSCs), which play a key role in the body’s homeostasis. Among our results, piceatannol reduced the number of senescent cells both after genotoxic stress that induced acute senescence and in senescent replicative cultures. Such senotherapeutics activity, moreover, promoted the recovery of cell proliferation and the stemness properties of MSCs. Altogether, our findings demonstrate piceatannol’s effectiveness in counteracting senescence by targeting its associated pathways and detecting and affecting P53-dependent and P53-independent senescence. Our study thus suggests that, given piceatannol’s various mechanisms to accomplish its pleiotropic activities, it may be able to counteract any senescent phenotypes.

Chestnut seeds are used for fresh consumption and for the industrial preparation of derivatives, such as chestnut flour. During industrial processing, large amounts of by-products are generally produced, such as leaves, flowers, shells and burs. In the present study, chestnut shells were extracted by boiling water in order to obtain polyphenol-rich extracts. Moreover, for the removal or non-phenolic compounds, a separation by preparative reverse phase chromatography in ten fractions was carried out. The richest fractions in terms of phenolic content were characterized by means of untargeted high-resolution mass spectrometric analysis together with a dedicated and customized data processing workflow. A total of 243 flavonoids, phenolic acids, proanthocyanidins and ellagitannins were tentatively identified in the five richest fractions. Due its high phenolic content (450.03 &micro, g GAE per mg of fraction), one tumor cell line (DU 145) and one normal prostate epithelial cell line (PNT2) were exposed to increasing concentration of fraction 3 dry extract for 24, 48 and 72 h. Moreover, for DU 145 cell lines, increase of apoptotic cells and perturbation of cell cycle was demonstrated for the same extract. Those outcomes suggest that chestnut industrial by-products could be potentially employed as a source of bioresources.

Resin-based composites are widely used as dental restorative materials due to their excellent properties. They must have high modulus, high hardness, and be chemically inert while minimizing moisture uptake. To fulfill these higher standard prerequisites and properties, continuous improvements in each of their components are required. This study develops novel composites with multiple biofunctions. Light-cured Bis-GMA/TEGDMA dental resin (RK)/layered double hydroxide intercalated with fluoride ions (LDH-F)/calcium bentonite (Bt) hybrid composites were prepared. The loading ratio of LDH-F to Bt was varied, ranging from 2.5/2.5 to 10/10 parts per hundred RK and structural, mechanical, and biological properties were studied. The incorporation of even small mass fractions (e.g., 2.5 wt % of LDH-F and 2.5 wt % of Bt) in RK dental resin significantly improved the mechanical properties of the pristine resin. The synthetized materials showed antibacterial and antibiofilm effects against three bacterial strains isolated from healthy volunteers&rsquo, saliva (Streptococcus spp., Bacteroides fragilis, and Staphylococcus epidermidis) without affecting its ability to induce dental pulp stem cells differentiation into odontoblast-like cells. The capability to balance between the antibiofilm activity and dental pulp stem cells differentiation in addition with improved mechanical properties make these materials a promising strategy in preventive and restorative dentistry.

Resveratrol (RSV) has been shown to exhibit many biological properties that can influence bone osteogenesis. However, RSV oral clinical treatment is limited due to its poor pharmacokinetics, low water solubility, and rapid metabolism. Therefore, it is necessary to develop a valid delivery system to release RSV directly into the target site. Electrospun drug-eluting fibers have gained great attention in the regenerative dentistry due to the ease of fabrication, the high surface to volume ratio and the drug-loading efficiency. The post-extraction preservation of the alveolar socket requires to operate on the bone remodeling processes both by stimulation of bone formation by osteoblasts and inhibition of osteoclast-mediated bone resorption. In this work, uniform defect-free fibers of poly(e-caprolactone) PCL and poly(lactic) acid (PLA) loading resveratrol were synthetized and characterized. In vitro assay demonstrated that the two membranes were able to release RSV in a tunable and sustained manner with different kinetic: PCL-RSV membrane showed an initial burst followed by a slow release, while PLA-RSV presented a much slower and continuous release over the time. Although both RSV-loaded materials showed similar in vitro osteoinductive capacity on human dental pulp stem cells, the differences on RSV release kinetic affected RANKL-induced osteoclastogenesis. Indeed, only the lower resveratrol-releasing membrane (PLA-RSV) was able both to induce osteoblast and to inhibit osteoclast differentiation, suggesting that this bioactive membrane could be used to preserve post-extraction alveolar ridge volume acting simultaneously on two fronts: first counteract bone resorption, second allows new bone formation.

In this study, chestnut shells (CS) were used in order to obtain bioactive compounds through different extraction procedures. The aqueous extracts were chemically characterized. The highest extraction yield and total phenolic content was obtained by conventional liquid extraction (CLE). Gallic and protocatechuic acids were the main simple phenols in the extract, with 86.97 and 11.20 mg/g chestnut shells dry extract (CSDE), respectively. Six tumor cell lines (DU 145, PC-3, LNCaP, MDA-MB-231, MCF-7, and HepG2) and one normal prostate epithelial cell line (PNT2) were exposed to increasing concentration of CSDE (1–100 µg/mL) for 24 h, and cell viability was evaluated using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide MTT assay. A reduced rate in cell viability was observed in DU 145, PC-3, LNCaP, and MCF-7 cells, while viability of the other assessed cells was not affected, except for PNT2 cells at a concentration of 100 μg/mL. Furthermore, CSDE—at concentrations of 55.5 and 100 µg/mL—lead to a significant increase of apoptotic cells in DU 145 cells of 28.2% and 61%, respectively. In conclusion, these outcomes suggested that CS might be used for the extraction of several polyphenols that may represent good candidates for alternative therapies or in combination with current chemotherapeutics.

Mitochondrial dysfunction seems to play a fundamental role in the pathogenesis of neurodegeneration in Huntington's disease (HD). We assessed possible neuroprotective actions of meldonium, a small molecule affecting mitochondrial fuel metabolism, in in vitro and in vivo HD models. We found that meldonium was able to prevent cytotoxicity induced by serum deprivation, to reduce the accumulation of mutated huntingtin (mHtt) aggregates, and to upregulate the expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) in mHTT-expressing cells. The PGC-1α increase was accompanied by the increment of mitochondrial mass and by the rebalancing of mitochondrial dynamics with a promotion of the mitochondrial fusion. Meldonium-induced PGC-1α significantly alleviated motor dysfunction and prolonged the survival of a transgenic HD Drosophila model in which mHtt expression in the nervous system led to progressive motor performance deficits. Our study strongly suggests that PGC-1α, as a master coregulator of mitochondrial biogenesis, energy homeostasis, and antioxidant defense, is a potential therapeutic target in HD.

BACKGROUND: Research on physiopathology of obesity may receive new hints from studies on skinny people (SP). These are individuals who show a poor or null gaining of body weight, in spite of high-calorie intake, by far exceeding the body requirements. AIM: To evaluate how circulating factors present in the SP sera may affect adipogenesis of mesenchymal stromal cells (MSCs). METHODS: We isolated MSCs from bone marrow of healthy donors with both normal body mass index (BMI) and caloric consumption. MSC cultures were primed with sera collected from SP or normal people (NP). Then biomolecular assays were performed to evaluate effect on proliferation, apoptosis, senescence, cell commitment, and differentiation. RESULTS: SP priming affected adipocyte cell commitment and reduced spontaneous adipogenesis. Moreover, an in-depth analysis of exogenous-induced adipocyte differentiation showed striking differences between differentiation in SP-primed samples compared with NP ones. In adipocytes from SP cultures we observed a reduced size of lipid droplets, an increased expression of adipose triglyceride lipase, along with high mitochondria content and ability to produce ATP in starvation condition. These data and the expression of UCP1 protein, indicated that SP pretreatment produced a bias toward brown adipocyte differentiation. CONCLUSION: Our data suggest that sera from SP may promote brown adipogenesis rather that white adipocyte differentiation. This finding could explain why SP present normal body composition in spite of an excess of caloric intake. We hypothesize that some circulating components present in the blood of these individuals may favor brown adipogenesis at expense of white adipocyte production.

Fluoride-releasing restorative dental materials can be beneficial to remineralize dentin and help prevent secondary caries. However, the effects of fluoride release from dental materials on the activity of dental pulp stem cells are not known. Here we investigate whether different fluoride release kinetics from dental resins supplemented with modified hydrotalcite (RK-F10) or fluoride-glass filler (RK-FG10) could influence the behavior of a human dental pulp stem cell subpopulation (STRO-1+ cells) known for its ability to differentiate toward an odontoblast-like phenotype. The 2 resins, characterized by similar physicochemical properties and fluoride content, exhibited different long-term fluoride release kinetics. Our data demonstrate that long-term exposure of STRO-1+ cells to a continuous release of a low amount of fluoride by RK-F10 increases their migratory response to transforming growth factor β1 (TGF-β1) and stromal cell–derived factor 1 (SDF-1), both important promoters of pulp stem cell recruitment. Moreover, the expression patterns of dentin sialoprotein ( dspp), dentin matrix protein 1 ( dmp1), osteocalcin ( ocn), and matrix extracellular phosphoglycoprotein ( mepe) indicate a complete odontoblast-like cell differentiation only when STRO-1+ cells were cultured on RK-F10. On the contrary, RK-FG10, characterized by an initial fluoride release burst and reduced lifetime of the delivery, did not elicit any significant effect on both STRO-1+ cell migration and differentiation. Taken together, our results highlight the importance of taking into account fluoride release kinetics in addition to fluoride concentration when designing new fluoride-restorative materials.

Curcumin, a nontoxic, naturally occurring polyphenol, has been recently proposed for the management of neurodegenerative and neurological diseases. However, a discrepancy exists between the well-documented pharmacological activities that curcumin seems to possess in vivo and its poor aqueous solubility, bioavailability, and pharmacokinetic profiles that should limit any therapeutic effect. Thus, it is possible that curcumin could exert direct regulative effects primarily in the gastrointestinal tract, where high concentrations of curcumin are present after oral administration. Indeed, a new working hypothesis that could explain the neuroprotective role of curcumin despite its limited availability is that curcumin acts indirectly on the central nervous system by influencing the “microbiota−gut−brain axis”, a complex bidirectional system in which the microbiome and its composition represent a factor which preserves and determines brain “health”. Interestingly, curcumin and its metabolites might provide benefit by restoring dysbiosis of gut microbiome. Conversely, curcumin is subject to bacterial enzymatic modifications, forming pharmacologically more active metabolites than curcumin. These mutual interactions allow to keep proper individual physiologic functions and play a key role in neuroprotection.

Lymphocytes are unique cells evolved to guard, in collaboration with other accessory cells (such as macrophages), the selfness of vertebrate organisms against the invasiveness of alien elements or macromolecules. Recognition of intruders is obtained either by cell-cell or cell-molecule interactions, in which sophisticated biochemical machineries, endowed in the lymphocyte plasma membrane, are set in action as important parts of defence mechanisms used by singles to defend their own individualities. As a consequence of the activity of all these surface molecular devices, a metabolic turmoil is triggered in these cells. Numerous intra- and extra-cellular biochemical signals are released, bringing about in a short time marked modifications of a number of important metabolic pathways such as those related to: 1) energy production; 2) control of electrolyte traffic in the cell; 3) transcription and translation of specific messenger RNA; 4) biosynthesis of tRNA and rRNA; 5) control of DNA replication and cell division1–10.

Copolymerisation of charged and neutral monomers is a well-tested strategy to introduce charged moieties in a polymeric chain and obtain polyelectrolyte polymers. We have synthesized new cationic and anionic polyelectrolytes by bulk radical copolymerisation of 2-hydroxyethyl methacrylate with [2-methacryloyloxyethyltrimethyl ammonium chloride (METAC)] or [2-acrylamido-2-methylpropane-sulphonic acid (AMPS)] monomers. The chemical structure of the synthesized copolymers was confirmed by FT-IR spectroscopy. Swelling studies on synthesized copolymers showed a high water content in the swollen state and a “smart behaviour” upon changes in external stimuli (pH and ionic strength). Cytotoxicity and cytocompatibility studies demonstrated that synthesized materials were not toxic. Moreover, the cationic copolymer showed good cell adhesion, whereas the anionic copolymer showed poor cell adhesion on its surface. X-ray photoelectron spectroscopy (XPS) showed that the disparate behaviour was due to the chemical nature of charged groups on the copolymer surfaces.

The mechanism by which estradiol (E2) acts on cell proliferation is still unclear. In this paper, we report the results of a series of experiments in an attempt to elucidate the effector pathway(s) involved in coupling the E2 receptors binding to cellular growth response in leiomyoma cells (LSMC). Under conditions of E2-dependent growth, E2 treatment of LSMC triggers rapid and transient activation of the MAP-kinase pathway. Interestingly, we demonstrate that the early downstream signal transduction events determined by E2-stimulation in quiescent LSMC, including the rapid protein tyrosine phosphorylation of a subset of intracellular proteins, such GAP, PI-3-K, and PLCg, and the concomitant activation of ancillary protein kinases, are related to E2-induced PDGF secretion. Moreover, we identify the PDGF, alone or in association with other growth factors, as the main growth factor involved in the proliferation response of LSMC to E2 stimulation. The addition of neutralizing antibodies anti-PDGF was able to inhibit the mitogenic activity present in LSMC conditioned media samples. On the other hand, E2 did not affect the constitutive expression as well as the ligand af®nity of PDGF receptors on LSMC plasmamembrane. Cell treatment with the antiestrogen ICI 182780 correlate both with a perturbation of E2-induced transductional circuit and with the disappearance of the mitogenic factor, PDGF, in LSMC conditioned media; the latter therefore, represents the main autocrine mediator of cell growth modulation, upregulated by E2 and down-regulated by antiestrogenic compound. Our experiments suggest that growth factor secretion is an initial and integral part of the signaling events mediated by the estradiol receptors, not related, at least in part, to E2 transcriptional modulation. The mechanism by which estradiol (E2) acts on cell proliferation is still unclear. In this paper, we report the results of a series of experiments in an attempt to elucidate the effector pathway(s) involved in coupling the E2 receptors binding to cellular growth response in leiomyoma cells (LSMC). Under conditions of E2-dependent growth, E2 treatment of LSMC triggers rapid and transient activation of the MAP-kinase pathway. Interestingly, we demonstrate that the early downstream signal transduction events determined by E2-stimulation in quiescent LSMC, including the rapid protein tyrosine phosphorylation of a subset of intracellular proteins, such GAP, PI-3-K, and PLCγ, and the concomitant activation of ancillary protein kinases, are related to E2-induced PDGF secretion. Moreover, we identify the PDGF, alone or in association with other growth factors, as the main growth factor involved in the proliferation response of LSMC to E2 stimulation. The addition of neutralizing antibodies anti-PDGF was able to inhibit the mitogenic activity present in LSMC conditioned media samples. On the other hand, E2 did not affect the constitutive expression as well as the ligand affinity of PDGF receptors on LSMC plasmamembrane. Cell treatment with the antiestrogen ICI 182780 correlate both with a perturbation of E2-induced transductional circuit and with the disappearance of the mitogenic factor, PDGF, in LSMC conditioned media; the latter therefore, represents the main autocrine mediator of cell growth modulation, upregulated by E2 and down-regulated by antiestrogenic compound. Our experiments suggest that growth factor secretion is an initial and integral part of the signaling events mediated by the estradiol receptors, not related, at least in part, to E2 transcriptional modulation. © 2001 Wiley-Liss, Inc.

An efficient regulation of fuel metabolism in response to internal and environmental stimuli is a vital task that requires an intact carnitine system. The carnitine system, comprehensive of carnitine, its derivatives, and proteins involved in its transformation and transport, is indispensable for glucose and lipid metabolism in cells. Two major functions have been identified for the carnitine system: (1) to facilitate entry of long-chain fatty acids into mitochondria for their utilization in energy-generating processes; (2) to facilitate removal from mitochondria of short-chain and medium-chain fatty acids that accumulate as a result of normal and abnormal metabolism. In cancer patients, abnormalities of tumor tissue as well as nontumor tissue metabolism have been observed. Such abnormalities are supposed to contribute to deterioration of clinical status of patients, or might induce cancerogenesis by themselves. The carnitine system appears abnormally expressed both in tumor tissue, in such a way as to greatly reduce fatty acid beta-oxidation, and in nontumor tissue. In this view, the study of the carnitine system represents a tool to understand the molecular basis underlying the metabolism in normal and cancer cells. Some important anticancer drugs contribute to dysfunction of the carnitine system in nontumor tissues, which is reversed by carnitine treatment, without affecting anticancer therapeutic efficacy. In conclusion, a more complex approach to mechanisms that underlie tumor growth, which takes into account the altered metabolic pathways in cancer disease, could represent a challenge for the future of cancer research.

To clarify the role and function of the N-myc product in cell differentiation and apoptosis, we used the antisense oligonucleotide technique to inhibit N-myc gene expression in neuroblastoma cells with different phenotypes: intermediate (I) and neuronal (N), or Schwann-glia (S), respectively. The results suggest that N-myc operates along different pathways. Inhibiting N-myc gene expression either results in suppression of cell proliferation or in induction of differentiation and/or apoptosis.

Bone marrow adipose tissue (BMAT) is different from fat found elsewhere in the body, and only recently have some of its functions been investigated. BMAT may regulate bone marrow stem cell niche and plays a role in energy storage and thermogenesis. BMAT may be involved also in obesity and osteoporosis onset. Given the paramount functions of BMAT, we decided to better clarify the human bone marrow adipogenesis by analyzing the role of the retinoblastoma gene family, which are key players in cell cycle regulation. Our data provide evidence that the inactivation of RB1 or RB2/P130 in uncommitted bone marrow stromal cells (BMSC) facilitates the first steps of adipogenesis. In cultures with silenced RB1 or RB2/P130, we observed an increase of clones with adipogenic potential and a higher percentage of cells accumulating lipid droplets. Nevertheless, the absence of RB1 or RB2/P130 impaired the terminal adipocyte differentiation and gave rise to dysregulated adipose cells, with alteration in lipid uptake and release. For the first time, we evidenced that RB2/P130 plays a role in bone marrow adipogenesis. Our data suggest that while the inactivation of retinoblastoma proteins may delay the onset of last cell division and allow more BMSC to be committed to adipocyte, it did not allow a permanent cell cycle exit, which is a prerequisite for adipocyte terminal maturation. © 2014 Landes Bioscience.

Objectives The aim of this work was the preparation of a new fluoride-releasing dental material characterized by a release of fluoride relatively constant over time without any initial toxic burst effect. This type of delivery is obtained by a matrix controlled elution and elicits the beneficial effect of a low amount of fluoride on human dental pulp stem cells (hDPSCs) towards mature phenotype. Methods The modified hydrotalcite intercalated with fluoride ions (LDH-F), used as filler, was prepared via ion exchange procedure and characterized by X-ray diffraction and FT-IR spectroscopy. The LDH-F inorganic particles (0.7, 5, 10, 20 wt.%) were mixed with a photo-activated Bis-GMA/TEGDMA (45/55 wt/wt) matrix and novel visible-light cured composites were prepared. The dynamic thermo-mechanical properties were determined by dynamic mechanical analyzer. The release of fluoride ions in physiological solution was determined using a ionometer. Total DNA content was measured by a PicoGreen dsDNA quantification kit to assess the proliferation rate of hDPSCs. Alkaline phosphatase activity (ALP) was measured in presence of fluoride resins. Results Incorporation of even small mass fractions ( e.g. 0.7 and 5 wt.%) of the fluoride LDH in Bis-GMA/TEGDMA dental resin significantly improved the mechanical properties of the pristine resin, in particular at 37 °C. The observed reinforcement increases on increasing the filler concentration. The release of fluoride ions resulted very slow, lasting months. ALP activity gradually increased for 28 days in hDPSCs cell grown, demonstrating that low concentrations of fluoride contributed to the cell differentiation. Conclusions The prepared composites containing different amount of hydrotalcite filler showed improved mechanical properties, slow fluoride release and promoted hDPSCs cell proliferation and cell differentiation.

Purpose The purpose of this work was to achieve detailed biomaterials characterization of a drug delivery system for local cancer treatment based on electrospun titanocene trichloride-loaded resorbable polycaprolactone (PCL) fibers. Methods The PCL fibers were characterized for their structural, morphologic and physical properties. The drug release kinetics of the titanocene complex was investigated at different concentrations, to obtain a set of correlations between structure and tuneable release. After exposing cancer cells directly onto the surface of PCL fibers, the anti-proliferative effects of titanocene-loaded PCL were assessed by: (i) counting viable cells via live/dead staining methods, and (ii) analyzing cell apoptosis. Results and Conclusions Titanocene concentration influenced fiber diameters reduced for PCL filled with titanocene. X-ray analysis suggested that the titanocene, encapsulated into the PCL fibers, is not allowed to crystallize and exists as amorphous aggregates into the fibers. The titanocene release curves presented two stages unrelated to PCL degradation: an initial burst release followed by a release linear with time, extending for a very long time. All of the titanocene-loaded fibers revealed sustained drug release properties suggesting their potential clinical applicability for the treatment of local cancer diseases.

Objectives Vascular endothelial growth factor (VEGF) is thought to play an important role in systemic sclerosis (SSc) pathogenesis. It was found to be upregulated in the serum and in the affected skin of scleroderma patients. However, its involvement in scleroderma lung disease is not clear. This study aimed to evaluate VEGF concentration in the bronchoalveolar lavage fluid (BALF) of scleroderma patients with interstitial lung disease, to correlate the cytokine levels in plasma and in the lung with pulmonary functional, radiological and cellular parameters, and with the progression of lung disease. Methods BALF and plasma VEGF concentrations were analysed by ELISA in 55 SSc patients with lung disease and 17 controls. Cytokine real-time PCR messenger RNA expression in alveolar macrophages was assessed. Lung involvement progression was evaluated after a 1-year follow-up. Results VEGF was found to be significantly lower in the BALF of scleroderma patients compared with controls. The lowest concentrations were observed in SSc patients with alveolitis. A decreased VEGF expression in alveolar macrophages was found in SSc patients with alveolitis. VEGF concentration in BALF correlated inversely with the ground glass score on high-resolution CT and with BALF neutrophil cell count. Moreover, SSc patients with a lower VEGF concentration showed a worsening in the interstitial score at follow-up. Conclusions Scleroderma interstitial lung disease is characterised by a VEGF deficiency. Lower concentrations were found in patients with progression of lung disease.

Growth factors and other regulatory molecules are required to direct differentiation of bone marrow-derived human mesenchymal stem cells (hMSC) along specific lineages. However, the therapeutic use of growth factors is limited by their susceptibility to degradation, and the need to maintain prolonged local release of growth factor at levels sufficient to stimulate hMSC. The aim of this study was to investigate whether a device containing heparan sulfate (HS), which is a co-factor in growth factor-mediated cell proliferation and differentiation, could potentiate and prolong the delivery of fibroblast growth factor-2 (FGF-2) and thus enhance hMSC stimulation. To this aim, we synthesized cationic polyelectrolyte polymers covalently and non-covalently anchored to HS and evaluated their effect on hMSC proliferation. Polymers non-covalently bound to HS resulted in the release of an HS/FGF-2 complex rather than FGF-2 alone. The release of this complex significantly restored hMSC proliferation, which was abolished in serum-free medium and only partially restored by the release of FGF-2 alone as occurred with polymer covalently bound to HS. We also demonstrate that exposure to HS/FGF-2 during early growth but not during post-confluence is essential for hMSC differentiation down the fibroblast lineage, which suggests that both factors are required to establish the correct stem cell commitment that is necessary to support subsequent differentiation. In conclusion, the delivery platform described here is a step towards the development of a new class of biomaterial that enables the prolonged, non-covalent binding and controlled delivery of growth factors and cofactors without altering their potency.

In the large intestine organic cation transporter type-2 (OCTN2) is recognized as a transporter of compounds such as carnitine and colony sporulation factor, promoting health of the colon intestinal epithelium. Recent reports suggest that OCTN2 expression in small intestine is under control of peroxisome proliferator-activated receptor-alpha (PPAR alpha). However, PPAR alpha contribution to colonic OCTN2 expression remains controversial. Here we examined the transcriptional regulation of colon OCTN2 gene by PPAR alpha. To exclude any additional modulation of other PPAR to OCTN2 expression, we used both in vivo and in vitro PPAR-null models and specific PPAR inhibitors. The PPAR gamma agonists thiazolidinediones increased both OCTN2 mRNA and protein expression in colonic epithelial cell lines independently by PPAR alpha expression. The induction was blocked only by PPAR gamma antagonists or by gamma ORF4, a PPAR gamma isoform with dominant negative activity, suggesting a PPAR gamma-dependent mechanism. A conserved noncanonical PPAR-responsive element was found by computational analysis in the first intron of human OCTN2 gene and validated by EMSA assay. Promoter-reporter assays further confirmed transcriptional functionality of the putative PPAR response element, whereas selective mutation caused complete loss of responsiveness to PPAR gamma activation. Finally, adenovirus-mediated overexpression of constitutively active PPAR gamma mutant increased colon OCTN2 expression in PPAR alpha(-/-) mice. Interestingly, animals overexpressing colon PPAR gamma showed a significant increase in plasma carnitine, thus demonstrating the functional contribution of large intestine to systemic carnitine homeostasis. This study reveals a PPAR gamma-dependent absorption machinery in colon that is likely involved in the health of colon epithelium, in the microbiota-host interactions and in the absorption of nutraceuticals and drugs.

Protein aggregates are a hallmark of polyglutamine diseases, including Huntington’s disease (HD). The transglutaminases, a class of Ca2+-dependent cross-linking enzymes, could be directly involved in the disease mechanisms [14,15]. In fact, we have shown that when tTGase is activated by a calcium ionophore, the expanded huntingtin fragment is more abundant and insoluble high molecular weight (Mw) aggregates in HD fibroblasts appear together with evidence of apoptosis. Interestingly, in chorea-acanthocytosis (ChAc) a rare autosomal-recessive disorder without CAG repeats, we found increased amounts of tTGase products in muscle and erythrocytes. Furthermore, immunohistochemistry demonstrated abnormal accumulation of tTGase products, as well as of proteinaceous bodies in ChAc muscles.

Recent studies suggest a close relationship between cell metabolism and apoptosis. We have evaluated changes in lipid metabolism on permeabilized hepatocytes treated with truncated Bid (tBid) in the presence of caspase inhibitors and exogenous cytochrome c. The measurement of beta-oxidation flux by labeled palmitate demonstrates that tBid inhibits beta-oxidation, thereby resulting in the accumulation of palmitoyl-coenzyme A (CoA) and depletion of acetyl-carnitine and acylcarnitines, which is pathognomonic for inhibition of carnitine palmitoyltransferase-1 (CPT-1). We also show that tBid decreases CPT-1 activity by a mechanism independent of both malonyl-CoA, the key inhibitory molecule of CPT-1, and Bak and/or Bax, but dependent on cardiolipin decrease. Overexpression of Bcl-2, which is able to interact with CPT-1, counteracts the effects exerted by tBid on beta-oxidation. The unexpected role of tBid in the regulation of lipid beta-oxidation suggests a model in which tBid-induced metabolic decline leads to the accumulation of toxic lipid metabolites such as palmitoyl-CoA, which might become participants in the apoptotic pathway.

We present preliminary experimental results concerning both single and stacked tunnel junctions in which one of the electrodes was formed by a superconductor/ferromagnet (S/F) bi-layer. In particular, in the stacked configuration a Nb/NiCu bi-layer was used as the intermediate electrode, and it was probed by tunneling on both sides. Tunnel junctions have been characterized in terms of current-voltage characteristics (IVC), and differential conductance. Preliminary steady-state injection-detection measurements performed in the stacked devices at T 4: 2 K are also presented and discussed. (C) 2003 Elsevier B.V. All rights reserved.

Fibrous encapsulation is known to occur to many prosthetic implants and is thought to be due to the cells not adhering adequately to the surface. For developing new materials able to enhance cellular adhesion by mimicking extracellular matrix components, polyelectrolyte polymers, characterized by tunable surface charges, have been proposed. Here we demonstrate that panoply of cell functions over a two-dimensional substratum is influenced by surface charge. We have at first generated structurally related polyelectrolyte substrata varying in their positive surface charge amount and subsequently evaluated a variety of behaviors of human primary fibroblasts seeded on these polymers. The proportion of adherent, spreading, and proliferating cells was increased significantly on cationic hydrophilic surfaces when compared with the neutral base surface. The extent of cell spreading correlated with cytoskeleton organization as assessed using immunofluorescence techniques. In the key experiment, the presence of cationic charges on cell adhesion-resistant neutral surface increased the synthesis of collagen I and III, the release of their metabolites, and the expression of their mRNA by fibroblasts. Interestingly, the scarce collagen deposits on neutral polymer consisted, for the most part, of collagen I while collagen III was present only in traces probably due to the secretion of metalloproteinase-2 by non-adherent fibroblasts. Taken together, these results show that polyelectrolyte films may promote the attachment of fibroblast cells as well as their normal secretory phenotype. Both effects could be potentially useful in integrating soft connective tissue to the implant, decreasing the chance of its fibrous encapsulation. J. Cell. Physiol. 198: 133–143, 2004. © 2003 Wiley-Liss, Inc.

Carnitine, gamma-trimethyl-beta-hydroxybutyrobetaine, is a small molecule widely present in all cells from prokaryotic to eukaryotic ones. It is the sole source of carbon and nitrogen in some bacteria; it serves as osmoprotectant in others. It is a carrier of acyl moieties, and exclusively of long-chain fatty acids for mitochondrial beta-oxidation in mammals. The conspicuously similar composition of the intracellular milieu among widely different species in relation to organic osmolyte systems involves the methylamine family to which carnitine belongs. This prompted us to examine the osmolytic properties of carnitine in an attempt to clarify the metabolic functions carnitine has acquired during evolution. An understanding of the metabolic functions of this organic compatible solute impinge on research involving this compound.

To investigate possible biochemical mechanisms underlying the ‘‘toxic gain of function’’ associated with polyglutamine expansions, the ability of guinea pig liver tissue transglutaminase to catalyze covalent attachments of various polyamines to polyglutamine peptides was examined. Of the polyamines tested, spermine is the most active substrate, followed by spermidine and putrescine. Formation of covalent cross links between polyglutamine peptides and polyamines yields high-Mr aggregates—a process that is favored with longer polyglutamines. In the presence of tissue transglutaminase, purified glyceraldehyde-3phosphate dehydrogenase (a key glycolytic enzyme that binds tightly to the polyglutamine domains of both huntingtin and dentatorubral‐pallidoluysian atrophy proteins) is covalently attached to polyglutamine peptides in vitro, resulting in the formation of high-Mr aggregates. In addition, endogenous glyceraldehyde-3-phosphate dehydrogenase of a Balb-c 3T3 fibroblast cell line overexpressing human tissue transglutaminase forms cross-links with a Q60 polypeptide added to the cell homogenate. Possibly, expansion of polyglutamine domains (thus far known to occur in the gene products associated with at least seven neurodegenerative diseases) leads to increased/aberrant tissue transglutaminase-catalyzed cross-linking reactions with both polyamines and susceptible proteins, such as glyceraldehyde-3-phosphate dehydrogenase. Formation of cross-linked heteropolymers may lead to deposition of high-Mr protein aggregates, thereby contributing to cell death. © 1998 Academic Press

Poly-L-lysine, a high molecular weight synthetic compound with a large number of free amino groups, was glycated with lactose through the Maillard reaction (MR). Polyclonal mouse and rabbit antibodies (Abs) were obtained and characterised by competitive ELISA (enzyme-linked immuno assay), immunoblotting and cytofluorimetric assays. Results demonstrated that the Abs react specifically with lactosylated food proteins, i.e. bovine serum albumin (BSA) and casein and do not cross react with the underivatised proteins. The immunoreactivity between Abs and lactose-BSA adducts was strongly inhibited by N-ϵ-deoxy-lactulosyl-L-lysine, indicating that this typical MR product is recognised specifically by our Abs. These reagents can be a useful tool to monitor the MR reaction between carbohydrates and lysine, which occurs during the thermal treatment of food.

Zero bias anomaly phenomena in conductance spectra relative to superconductor/normal metal{endash}constriction{endash}normal metal ({ital S}{sub 1}/{ital N}{sub 1}-{ital c}-{ital N}{sub 2}) junctions are interpreted in terms of the proximity effect. Attention is focused in particular on the influence of the magnetic field on the conductance in point contact junctions and microjunctions with nonhomogeneous base electrodes ({ital S}{sub 1}/{ital N}{sub 1}). We find that the highly transmissive nature of the {ital N}{sub 1}-{ital c}-{ital N}{sub 2} interface is fundamental in determining the weak dependence of zero bias conductance on the applied magnetic field. The predictions of our model are compared with experimental data on Nb based junctions by Xiong {ital et} {ital al}. [Phys. Rev. Lett. {bold 71}, 1907 (1993)], supplying helpful information on the actual morphology of the structures. {copyright} {ital 1996 The American Physical Society.}

Both alpha- and beta-endorphin were shown to incorporate radioactive polyamines during incubation in the presence of purified transglutaminase and Ca2+, spermine acting as the best acyl acceptor substrate. The endorphin labelling is dependent on the time of exposure to the enzyme and on the substrate concentration. In the absence of acyl acceptor polyamines, isotopically labelled beta-endorphin gives rise to high molecular weight radioactive polymer(s). Moreover, when different proteins acting as transglutaminase substrates are added, beta-endorphin produces heterologous structures. These data indicate that the glutamine-71 of the beta-lipotropin chain, contained in both alpha- and beta-endorphin, functions as acyl donor substrate for the enzyme and that at least one beta-endorphin lysyl residue can serve as acyl acceptor.

Experimental results on stable point-contact Nb/Y-Ba-Cu-O structures are reported. Reproducible current-voltage characteristics showing Josephson coupling are obtained. Measurements of dV/dI give an estimate for the energy-gap value of the high-T/sub c/ superconductor Y-Ba-Cu-O ..delta.. = 19.5 +- 2.0 mV, corresponding to a ratio (2..delta..(0)/k/sub B/T/sub c/) = 4.8 in agreement with previous results obtained by a different technique.

SPECIFIC AIMSOxidizing free radicals reduce the chaperone activity of lens α-crystallin and increase the susceptibility of lens proteins to serve as substrate for transglutaminase (TGase) activity, thereby leading to the protein cross-linking and cataractogenesis typical of aging and diabetes. To evaluate whether L-carnitine protects α-crystallin function in its ability to prevent aberrant protein associations and inhibits TGase-induced protein cross-linking, we measured the molecular chaperone activity of α-crystallin and isopeptide cross-links in rat lenses exposed in vitro to H2O2 in the presence and absence of L-carnitine and in control (untreated) lenses.PRINCIPAL FINDINGS1. L-carnitine prevents depletion of free carnitine but not of reduced glutathione (GSH), increases acetyl-L carnitine concentration, and preserves cell integrity in lens stressed with H2O2Concentrations of free carnitine and GSH (which is highly represented in lens tissue and protects from in vitro diabetic cataract) were unchanged...

Two fragments of SV-IV, one of the major proteins secreted from the rat seminal vesicle epithelium, were produced in vitro by protein cleavage with CNBr at level of the single methionine residue (Met-70) occurring in its polypeptide chain. After their purification by reversed-phase chromatography, SV-IV/A (71-70 fragment) and SV-IV/B (71–90 fragment) were assayed as transglutaminase substrates, and their anti-inflammatory, anti-thrombotic and immunosuppressive properties were evaluated in comparison with native SV-IV. Both fragments retained the SV-IV ability to act as transglutaminase substrates in vitro; fast atom bombardment mass spectrometry analyses of the reaction products pointed to Gln-9 and Gln-86 as acyl donor sites, and to Lys-59, -79 and -80 as acyl acceptor sites. In contrast, only SV-IV/A was shown to possess, like SV-IV, the property of inhibiting both the intensity of the carrageenin-induced rat foot edema and the platelet aggregation induced in vivo by different agents. Finally, the two protein fragments were found to be completely unable to inhibit both the mitogen-induced proliferation of human T cells and the mixed lymphocyte reaction.

Fusaproliferin (FP) is a sesterterpene mycotoxin produced by some species of Fusarium, common contaminants of maize. Toxicity assays on Artemia salina L. brine shrimp larvae using FP and its derivatives showed that toxic activity of the metabolite increased after acetylation of the two hydroxyl groups and was reduced when the naturally occurring acetyl group was removed. In chick embryotoxicity bioassays, severe teratogenic effects were observed in 20% of the embryos exposed at 5 mM FP on day 1. In particular FP was responsible for cephalic dicotomy, macrocephaly, and limb asymmetry. At the same concentration, FP also caused pathological changes such as hemorrhaging on the surface of the wings, skull, and legs. Incomplete closure of the umbilicus was seen in 30% of embryos inoculated with 5 mM of FP on day 1 and in 20% of those inoculated on day 11. Keywords: Fusaproliferin; Fusarium proliferatum; chick; embryotoxicity; teratogenic; mycotoxin