Synthesis of 9-Hydroxystearic Acid Derivatives and Their Antiproliferative Activity on HT 29 Cancer Cells.

9-Hydroxystearic acid (9-HSA) is an endogenous cellular lipid that possesses antiproliferative and selective effects against cancer cells. A series of derivatives were synthesized in order to investigate the effect of the substituent in position 9 and on the methyl ester functionality on the biological activity. The two separate enantiomers of methyl 9-hydroxystearate and of methyl 9-aminostearate showed antiproliferative activity against the HT29 cell line. This indicates the importance of position 9 groups being able to make hydrogen bonding with the molecular target. Further, this effect must be preserved when the carboxy group of 9-HSA is esterified. The biological tests showed that the amines, contrarily to methyl esters, resulted in cytotoxicity. A deep investigation on the effect of methyl (R)-9-hydroxystearate on HT29 cells showed an antiproliferative effect acting through the CDKN1A and MYCBP gene expression.

Unprecedented Behavior of (9 R)-9-Hydroxystearic Acid-Loaded Keratin Nanoparticles on Cancer Cell Cycle.

Histone deacetylases, HDACs, have been demonstrated to play a critical role in epigenetic signaling and were found to be overexpressed in several type of cancers; therefore, they represent valuable targets for anticancer therapy. 9-Hydroxystearic acid has been shown to bind the catalytic site of HDAC1, inducing G0/G1 phase cell cycle arrest and activation of the p21WAF1 gene, thus promoting cell growth inhibition and differentiation in many cancer cells. Despite the ( R) enantiomer of 9-hydroxystearic acid (9R) displaying a promising in vitro growth-inhibitory effect on the HT29 cell line, its scarce water solubility and micromolar activity require novel solutions for improving its efficacy and bioavailability. In this work, we describe the synthesis and in vitro biological profiling of 9R keratin nanoparticles (9R@ker) obtained through an in-water drug-induced aggregation process. The anticancer activity of 9R@ker was investigated in the HT29 cell line; the results indicate an increased fluidity of cell membrane and a higher intracellular ROS formation, resulting in an unexpected S phase cell cycle arrest (25% increase as compared to the control) induced by 9R@ker with respect to free 9R and an induction of cell death.

Purification of active recombinant human histone deacetylase 1 (HDAC1) overexpressed in Escherichia coli.

OBJECTIVE: We attempted to overexpress Human Histone Deacetylase 1 (HDAC1) in Escherichia coli. RESULTS: A synthetic gene coding for HDAC1, and optimised for E. coli codon usage, was cloned into pBADHisB, generating pBAD-rHDAC1. This construct was used to transform E. coli TOP10, and the target protein was overexpressed and partially purified. According to its elution volume from a Superdex 200 column, the partially purified rHDAC1 was obtained in aggregated form, i.e., as an octamer. The dissociation of octameric HDAC1 was tested using several agents, among which sodium dodecyl sulfate was competent in partially dissociating rHDAC1 aggregates. When the enzyme activity was tested in vitro using 3H-acetyl-labelled histones both protein samples, aggregated and dissociated, were active. Hence, our results suggest that E. coli represents an alternative system for the production of the recombinant HDAC1. CONCLUSIONS: We described a procedure for the overexpression in E. coli of recombinant HDAC1, the purification of which in active form can be successfully performed, although yielding an octameric aggregate.

Correction to: Parma consensus statement on metabolic disruptors.

CORRECTION: After publication of the article [1], it has been brought to our attention that the thirteenth author of this article has had their name spelt incorrectly. In the original article the spelling "Laura Rizzir" was used. In fact the correct spelling should be "Laura Rizzi".

Synthesis, cytotoxicity and anti-cancer activity of new alkynyl-gold(I) complexes.

Alkynyl(triphenylphosphine)gold(i) complexes carrying variously substituted propargylic amines have been synthesized and fully characterized in solution and solid state. High levels of toxicity (i.e. micromolar range) were recognized for a series of cancer cell lines with particular emphasis on HT29, IGROV1, HL60 and I407. In particular the lead compound 3ab was identified as the most active compound in all cell lines (IC50: 1.7-7.9 µM).

Parma consensus statement on metabolic disruptors.

A multidisciplinary group of experts gathered in Parma Italy for a workshop hosted by the University of Parma, May 16-18, 2014 to address concerns about the potential relationship between environmental metabolic disrupting chemicals, obesity and related metabolic disorders. The objectives of the workshop were to: 1. Review findings related to the role of environmental chemicals, referred to as "metabolic disruptors", in obesity and metabolic syndrome with special attention to recent discoveries from animal model and epidemiology studies; 2. Identify conclusions that could be drawn with confidence from existing animal and human data; 3. Develop predictions based on current data; and 4. Identify critical knowledge gaps and areas of uncertainty. The consensus statements are intended to aid in expanding understanding of the role of metabolic disruptors in the obesity and metabolic disease epidemics, to move the field forward by assessing the current state of the science and to identify research needs on the role of environmental chemical exposures in these diseases. We propose broadening the definition of obesogens to that of metabolic disruptors, to encompass chemicals that play a role in altered susceptibility to obesity, diabetes and related metabolic disorders including metabolic syndrome.

Effects of ionic liquids on membrane fusion and lipid aggregation of egg-PC liposomes.

In this study we have explored the effects of different groups of ionic liquids (ILs) on membrane fusion. The ILs used contain different head groups: N-methylimidazolium, 3-methylpyridinium and N-methylpyrrolidinium; short alkyl or ether functionalized side chains (with one or two ethoxy functionalities), paired with chloride anion. These ILs have been compared with 1-dodecyl-3-methylimidazolium bromide as example of a highly lipophilic IL. The effect of ILs on membrane fusion was investigated through pyrene steady state fluorescence probing, using the IE factor and excimer/monomer ratio (IE/IM) as parameters. The ratio between the vibronic bands of pyrene (I1/I3 ratio) has been used to monitor the effect of ILs on the aggregation properties of egg-PC liposomes. The effect of different ILs' families was evident; the pyridinium ILs induced a greater extent of fusion than pyrrolidinium and imidazolium ILs having the same side chain. Marginal effect could be attributed to different anions. ILs with short alkyl chains were usually more effective than ether functionalized ones. The aggregation behaviors of ILs having dioxygenated chains have been measured in buffer solution.

A peptidic hydrogel that may behave as a "Trojan Horse".

A physical hydrogel prepared with the low-molecular-weight hydrogelator (LMWHG) CH2(C3H6CO-L-Phe-D-Oxd-OH)2 and water/ethanol mixture was applied as a potential "Trojan Horse" carrier into cells. By SEM and XRD analysis we could demonstrate that a fibrous structure is present in the xerogel, making a complex network. The gelator is derived from α-amino acids (Thr, Phe) and a fatty acid (azelaic acid) and is biocompatible: it was dosed to IGROV-1 cells, which internalized it, without significantly affecting the cell proliferation. To check the internalization process by confocal microscopy, fluorescent hydrogels were prepared, introducing the fluorescent dansyl moiety into the mixture.

Structural changes in a protein fragment from abalone shell during the precipitation of calcium carbonate.

Mineralized tissues grow through biologically controlled processes in which specific macromolecules are involved. Some of these molecules, which are present in very low concentrations and are difficult to localize and characterize, become entrapped inside the mineralized tissue. Herein, a protein fragment, GP, which was obtained by the alkaline digestion of the green sheet of the abalone shell, is used as a probe to study the changes in molecular structure that occur during the precipitation of calcium carbonate. This important goal was achieved by exploiting a fluorescent tag in GP. The experimental results that were obtained by using spectroscopic-, chromatographic-, and microscopic techniques indicate that GP controls the precipitation kinetics and morphology of calcium carbonate crystals, and that it only undergoes structural reorganization when entrapped inside calcium carbonate crystals. To the best of our knowledge, this report represents one of the first studies on the conformational changes of a protein fragment that is involved in biomineralization processes on moving from the solution phase into the mineral phase.

Mechanism and stereoselectivity of HDAC I inhibition by (R)-9-hydroxystearic acid in colon cancer.

9-Hydroxystearic acid (9-HSA) belongs to the endogenous lipid peroxidation by-products that decrease in tumors, causing as a consequence the loss of one of the control mechanisms on cell division. It acts as a histone deacetylase (HDAC, E.C 3.5.1.98) inhibitor, and the interaction of the two enantiomers of 9-HSA with the catalytic site of the enzyme, investigated by using a molecular modelling approach, has been reported to be different. In this work we tested out this prediction by synthesizing the two enantiomers (R)-9-HSA (R-9) and (S)-9-HSA (S-9) starting from the natural source methyl dimorphecolate obtained from Dimorphotheca sinuata seeds and investigating their biological activity in HT29 cells. Both enantiomers inhibit the enzymatic activity of HDAC1, HDAC2 and HDAC3, R-9 being more active; R-9 and S-9 inhibitory effect induces an increase in histone H4 acetylation. We also demonstrate that the antiproliferative effect brought about by R-9 is more pronounced as well as we observe increase of p21 transcription and protein content, while the expression of cyclin D1 is decreased. Starting from these observations it can be hypothesized that the interaction of R-9 with HDAC1 induce conformational changes in the enzyme causing loss of its interaction with other proteins, like cyclin D1 itself.

The interstitial crystal-nucleating sheet in molluscan Haliotis rufescens shell: a bio-polymeric composite.

The interstitial green sheets in abalone shell nacre are shown to be bifacially differentiated trilaminate polymeric complexes, with glycoprotein layers sandwiching a central core containing chitin. They share some common feature with the organic matrix layers between the aragonite tablets in the nacre and the periostracum, and show similarities to the myostracum. Thus, although the green sheet is reported to be unique to the abalone shell, it represents an interesting model for the study of molluscan shell biomineralization processes. Indeed, during shell formation, prismatic and spherulitic aragonite precedes and follows the deposition of the interstitial green polymeric composite sheets, and there is evidence to suggest that these sheets demark the interruption of nacre synthesis and serve to nucleate the resumption of calcium carbonate crystal growth. The green polymeric interstitial sheet purified from the abalone shell was investigated by spectroscopic and imaging techniques: FTIR, confocal microscopy, scanning and transmission electron microscopy, and by pyrolysis combined with GC-MS. Structural and compositional differences are observed between the surfaces of the two sides of the interstitial polymeric composite sheets. Moreover, comparative crystallization experiments on the green sheet sides also reveal asymmetry with respect to the nucleation of calcium carbonate. These findings suggest that these bifacially differentiated interstitial composites may play an active role in the mineral assembly processes, with one of the surfaces acting as a crystal nucleator.

Introduction of oxygenated side chain into imidazolium ionic liquids: evaluation of the effects at different biological organization levels.

The biological effects of a class of oxygenated imidazolium ionic liquids were studied in comparison with alkyl imidazolium salts (BMIM BF4 and BMIM N(CN)2).The cellular and subcellular effects were evaluated on rat pheochromocytoma PC12 cell lines, through MTT test, lactate dehydrogenase release and acetylcholinesterase inhibition; the eco-toxicological responses were assessed through the acute toxicity tests towards Daphnia magna and Vibrio fischeri. The introduction of ethoxy moieties in the lateral chain of imidazolium cations reduced the biological effects in all the tests. The acute toxicity towards D. magna was not affected by the number of ethoxy units, but the crustacean seemed to be sensitive to the type of anion; on the contrary, a further addition of ethoxy moieties increased the toxicity towards V. fischeri, M(OE)4MIM N(CN)2 being the most toxic oxygenated ionic liquid. In the cytotoxicity assays the salts with oxygenated cations resulted ineffective compared to BMIMs, independently from the anion and the number of ethoxy units in the lateral chain. In order to estimate the influence on membrane fluidity, an analysis of fluorescence anisotropy was done and it indicated that BMIM BF4, the most toxic ionic liquid among the tested ones, led to a destabilization of the model membranes at any molarity.

Characterization of cholinesterase activity in three bivalves inhabiting the North Adriatic sea and their possible use as sentinel organisms for biosurveillance programmes.

Anticholinesterases constitute a major portion of modern synthetic insecticides and the assessment of cholinesterase (ChE) inhibition is widely used as a specific biomarker for evaluating the exposure of non-target organisms to these pollutants. To evaluate the possible exposure of the North Adriatic sea coastal environment to residues of agriculture practices, we ascertained whether the oyster, Ostrea edulis and the clam, Tapes philippinarum, highly valuable resources commercially harvested in the area, could be selected as sentinel species. We characterized the ChE biochemical properties in their gills, and for comparison the analysis was carried out also in gills of the mussel, Mytilus galloprovincialis, extensively used as a biological indicator of water quality. In the oysters and mussels, the ChE activity was a function of increasing concentrations of substrate in the range 0.01-0.5 mM. K(m) values were 93+/-15 and 77+/-8 microM for O. edulis and M. galloprovincialis, respectively. No detectable ChE activity was found in gills of T. philippinarum, at any tested condition. Significant inhibition of ChE activity by eserine, carbaryl, and ethyl-paraoxon was observed in gills of O. edulis and M. galloprovincialis. Although field validation is needed, the present study suggests that O. edulis may be employed as a biological indicator for assessing pesticide contamination, whilst T. philippinarum does not seem useful for this purpose.

Conformational features of a synthetic model of the first extracellular loop of the angiotensin II AT1A receptor.

The angiotensin II AT1A receptor belongs to the G-protein coupled receptors (GPCRs). Like other membrane proteins, GPCRs are not easily amenable to direct structure determination by the currently available methods. The peptide encompassing the putative first extracellular loop of AT1A (residues Thr88-Leu100, el1) has been synthesized along with a cyclic model where the linear peptide has been covalently linked to a template designed to keep the distance between the peptide termini as expected in the receptor. The conformational features of the two molecules have been studied using circular dichroism and NMR techniques. The region W94PFG97 forms a type-II beta-turn and undergoes a Trp-Pro peptide bond cis-trans isomerization in both peptides confirming that these characteristics are intrinsic to el1. In addition, the presence of the spacer seems to modulate the flexibility of the peptide.

Cd2+ and Hg2+ affect glucose release and cAMP-dependent transduction pathway in isolated eel hepatocytes.

Isolated hepatocytes of the European eel (Anguilla anguilla) have been used as experimental model to characterize the effects of Cd(2+) and Hg(2+) on either basal or epinephrine-stimulated glucose release. Cd(2+) strongly reduced glucose output from cells perifused in BioGel P4 columns and challenged with epinephrine, with a maximum inhibition of 95% reached at 10 microM (IC(50) 0.04 microM). The epinephrine-stimulated glucose output was also reduced by Hg(2+), although a significant inhibition of about 60% was achieved only at 10 microM (IC(50) 5 microM). The possible influence of Cd(2+) and Hg(2+) on adenylyl cyclase/cAMP transduction pathway has been investigated, since this system is known to play a pivotal role in the regulation of fish liver glycogen breakdown and consequent glucose release. Micromolar concentrations of both heavy metals significantly reduced the epinephrine-modulated cAMP levels in isolated eel hepatocytes, in good agreement with the reduction of glucose output. Cd(2+) and Hg(2+) also significantly reduced basal and epinephrine-stimulated adenylyl cyclase activity in liver membrane preparations. A competitive inhibition with respect to Mg(2+) was shown by Cd(2+) and Hg(2+), which significantly reduced the affinity of the allosteric activator for the adenylyl cyclase system. Apparent Km for Mg(2+) was 4.35 mM in basal conditions, and increased to 9.1 and 7.1 mM in the presence of 10 microM Cd(2+) and Hg(2+), respectively. These results indicate that Cd(2+) and Hg(2+) may impair a crucial intracellular transduction pathway involved in the adrenergic control of glucose metabolism, but also in several other routes of hormonal regulation of liver functions.

Conformational flexibility of three cytoplasmic segments of the angiotensin II AT1A receptor: a circular dichroism and fluorescence spectroscopy study.

The conformation of three synthetic peptides encompassing the proximal and distal half of the third intracellular loop (Ni3 and Ci3) and a portion of the cytoplasmic tail (fCT) of the angiotensin II AT1A receptor has been studied using circular dischroism and fluorescence spectroscopies. The results show that the conformation of the peptides is modulated in various ways by the environmental conditions (pH, ionic strength and dielectric constant). Indeed, Ni3 and fCT fold into helical structures that possess distinct stability and polarity due to the diverse forces involved: mainly polar interactions in the first case and a combination of polar and hydrophobic interactions in the second. The presence of these various features also produce distinct intermolecular interactions. Ci3, instead, exists as an ensemble of partially folded states in equilibrium. Since the corresponding regions of the angiotensin II AT1A receptor are known to play an important role in the receptor function, due to their ability to undergo conformational changes, these data provide some new clues about their different conformational plasticity.