75 research outputs found
3D-QSAR using pharmacophore-based alignment and virtual screening for discovery of novel MCF-7 cell line inhibitors
No full textThe development of a novel approach for the prediction of antiestrogenic activity is described, bringing up to date a previous pharmacophore study. Software Phase has been used to derive a 3D-QSAR model based, as alignment rule, on a pharmacophore built on three compounds highly active against MCF-7 cell line. Five features comprised the pharmacophore: two hydrogen-bond acceptors, one hydrogen-bond donor, and two aromatic rings. The sequential 3D-QSAR yielded a test set q2 equal to 0.73 and proved to be predictive with respect to an external test set of 21 compounds (r2 = 0.69). The model was used to detect new MCF-7 inhibitors through 3D-database searching and identified fourteen compounds that were subsequently tested in vitro against the MCF-7 human breast adenocarcinoma cell line. Eleven out of the fourteen compounds exhibited inhibitory activity with IC50 values ranging between 30 and 186 μM. The results of the study confirmed the fundamental validity of the chosen approach as a hit discovery tool. © 2013 Elsevier Masson SAS. All rights reserved
Calstorin, a new Ca2+ binding protein of the microsome lumen which is abundant in the rat brain.
No full textA new Ca2+ binding protein (apparent Mr: 54 KDa; pI: 4.37; Stains All positive) which, based on N terminal sequence and immunological criteria, appears different from calsequestrin, calreticulin and the chaperonins, has been identified in the rat brain and recovered primarily in the microsome fraction. Carbonate extraction and trypsin digestion experiments suggest the protein to be located within the microsome lumen. Its expression levels are considerable especially in the cerebellum (65% with respect to calreticulin). 45Ca binding experiments on 2D blots suggest the protein to be of high capacity (higher than calreticulin) and low affinity (apparent Kd: 3 mM). These properties are typical of the proteins participating in the storage of Ca2+ within rapidly exchanging organelles. The tentative name of calstorin (calcium-storage-protein, CST) is therefore proposed
Tetrahydrofuran Acetogenins from Laurencia glandulifera
No full textFive new C(15) acetogenin en-ynes (1-5) with a rare tetrahydrofuran moiety and a linear biosynthetic precursor (6) were isolated from an organic extract of Laurencia glandulifera, collected from the island of Crete in the south Aegean Sea. The structures of the new natural products, as well as their relative configuration, were established by means of spectroscopic data analysis. The cytotoxicity of the isolated natural products was evaluated against five human tumor cell lines
Promoter Elements and Factors Involved In Hepatic Transcription of the Human Apoa-i Gene Positive and Negative Regulators Bind To Overlapping Sites
No full textDNase I footprinting analysis of the proximal apoA-I promoter sequences with rat liver nuclear extracts identified four protected regions: A, -22 to +17; B, -128 to -77; C, -175 to -148; and D, -220 to -190. Region D (-220 to -190) binds at least two distinct activities, designated AID1 and AID2, respectively, which can be separated by ion exchange chromatography. Region C (-175 to -148) forms five DNA protein complexes. Three of the complexes (2, 4, and 5) originate from the binding of more than one heat-stable nuclear factor, and two (1 and 3), from the binding of two heat-labile factors. The heat-stable factors bind in the -175 to -148 region and can be distinguished from C/EBP, which recognizes the same region, with DNA binding gel electrophoretic assays. Both factors 1 and 3 bind in the -168 to -148 apoA-I region. Despite the lack of a CCAAT motif in this region, the binding of factor 1 is competed out by oligonucleotides containing the binding sites of NFY and NFY*. Mutagenesis of the promoter region showed that mutations in the -171 to -166 and -158 to -153 regions diminished the binding of the heat-stable factors and reduced hepatic transcription to 14 and 8% of control, respectively. In contrast, a mutation in the -164 to -159 region abolished the binding of factor 1 and was associated with a 4.6-fold increase in hepatic transcription. These findings suggest that the heat-stable factors act as positive regulators, whereas factor 1 acts as a negative regulator in apoA-I gene transcription
Pharmacophore modeling for qualitative prediction of antiestrogenic activity
No full textA ligand-based pharmacophore approach for the prediction of antiestrogenic activity to be used as an in silico screening tool for bioactive compounds including natural products was developed using Catalyst HypoGen. The generated pharmacophore hypothesis (HYPO-7) consisted of five features, namely, one hydrophobic (HY1), two hydrophobic aromatic (HY2), one hydrogen-bond acceptor (HBA), and one hydrogen-bond donor (HBD). HYPO-7 successfully predicted the lack of cytotoxicity of a number of new metabolites isolated from the red alga Laurencia glandulifera. Furthermore, a screening of the Asinex Gold Collection database was performed by coupling HYPO-7 with a docking filtration, which resulted in a restricted set of 12 new scaffolds to be investigated as potential SERMs. The inhibitory activity of these compounds was evaluated in vitro using MCF7 human breast adenocarcinoma cell line. Ten out of the twelve compounds exhibited inhibitory activity with IC50 values between 26 and 188 μM. This result shows that application of HYPO-7 could assist in the selection of potentially active compounds, thus expediting the hit discovery process. © 2009 American Chemical Society
Hypoxia-inducible factor-1α increase is an early and sensitive marker of lung cells responding to benzo[a] pyrene
No full textHypoxia inducible factor-1α (HIF-1α) is a central regulator of tumor survival and metastasis, responsible for metabolic adaptation to hypoxic conditions and promotion of angiogenesis. It has been also shown to respond to non-hypoxic stimuli, such as growth factors and moderate oxidative stress. We examined the protein levels of HIF-1α in A549 human lung cells exposed to the typical carcinogen benzo[a]pyrene (B[a]P). Our results revealed that B[a]P, at low, non-cytotoxic concentrations, induced a transient increase of nuclear HIF-1α and its target, GLUT1. HIF-1α upregulation was partly mediated by Akt kinase and coincided with increased nuclear levels of the redox-sensitive marker, nuclear factor erythroid 2-related factor-2 (NrF-2). B[a]P-induced HIF-1α was also detected during serum depletion or treatment with the hypoxia-mimicking agent, CoCl2. In addition, exposure of A549 cells to B[a]P containing diesel exhaust particles enhanced HIF-1α accumulation, probably due to the presence of additional carcinogenic compounds. B[a]P-induced increase of HIF-1α was further confirmed in normal rat and human lung fibroblasts. Our findings indicate that HIF-1α stimulation may act as an early and sensitive marker of exposure to low, non-cytotoxic concentrations of B[a]P and/or other carcinogens. © 2012 Begell House, Inc
Induction of Akt by endogenous neurosteroids and calcium sequestration in P19 derived neurons
No full textNeuronal cell death caused by pathophysiological over-activation of glutamate receptors and the subsequent Ca2+ overloading, has been implicated in neurodegeneration after stroke, cerebral trauma and epileptic seizures. Recent findings suggest that certain progesterone metabolites (neurosteroids) such as allopregnanolone and dehydroepiandrosterone can protect neuronal cells from such insults. In the present study, murine P19 cells were induced to differentiate into postmitotic neurons expressing specific neuronal markers, including GABAA and NMDA receptors. Activation of NMDA receptors in P19-N neurons resulted in excitotoxic cell death, which involved suppression of the phosphorylation of the survival kinase PKB/ Akt. Allopregnanolone and DHEA induced a rapid and prolonged phosphorylatio0n of the Akt kinase and they were able to reverse the NMDAinduced suppression of the PI3-K/Akt pathway. The specificity of the neuroprotective effects of these neurosteroids was confirmed by the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin, as well as by the GABAA receptor antagonist, bicuculline. The neurotoxic effect of NMDA on P19-N neurons was directly correlated with increased Ca2+ entry, since the addition of EGTA or BAPTA-AM, significantly suppressed the NMDA-induced decrease of phospho-Akt and subsequent neuronal death. These results suggest that neurosteroids are able to act as survival factors on P19-N neurons, promoting the activation of the PI3-K/Akt pathway through a calcium-entry dependent mechanism. © Springer 2008
Developmental and age-related alterations of calcium homeostasis in human fibroblasts
No full textCalcium homeostasis, in terms of both cytosolic calcium concentration ([Ca2+](i)) and capacitative calcium entry, has been investigated in human skin and lung fibroblasts at different developmental and ageing stages by employing the Fura-2 based measurements. [Ca2+](i) in foetal lung or skin cells were nearly similar. However, significant changes were observed between foetal and adult fibroblasts and interestingly in opposite directions depending on the tissue of origin. In particular, in adult lung cells [Ca2+](i) was more than three-fold higher compared to adult skin fibroblasts, a difference which may contribute to tissue-specific functions. Capacitative Ca2+ entry, i.e. the transient [Ca2+](i) increase induced by re-addition of extracellular calcium after depletion of thapsigargin-sensitive calcium stores, was found to exhibit the same pattern of differences during foetal-to-adult transition (it is two-fold higher in adult lung cells than in adult skin cells). At variance, we found capacitative Ca2+ entry to be significantly attenuated during in vivo or in vitro ageing of fibroblasts; while minor alterations of [Ca2+](i) were observed. These findings indicate that capacitative calcium entry rather than [Ca2+](i), is mainly affected during the ageing process. (C) 2002 Elsevier Science Inc. All rights reserved
Attenuation of oxidative stress in HL-1 cardiomyocytes improves mitochondrial function and stabilizes Hif-1α
No full textHL-1 cardiomyocytes were subjected to simulated hypoxia, in the presence of cobalt chloride, which resulted in reduction of cell viability and induction of DNA laddering, indicating the activation of the apoptotic cascade. In the presence of trolox, ascorbic acid, melatonin and the hybrid compound of trolox and lipoic acid (LaT 3a), cell viability was increased, with LaT 3a exhibiting the best effect. Antioxidant treatment restored ATP levels, abolished laddering of DNA, abrogated MPTP opening, Bax translocation to the mitochondria and cytochrome c release to the cytoplasm. Moreover, severe hypoxia, was found to destabilize hypoxia inducible factor-1α (Hif-1α) mRNA. Reduction of oxidative stress attenuated this effect, implying a possible anti-apoptotic action of the master regulator of hypoxia response. Our data suggest that antioxidants can maintain cell function and survival by inhibiting the mitochondrial apoptotic pathway and stabilizing Hif-1α. © 2005 Taylor & Francis
Amyloid-β Protein Precursor Regulates Depolarization-Induced Calcium-Mediated Synaptic Signaling in Brain Slices
No full textBackground: Coordinated calcium influx upon neuronal depolarization activates pathways that phosphorylate CaMKII, ERKs, and the transcription factor CREB and, therefore, expression of pro-survival and neuroprotective genes. Recent evidence indicates that amyloid-β protein precursor (AβPP) is trafficked to synapses and promotes their formation. At the synapse, AβPP interacts with synaptic proteins involved in vesicle exocytosis and affects calcium channel function. Objective: Herein, we examined the role of AβPP in depolarization-induced calcium-mediated signaling using acute cerebral slices from wild-type C57bl/6 mice and AβPP-/-C57bl/6 mice. Methods: Depolarization of acute cerebral slices from wild-type C57bl/6 and AβPP-/-C57bl/6 mice was used to induce synaptic signaling. Protein levels were examined by western blot and calcium dynamics were assessed using primary neuronal cultures. Results: In the absence of AβPP, decreased pCaMKII and pERKs levels were observed. This decrease was sensitive to the inhibition of N-and P/Q-type Voltage Gated Calcium Channels (N-and P/Q-VGCCs) by ω-conotoxin GVIA and ω-conotoxin MVIIC, respectively, but not to inhibition of L-type VGCCs by nifedipine. However, the absence of AβPP did not result in a statistically significant decrease of pCREB, which is a known substrate of pERKs. Finally, using calcium imaging, we found that down regulation of AβPP in cortical neurons results in a decreased response to depolarization and altered kinetics of calcium response. Conclusion: AβPP regulates synaptic activity-mediated neuronal signaling by affecting N-and P/Q-VGCCs. © 2020-IOS Press and the authors. All rights reserved
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