Inhibition of fatty acid synthase in melanoma cells activates the intrinsic pathway of apoptosis

Fatty acid synthase (FASN) is the metabolic enzyme responsible for the endogenous synthesis of the saturated long-chain fatty acid, palmitate. In contrast to most normal cells, FASN is overexpressed in a variety of human cancers, including cutaneous melanoma, in which its levels of expression are associated with tumor invasion and poor prognosis. We have previously shown that FASN inhibition with orlistat significantly reduces the number of spontaneous mediastinal lymph node metastases following the implantation of B16-F10 mouse melanoma cells in the peritoneal cavity of C57BL/6 mice. In this study, we investigate the biological mechanisms responsible for the FASN inhibition-induced apoptosis in B16-F10 cells. Both FASN inhibitors, cerulenin and orlistat, significantly reduced melanoma cell proliferation and activated the intrinsic pathway of apoptosis, as demonstrated by the cytochrome c release and caspase-9 and -3 activation. Further, apoptosis was preceded by an increase in both reactive oxygen species production and cytosolic calcium concentrations and independent of p53 activation and mitochondrial permeability transition. Taken together, these findings demonstrate the mitochondrial involvement in FASN inhibition-induced apoptosis in melanoma cells. Laboratory Investigation (2011) 91, 232-240; doi:10.1038/labinvest.2010.157; published online 30 August 2010

Using Computer-aided Drug Design and Medicinal Chemistry Strategies in the Fight Against Diabetes

The aim of this work is to present a simple, practical and efficient protocol for drug design, in particular Diabetes, which includes selection of the illness, good choice of a target as well as a bioactive ligand and then usage of various computer aided drug design and medicinal chemistry tools to design novel potential drug candidates in different diseases. We have selected the validated target dipeptidyl peptidase IV (DPP-IV), whose inhibition contributes to reduce glucose levels in type 2 diabetes patients. The most active inhibitor with complex X-ray structure reported was initially extracted from the BindingDB database. By using molecular modification strategies widely used in medicinal chemistry, besides current state-of-the-art tools in drug design (including flexible docking, virtual screening, molecular interaction fields, molecular dynamics. ADME and toxicity predictions), we have proposed 4 novel potential DPP-IV inhibitors with drug properties for Diabetes control, which have been supported and validated by all the computational tools used herewith.

Isolation and structural characterization of a new fibrin(ogen)olytic metalloproteinase from Bothrops moojeni snake venom

A proteinase, named BmooMP alpha-I, from the venom of Bothrops moojeni, was purified by DEAE-Sephacel, Sephadex G-75 and heparin-agarose column chromatography. The enzyme was purified to homogeneity as judged by its migration profile in SDS-PAGE stained with coomassie blue, and showed a molecular mass of about 24.5 kDa. Its complete cDNA was obtained by RT-PCR and the 615 bp codified for a mature protein of 205 amino acid residues. The multiple alignment of its deduced amino acid sequence and those of other snake venom metalloproteinases showed a high structural similarly, mainly among class P-IB proteases. The enzyme cleaves the A alpha-chain of fibrinogen first, followed by the B beta-chain, and shows no effects on the gamma-chain. On fibrin, the enzyme hydrolyzed only the beta-chain, leaving the gamma-dimer apparently untouched. It was devoid of phospholipase A(2), hemorrhagic and thrombin-like activities. Like many venom enzymes, it is stable at pH values between 4 and 10 and stable at 70 degrees C for 15 min. The inhibitory effects of EDTA on the fibrinogenolytic activity suggest that BmooMP alpha-I is a metalloproteinase and inhibition by beta-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Aprotinin and benzamidine, specific serine proteinase inhibitors, had no effect on BmooMP alpha-I activity. Since the BmooMP alpha-I enzyme was found to cause defibrinogenation when administered i.p. on mice, it is expected that it may be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis. (C) 2007 Elsevier Ltd. All rights reserved.

Molecular cloning and biochemical characterization of a myotoxin inhibitor from Bothrops alternatus snake plasma

Phospholipases A(2) (PLA(2)s) are important components of Bothrops snake venoms, that can induce several effects on envenomations such as myotoxicity, inhibition or induction of platelet aggregation and edema. It is known that venomous and non-venomous snakes present PLA(2) inhibitory proteins (PLIs) in their blood plasma. An inhibitory protein that neutralizes the enzymatic and toxic activities of several PLA2s from Bothrops venoms was isolated from Bothrops alternatus snake plasma by affinity chromatography using the immobilized myotoxin BthTX-I on CNBr-activated Sepharose. Biochemical characterization of this inhibitory protein, denominated alpha BaltMIP, showed it to be a glycoprotein with Mr of similar to 24,000 for the monomeric subunit. CD spectra of the PLA(2)/inhibitor complexes are considerably different from those corresponding to the individual proteins and data deconvolution suggests that the complexes had a relative gain of helical structure elements in comparison to the individual protomers, which may indicate a more compact structure upon complexation. Theoretical and experimental structural studies performed in order to obtain insights into the structural features of aBaltMIP indicated that this molecule may potentially trimerize in solution, thus strengthening the hypothesis previously raised by other authors about snake PLIs oligomerization. (C) 2010 Elsevier Masson SAS. All rights reserved.

The effect of repeated prepulse and reflex stimulus presentations on startle prepulse inhibition

The magnitude of a startle reflex is inhibited if the reflex-eliciting stimuli is preceded by a prepulse stimulus at a short lead interval. Previous research in humans has shown that the extent of prepulse inhibition decreases over repeated presentations of reflex stimuli and prepulse-reflex stimulus pairings. The present study (N=70) investigated the effect of repeated presentations of prepulse stimuli, reflex stimuli, or prepulse-reflex stimulus pairings on prepulse inhibition. Five groups of subjects were presented during habituation training with either (a) reflex stimuli, (b) prepulse-reflex stimulus pairings, (c) a random sequence of prepulse and reflex stimuli, (d) prepulse stimuli, or (e) experimentally irrelevant light stimuli. Prepulse inhibition was reduced if startle stimuli were presented during habituation ((a), (b), (c)), but not after repeated presentation of the prepulse or the light stimulus ((d), (e)). The reduction in prepulse inhibition was abolished after dishabituation of the startle reflex. The present results indicate that habituation of the startle reflex can result in a reduction of prepulse inhibition. (C) 1998 Elsevier Science B.V.

Inhibition of phenotype modulation, growth, and migration of vascular smooth muscle cells by a guanosine-rich 30-mer phosphorothioate oligodeoxynucleotide

The testing of a 30-mer dG-rich phosphorothioate oligodeoxynucleotide (LG4PS) for effects on the behaviour of vascular smooth muscle cells (VSMC) in vitro and in vivo is described. LG4PS at 0.3 mu M inhibited significantly the phenotype modulation of freshly isolated rabbit VSMC, and cell outgrowth from pig aortic explants was inhibited similar to 80% by 5 mu M LG4PS. The growth of proliferating rabbit and pig VSMC was inhibited similar to 70% by 0.3 mu M and 5 mu M LG4PS, respectively. Though less marked, the antiproliferative effects of LG4PS on human VSMC were comparable to those obtained with heparin. The cytotoxic effects of LG4PS on VSMC in vitro were low. Despite these promising results, adventitial application of 2-200 nmol LG4PS in pluronic gel failed to reduce vascular hyperplasia in balloon-injured rabbit carotid arteries, and the highest dose caused extensive mortality. (C) 1997 Academic Press Limited.

Ethanol inhibition of brain ornithine decarboxylase activity in the postnatal rat

The purpose of this study was to determine the relationship between ornithine decarboxylase activity (ODC; a marker for perturbed cell development), the blood alcohol level, and alcohol-induced microencephaly in the developing rat brain after binge treatment with ethanol vapour. By manipulating ethanol flow we were able to adjust vapour concentrations (24-65 mg ethanol/l air) such that an acute exposure of ethanol vapour for 3 h resulted in a range of blood alcohol levels (2.3-5.5 mg/ml). Acute studies showed that ethanol dose-dependently inhibited rat hippocampal and cerebellar ODC activity at PND4-PND10. There was a significant correlation between the blood alcohol level and degree of inhibition at all ages tested. Chronic treatment from PND4 to PND9 caused a significant decrease in both brain to body weight ratio and in hippocampal and cerebellar ODC activities at PND10. These results indicate that ethanol-induced disruption in ODC could play a significant role in ethanol's teratogenic effects during early postnatal development. (C) 1998 Elsevier Science Inc.

Nitric oxide inhibition of the rat olfactory cyclic nucleotide-gated cation channel

The effects of nitric oxide (NO) and other cysteine modifying agents were examined on cyclic nucleotide-gated (CNG) cation channels from rat olfactory receptor neurons. The NO compounds, S-nitroso-cysteine (SNC) and 3-morpholino-sydnonomine (SIN-1), did not activate the channels when applied for up to 10 min. The cysteine alkylating agent, N-ethylmaleimide (NEM), and the oxidising agent, dithionitrobensoate (DTNB), were also without agonist efficacy. Neither SNC nor DTNB altered the cAMP sensitivity of the channels. However, 2-min applications of SIN-1, SNC and DTNB inhibited the cAMP-gated current to approximately 50% of the control current level. This inhibition showed no spontaneous reversal for 5 min but was completely reversed by a 2-min exposure to DTT. The presence of cAMP protected the channels against NO-induced inhibition. These results indicate that inhibition is caused by S-nitrosylation of neighboring sulfhydryl groups leading to sulfhydryl bond formation. This reaction is favored in the closed channel state. Since recombinantly expressed rat olfactory alpha and beta CNG channel homomers and alpha/beta heteromers are activated and not inhibited by cysteine modification, the results of this study imply the existence of a novel subunit or tightly bound factor which dominates the effect of cysteine modification in the native channels. As CNG channels provide a pathway for calcum influx, the results may also have important implications for the physiological role of NO in mammalian olfactory receptor neurons.

Focused antithrombotic therapy: Novel anti-platelet salicylates with reduced ulcerogenic potential and higher first-pass detoxification than aspirin in rats

The use of aspirin as an anti-platelet drug is limited by its propensity to induce gastric injury and by its adverse effect on vascular prostacyclin formation. Two phenolic non-steroidal anti-inflammatory drugs (salicyclic acid and diflunisal) were modified by esterification with a series of O-acyl moieties. The short-term ulcerogenic in vitro and in vivo anti-platelet properties, pharmacodynamic profiles, and extent of hepatic extraction of these phenolic esters were compared with aspirin (acetylsalicylic acid). The more lipophilic esters (longer carbon chain length in O-acyl group) show significantly less gastrotoxicity in stressed rats than does aspirin after a single oral dose. The in vitro and in vivo anti-platelet studies show that these phenolic esters inhibited (1) arachidonate-triggered human platelet aggregation and (2) thrombin-stimulated rat serum thromboxane Ag production by platelets in the clotting process almost as effectively as aspirin. The hepatic extractions of these O-acyl derivatives are significantly higher than those of aspirin. The pharmacodynamic studies show that these O-acyl derivatives of salicylic acid and diflunisal probably bind to, or combine with, the same site on the platelet cyclooxygenase as aspirin. Replacing the O-acetyl group with longer chain O-acyl moiety in this series of phenolic esters markedly reduced the potential of these agents to induce short-term gastric injury but did not lessen their activity as inhibitors of platelet aggregation. These non-acetyl salicylates may therefore represent a novel class of anti-platelet drugs with less ulcerogenic potential.

Inhibition of NK cells by murine CMV-encoded class I MHC homologue m144

Murine cytomegalovirus (CMV)-encoded protein m144 is homologous to class I MHC heavy-chain and is thought to regulate NK-cell-mediated immune responses in vivo. To examine the effects of m144 on Nh cytotoxicity in vitro, various cell lines were transfected with wild-type m144 or a chimeric construct in which the cytoplasmic domain of m144 was replaced with green fluorescence protein. Burkitt lymphoma line Raji expressed a significant level of m144 as determined by anti-m144 mAb binding or the green fluorescence of the fusion protein. The level of m144 expression was relatively low compared with that of transfected murine class I MHC Dd. However, m144 on Raji cells partially inhibited antibody-dependent cell-mediated cytotoxicity of IL-2-activated NK cells. NK cells from the CMV-susceptible BALB/c as well as those from the resistant C57BL/6 mice were inhibited by m144. Antibodies against the known murine NK inhibitory receptors Ly-49A, C, G, and I did not affect the inhibitory effect of m144. These results suggest that the murine CMV class I MHC homologue m144 partially inhibits MZ cells by interacting with a novel inhibitory receptor. (C) 1999 Academic Press.

Inhibition of settlement and metamorphosis of the ascidian Herdmania curvata by non-geniculate coralline algae

The surfaces of non-geniculate coralline algae (NCA) are known to induce the settlement and metamorphosis of disparate marine taxa. In this study we investigate the responsiveness of larvae of Herdmania curvata (Ascidiacea: Stolidobranchia) to three species of NCA (Neo-goniolithon brassica-florida, Hydrolithon onkodes, and Lithothamnium prolifer) that cohabit the slope and crest of Heron Reef, Great Barrier Reef. H. curvata larvae were first exposed to these NCA at or within 2 h of hatching, which is 1 to 2 h prior to attaining competence, and then cultured continuously with the NCA for 12 to 14 h. Rates of settlement and metamorphosis of H, curvata cultured in laboratory chambers in the presence of the different NCA were significantly lower than spontaneous rates in seawater. The limited settlement in treatments containing NCA were confined entirely to the chamber periphery, and settlement never occurred on the surface of the NCA. The inhibitory effect was dose-dependent and was stronger in H. brassica-florida and H. onkodes than in L. prolifer. Larvae that did not settle in treatments with NCA had rounded anterior trunks and, in extreme cases, kinked tails with rounded and dissociated tail muscle cells. In some individuals, we observed the anterior chemosensory papillae being sloughed off the larval body. Morphological analysis of trunk ectodermal and mesenchymal nuclei of larvae cultured in the presence of the NCA revealed that general necrotic cell death was occurring. Importantly, H. curvata larvae that were exposed to NCA could not subsequently be induced to metamorphose in KCl-elevated seawater, whereas larvae not exposed to NCA metamorphosed at high rates in KCl-elevated seawater.

Inhibition of transmitter release and long-term depression in the avian hippocampus

Long-term depression has recently been shown to occur at glutamatergic synapses in the avian hippocampus and requires activation of calcium/calmodulin-dependent protein kinase II in the nerve terminal. Here using whole cell and intracellular recordings from brain slices, we show that the N-type calcium channel contributes significantly to glutamate release in the avian hippocampus. Activation of the metabotrobic gamma-aminobutyric acid (GABA)(B) receptor by the specific agonist baclofen blocks synaptic transmission. The action of baclofen was associated with a change in paired pulse facilitation indicating that it resulted from a reduction in the probability of transmitter release, In contrast, no change in paired pulse facilitation was observed following the induction of long-term depression. These results show that activation of GABA(B) receptors and long-term depression reduce transmitter release by distinct mechanisms. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.