Biochemical characterization and homology modeling of a purine-specific ribonucleoside hydrolase from the archaeon Sulfolobus solfataricus: Insights into mechanisms of protein stabilization

We report the biochemical and structural characterization of the purine-specific ribonucleoside hydrolase from the archaeon Sulfolobus solfataricus (SsIAG-NH). SsIAG-NH is a homodimer of 70 kDa specific for adenosine, guanosine and inosine. SsIAG-NH is highly thermophilic and is characterized by extreme thermodynamic stability (T_m, 107 °C), kinetic stability and remarkable resistance to guanidinium chloride-induced unfolding. A disulfide bond that, on the basis of SDS-PAGE is positioned intersubunits, plays an important role in thermal stability. SsIAG-NH shares 43% sequence identity with the homologous pyrimidine-specific nucleoside hydrolase from S. solfataricus (SsCU-NH). The comparative sequence alignment of SsIAG-NH, SsCU-NH, purine non-specific nucleoside hydrolase from Crithidia fasciculata and purine-specific nucleoside hydrolase from Trypanosoma vivax shows that, only few changes in the base pocket are responsible for different substrate specificity of two S. solfataricus enzymes. The structure of SsIAG-NH predicted by homology modeling allows us to infer the role of specific residues in substrate specificity and thermostability.     

Guanosine effect on cholesterol efflux and apolipoprotein E expression in astrocytes

The main source of cholesterol in the central nervous system (CNS) is represented by glial cells, mainly astrocytes, which also synthesise and secrete apolipoproteins, in particular apolipoprotein E (ApoE), the major apolipoprotein in the brain, thus generating cholesterol-rich high density lipoproteins (HDLs). This cholesterol trafficking, even though still poorly known, is considered to play a key role in different aspects of neuronal plasticity and in the stabilisation of synaptic transmission. Moreover, cell cholesterol depletion has recently been linked to a reduction in amyloid beta formation. Here we demonstrate that guanosine, which we previously reported to exert several neuroprotective effects, was able to increase cholesterol efflux from astrocytes and C6 rat glioma cells in the absence of exogenously added acceptors. In this effect the phosphoinositide 3 kinase/extracellular signal-regulated kinase 1/2 (PI3K/ERK1/2) pathway seems to play a pivotal role. Guanosine was also able to increase the expression of ApoE in astrocytes, whereas it did not modify the levels of ATP-binding cassette protein A1 (ABCA1), considered the main cholesterol transporter in the CNS. Given the emerging role of cholesterol balance in neuronal repair, these effects provide evidence for a role of guanosine as a potential pharmacological tool in the modulation of cholesterol homeostasis in the brain.     

Selective Disruption of Insulin-like Growth Factor-1 (IGF-1) Signaling via Phosphoinositide-dependent Kinase-1 Prevents the Protective Effect of IGF-1 on Human Cancer Cell Death

Insulin-like growth factor-1 (IGF-1) signaling system exerts a broad antiapoptotic function and plays a crucial role in resistance to anticancer therapies. Exposure of MCF-7 breast cancer cells to IGF-1 rapidly and transiently induced tyrosine phosphorylation and activation of phosphoinositide-dependent kinase-1 (PDK1). This was paralleled by Akt/protein kinase B and protein kinase C-ζ phosphorylation, at Thr³⁰⁸ and Thr⁴¹⁰, respectively. IGF-1 treatment also enhanced PDK1 interaction with IGF-1 receptor (IGF-1R) in intact MCF-7 cells. Pulldown assays revealed that PDK1 bound IGF-1R in vitro and that the region encompassing amino acids 51–359 of PDK1 was necessary for the interaction. Synthetic peptides corresponding to IGF-1R C terminus amino acids 1295–1337 (C43) and to PDK1 amino acids 114–141 reduced in vitro IGF-1R/PDK1 interaction in a concentration-dependent manner. Loading of fluoresceinated-C43 (fluorescein isothiocyanate (FITC)-C43) into MCF-7 cells significantly reduced IGF-1R/PDK1 interaction and phosphorylation of PDK1 substrates. Moreover, FITC-C43 intracellular loading reverted the protective effect of IGF-1 on growth factor deprivation-induced cell death. Finally, the inhibition of IGF-1R/PDK1 interaction and signaling by FITC-C43 was accompanied by 2-fold enhanced killing capacity of cetuximab in human GEO colon adenocarcinoma cells and was sufficient to restore cell death in cetuximab-resistant cell clones. Thus, disruption of PDK1 interaction with IGF-1R reduces IGF-1 survival effects in cancer cells and may enhance cell death by anticancer agents.     

The triazatruxene derivative azatrux binds to the parallel form of the human telomeric G-quadruplex under molecular crowding conditions: biophysical and molecular modeling studies

The present study has employed a combination of spectroscopic, calorimetric and computational methods to explore the binding of the three side-chained triazatruxene derivative, termed azatrux, to a human telomeric G-quadruplex sequence, under conditions of molecular crowding. The binding of azatrux to the tetramolecular parallel [d(TGGGGT)]₄ quadruplex in the presence and absence of crowding conditions, was also characterized. The data indicate that azatrux binds in an end-stacking mode to the parallel G-quadruplex scaffold and highlights the key structural elements involved in the binding. The selectivity of azatrux for the human telomeric G-quadruplex relative to another biologically relevant G-quadruplex (c-Kit87up) and to duplex DNA was also investigated under molecular crowding conditions, showing that azatrux has good selectivity for the human telomeric G-quadruplex over the other investigated DNA structures.     

A novel support for laccase immobilization: cellulose acetate modified with ionic liquid and application in biosensor for methyldopa detection

A material based on cellulose acetate (CA) and the room temperature ionic liquid 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (BMI·N(Tf)(2)) was developed and characterized by scanning electron microscopy, electron dispersive spectroscopy and infrared analysis. Laccase (Lac) from Aspergillus oryzae was immobilized in this material to investigate the behavior of methyldopa by square-wave voltammetry. Under optimized conditions, the Lac biosensor based on CA/BMI·N(Tf)(2) exhibited an excellent electrocatalytic performance: the analytical curve showed good linear range for methyldopa concentrations from 34.8 to 370.3 μM with a detection limit of 5.5 μM. This sensor demonstrated acceptable stability (ca. 60 days; at least 350 determinations), good repeatability and reproducibility (relative standard deviations of 1.5 and 4.3%, respectively). The recovery study of methyldopa in pharmaceutical formulations ranged from 94.1 to 105.9%. The determination of this substance using the biosensor compared favorably with that using a spectrophotometry procedure at the 95% confidence level, and indicated potential application to methyldopa determination in pharmaceutical samples.     

Productive infection of bovine papillomavirus type 2 in the placenta of pregnant cows affected with urinary bladder tumors

Papillomaviruses (PVs) are believed to be highly epitheliotropic as they usually establish productive infections within stratified epithelia. In vitro, various PVs appear to complete their entire life-cycle in different trophoblastic cell lines. In this study, infection by and protein expression of bovine papillomavirus type 2 (BPV-2) in the uterine and chorionic epithelium of the placenta has been described in four cows suffering from naturally occurring papillomavirus-associated urothelial bladder tumors. E5 oncoprotein was detected both by Western blot analysis and immunohistochemically. It appears to be complexed and perfectly co-localized with the activated platelet-derived growth factor ß receptor (PDGFßR) by laser scanning confocal microscopy. The activated PDGFßR might be involved in organogenesis and neo-angiogenesis rather than in cell transformation during pregnancy. The major capsid protein, L1, believed to be only expressed in productive papillomavirus infection has been detected by Western blot analysis. Immunohistochemical investigations confirmed the presence of L1 protein both in the cytoplasm and nuclei of cells of the uterine and chorionic epithelium. Trophoblastic cells appear to be the major target for L1 protein expression. Finally, the early protein E2, required for viral DNA replication and known to be expressed during a productive infection, has been detected by Western blot and immunohistochemically. Electron microscopic investigations detected viral particles in nuclei of uterine and chorionic epithelium. This study shows that both active and productive infections by BPV-2 in the placenta of pregnant cows can occur in vivo.     

Chemical cues involved in the attraction of the oligolectic bee Hoplitis adunca to its host plant Echium vulgare

Host recognition is a key process in oligolectic bees but the mechanisms through which they find and recognize appropriate pollen host plant are not entirely clear. Hoplitis adunca is a monolectic bee collecting pollen only from Echium spp. (Boraginaceae). We aimed to test whether Echium vulgare floral scent plays a major role in the attraction of H. adunca females, and to identify components of E. vulgare scent that may be involved in this specific attraction. We used a combination of behavioral and chemical (GC/GC–MS, PTR-MS) analyses. In order to identify the chemical cues likely to be involved in the specific attraction of H. adunca, we compared the scent of fresh flowers, nectar, pollen, and whole plants of E. vulgare and Anchusa officinalis, another Boraginaceae, which does not attract H. adunca. H. adunca females were attracted to the scent of E. vulgare flowers when offered against a blank or against the scent of A. officinalis flowers. However, H. adunca females were not attracted to the scent of A. officinalis flowers when offered against a blank. The emission spectra of the two plant species differed markedly, as did the emission spectra of various flower components (pollen, nectar and whole flowers) within a species. Pollen presented a low volatile release, but emitted significantly higher amounts of mass 55 (butanal, 1,3-butadiene, or other volatiles of molecular mass 54), and mass 83 (hexanal, hexenols, hexenyl acetate, or other volatiles of molecular mass 82) in E. vulgare than in A. officinalis. Nectar produced a particular emission spectrum with high emission rates of masses 109 and 123. Mass 109 may likely correspond to 1,4-benzoquinone, a volatile specifically measured in E. vulgare in parallel studies to this one. The flower emission spectrum was mainly a combination of the pollen and the nectar scents, although it also contained additional volatile compounds such as those of mass 63 or mass 81. As for terpenes, E. vulgare emitted limonene, longicyclene, junipene, trans-caryophyllene and α-humulene, that were not detected in A. officinalis, and the most emitted monoterpenes were α-pinene, junipene and limonene whereas the most emitted terpenoid by A. officinalis was α-pinene. After identifying these chemical cues, olfactory/behavioural assays with specific volatiles and combinations of volatiles are necessary to understand the chemical interactions of the H. adunca-E. vulgare system.     

Effect of lipid membranes on the apparent pK of the local anesthetic tetracaine spin label and titration studies

Electrometric titrations and spin label data demonstrate changes in the experimentally determined apparent pK of an ionizable drug in the presence of membranes. This effect is attributed to the difference in partition coefficients for the charged and uncharged forms of the drug. Investigation of the binding of a local anesthetic, tetracaine, to egg phosphatidylcholine membranes indicates that the drug apparent pK decreases in the presence of membranes, the decrease being a function of membrane concentration. The agreement between titration and spin label studies is very good and could be simulated by calculating membrane-bound and free populations of charged and uncharged tetracaine from the independently-measured partition coefficients for the two forms.