Hybrid molecules based on fullerene C60 and 5Z,9Z-dienoic acids: Synthesis and cytotoxic activity

The present research project details synthesis of new hybrid methanofullerenes based on acetylene and triazole esters of malonic acid containing 5Z,9Z-dienoic acids and fullerene C₆₀ under Bingel-Hirsch conditions, including study of the cytotoxic activity with respect to Jurkat, K562, U937 and HL60 tumor cell lines. Hybrid methanofullerenes containing acetylenic fragments, unlike triazole substituents, were found to exhibit higher cytotoxicity, but are characterized by lower selectivity of action in relation to healthy cells.     

Synthesis,biological evaluation and structure-activity relationships of 5-arylidene tetramic acids with antibacterial activity against methicillin-resistant Staphylococcus aureus

The steady rise of the antimicrobial resistance is a major global threat to human health that requires the urgent need for novel antibiotics. In this work we report the synthesis of a small library of 3-subsituted-5-arylidene tetramic acids in order to investigate the scope of our previously established methodology via an intermediate oxazolone and their antimicrobial activity. From this series of 14 tetramic acids, 11 derivatives are novel and one of them is a Schiff base, which was structurally characterized with single-crystal X-ray analysis and NMR spectroscopy. The compounds incorporating a lipophilic acyl group at carbon-3 of the ring showed moderate to high activity with minimum inhibitory activity of 4–32 μg/mL against methicillin-resistant Staphylococcus aureus (MRSA), accompanied by no human cell toxicity and hemolytic activity within the tested concentration range. The substituent at para position of the aryl ring seemed to have no or little effect on the antimicrobial activity of these compounds.     

Lineage-Restricted Regulation of SCD and Fatty Acid Saturation by MITF Controls Melanoma Phenotypic Plasticity

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Adipose triglyceride lipase regulates eicosanoid production in activated human mast cells

Human mast cells (MCs) contain TG-rich cytoplasmic lipid droplets (LDs) with high arachidonic acid (AA) content. Here, we investigated the functional role of adipose TG lipase (ATGL) in TG hydrolysis and the ensuing release of AA as substrate for eicosanoid generation by activated human primary MCs in culture. Silencing of ATGL in MCs by siRNAs induced the accumulation of neutral lipids in LDs. IgE-dependent activation of MCs triggered the secretion of the two major eicosanoids, prostaglandin D2 (PGD2) and leukotriene C4 (LTC4). The immediate release of PGD2 from the activated MCs was solely dependent on cyclooxygenase (COX) 1, while during the delayed phase of lipid mediator production, the inducible COX-2 also contributed to its release. Importantly, when ATGL-silenced MCs were activated, the secretion of both PGD2 and LTC4 was significantly reduced. Interestingly, the inhibitory effect on the release of LTC4 was even more pronounced in ATGL-silenced MCs than in cytosolic phospholipase A₂-silenced MCs. These data show that ATGL hydrolyzes AA-containing TGs present in human MC LDs and define ATGL as a novel regulator of the substrate availability of AA for eicosanoid generation upon MC activation.     

Novel Interaction of the Z-DNA Binding Domain of Human ADAR1 with the Oncogenic c-Myc Promoter G-Quadruplex

Both G-quadruplex and Z-DNA can be formed in G-rich and repetitive sequences on genome, and their formation and biological functions are controlled by specific proteins. Z-DNA binding proteins, such as human ADAR1, have a highly conserved Z-DNA binding domain having selective affinity to Z-DNA. Here, our study identifies the Z-DNA binding domain of human ADAR1 (hZα_ADAR1) as a novel G-quadruplex binding protein that recognizes c-myc promoter G-quadruplex formed in NHEIII₁ region and represses the gene expression. An electrophoretic migration shift assay shows the binding of hZα_ADAR1 to the intramolecular c-myc promoter G-quadruplex-forming DNA oligomer. To corroborate the binding of hZα_ADAR1 to the G-quadruplex, we conducted CD and NMR chemical shift perturbation analyses. CD results indicate that hZα_ADAR1 stabilizes the parallel-stranded conformation of the c-myc G-quadruplex. The NMR chemical shift perturbation data reveal that the G-quadruplex binding region in hZα_ADAR1 was almost identical with the Z-DNA binding region. Finally, promoter assay and Western blot analysis show that hZα_ADAR1 suppresses the c-myc expression promoted by NHEIII₁ region containing the G-quadruplex-forming sequence. This finding suggests a novel function of Z-DNA binding protein as a regulator of G-quadruplex-mediated gene expression.     

Phosphatases modulate transmission and serotonin facilitation at synapses: Studies with the inhibitor okadaic acid

We examined the role of phosphatases in synaptic transmission using the permeant phosphatase inhibitor okadaic acid (OA). In the crayfish neuromuscular junction (NMJ), postsynaptic effects including increases in input resistance occurred at doses greater than 5 μM OA. At lower doses (0.5–5 μM) the effects were solely presynaptic and transmitter release increased over three-fold despite small reductions in amplitude and duration of presynaptic action potentials. Potentiating effects of serotonin on transmitter release, Which depend on phosphorylation, were increased by OA. Frequency facilitation was reduced but its decay was not affected. In frog NMJs, OA increased spontaneous and evoked release two-fold through presynaptic mechanisms. An inactive analog of OA, OA tetra-acetate, had no effect on transmitter release at frog and crayfish NMJ. Therefore, phosphatases have a strong modulating influence on synaptic transmission.     

Multiple forms of O-methyltransferase involved in the microbial conversion of abietic acid into methyl abietate by Mycobacterium sp.

Six out of seven tested strains of mycobacteria transformed abietic acid to methyl abietate in shake culture. The conversion carried out by Mycobacterium sp. MB 3683 was induced by the substrate and stimulated by methionine. Fractionation of the cell extract of Mycobacterium sp. MB 3683 on DEAE cellulose, Ultrogel AcA 44 and MONO Q resulted in the separation of three distinct methyltransferase activities which could also esterify palmitic acid. The separated forms of the methyltransferase exhibited different activities towards these two substrates.     

Adenosine and Glutamate Modulate Each Other's Release from Rat Hippocampal Synaptosomes

Abstract: In rat hippocampal synaptosomes, adenosine decreased the K⁺ (15 mM) or the kainate (1 mM) evoked release of glutamate and aspartate. An even more pronounced effect was observed in the presence of the stable adenosine analogue, R-phenylisopropyladenosine. All these effects were reversed by the selective adenosine A₁ receptor antagonist 8-cyclo-pentyltheophylline. In the same synaptosomal preparation, K⁺ (30 mM) strongly stimulated the release of the preloaded [³H]adenosine in a partially Ca²⁺-dependent and tetrodotoxin (TTX)-sensitive manner. Moreover, in the same experimental conditions, both l -glutamate and l -aspartate enhanced the release of [³H]adenosine derivatives ([³H]ADD). The gluta-mate-evoked release was dose dependent and appeared to be Ca²⁺ independent and tetrodotoxin insensitive. This effect was not due to metabolism because even the nonmetabolizable isomers d -glutamate and d -aspartate were able to stimulate [³H]ADD release. In contrast, the specific glutamate agonists N-methyl-d -aspartate, kainate, and quisqualate failed to stimulate [³H]ADD release, suggesting that glutamate and aspartate effects were not mediated by known excitatory amino acid receptors. Moreover, NMDA was also ineffective in the absence of Mg²⁺ and l -glutamate-evoked release was not inhibited by adding the specific antagonists 2-amino-5-phosphonovaleric acid or 6–7-dinitroquinoxaline-2, 3-dione. The stimulatory effect did not appear specific for only excitatory amino acids, as γ-anunobutyric acid stimulated [³H]ADD release in a dose-related manner. These results suggest that, at least in synaptosomal preparations from rat hippocampus, adenosine and glutamate modulate each other's release. The exact mechanism of such interplay, although still, unknown, could help in the understanding of excitatory amino acid neurotoxicity.     

Septal and Hippocampal Glutamate Receptors Modulate the Output of Acetylcholine in Hippocampus: A Microdialysis Study

Abstract: In the present study, glutamate receptor agonists and antagonists were administered by retrograde microdialysis into either the medial septum/vertical limb of the diagonal band (MS/vDB), or hippocampus, and the output of acetylcholine (ACh) was measured in the hippocampus by using intracerebral microdialysis. Perfusion with N -methyl- d -aspartate (NMDA) and ( S )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) in the MS/vDB caused an increase in ACh output in the hippocampus. This increase was completely blocked by coadministration of their respective antagonists d (−)-2-amino-5-phosphonopentanoic acid ( d -AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Perfusion in the MS/vDB with kainic acid also caused an increase in ACh output, but coadministration of CNQX attenuated the increase only partially. Perfusion with d -AP5 or CNQX alone in the septal probe did not affect ACh output from the hippocampus. In contrast to the results of septal administration of NMDA and AMPA, local perfusion with the same drugs in the hippocampus caused a decrease in ACh output. Whereas the results of septal administration of drugs indicate that septal cholinergic neurons probably receive excitatory glutamatergic innervation, the decrease in ACh output caused by administration of NMDA and AMPA in the hippocampus is poorly understood.     

Purification and properties of pyruvate kinase from Thermoplasma acidophilum

Thermoplasma acidophilum is a thermoacidophilic archaebacterium occupying a paradoxical place in phylogenetic trees (phenotypically it is a thermoacidophile but phylogenetically it classifies with the methanogens). To better understand its phylogeny, the pyruvate kinase from this organism is being investigated as a molecular marker. The enzyme has been purified and has a native M(r) of 250,000. It consists of four, apparently identical subunits each of M(r) 60,000. No remarkable kinetic differences have been found between this thermophilic enzyme and its mesophilic counterparts other than its greater thermostability. Its amino acid composition has been determined and some partial sequencing has been done.