Molecular modeling studies toward the structural optimization of new cyclopeptide-based HDAC inhibitors modeled on the natural product FR235222

The natural cyclopeptide FR235222 is a potent HDAC inhibitor displaying relevant multiple anticancer effects and is considered an attractive lead compound for the generation of new and more effective antitumor therapeutics. Recently, we have synthesized a small collection of FR235222 simplified analogues which showed interesting biological activities. These results encouraged us to further explore the structural determinants responsible for the activity of this class of HDAC inhibitors in order to gain guidelines for the rational design of new derivatives with putative higher affinity for this target. In the present paper, we report the results obtained, docking these ligands in the binding pocket of HDLP, an HDAC homologue.     

Nucleotide sequence of the pirital virus (family Arenaviridae) small genomic segment

Pirital virus is a newly discovered South American member of the family Arenaviridae. We determined that the complete nucleotide sequence of the small genomic segment of Pirital virus is 3393 nt long, and encodes the viral nucleoprotein (N) and glycoprotein precursor (GPC) (561 aa and 509 aa, respectively) in nonoverlapping open reading frames of opposite polarities. The N and GPC genes are separated by an intergenic region that is 80 nt long; the predicted secondary structure of this region includes a single hairpin stabilized by 11 G-C and 8 A-U base pairs. Independent analyses of N and GPC amino acid sequence data confirmed that Pirital virus is related to Pichindé virus and belongs to the lineage A of the New World (Tacaribe complex) arenaviruses. The analysis of genetic distances between Pirital virus and other arenaviruses confirmed that Pirital virus is a distinct species within the family Arenaviridae.     

MPP+-induced cytotoxicity in neuroblastoma cells: Antagonism and reversal by guanosine

Guanosine exerts neuroprotective effects in the central nervous system. Apoptosis, a morphological form of programmed cell death, is implicated in the pathophysiology of Parkinson’s disease (PD). MPP⁺, a dopaminergic neurotoxin, produces in vivo and in vitro cellular changes characteristic of PD, such as cytotoxicity, resulting in apoptosis. Undifferentiated human SH-SY5Y neuroblastoma cells had been used as an in vitro model of Parkinson’s disease. We investigated if extracellular guanosine affected MPP⁺-induced cytotoxicity and examined the molecular mechanisms mediating its effects. Exposure of neuroblastoma cells to MPP⁺ (10 μM–5 mM for 24–72 h) induced DNA fragmentation in a time-dependent manner (p < 0.05). Administration of guanosine (100 μM) before, concomitantly with or, importantly, after the addition of MPP⁺ abolished MPP⁺-induced DNA fragmentation. Addition of MPP⁺ (500 μM) to cells increased caspase-3 activity over 72 h (p < 0.05), and this was abolished by pre- or co-treatment with guanosine. Exposure of cells to pertussis toxin prior to MPP⁺ eliminated the anti-apoptotic effect of guanosine, indicating that this effect is dependent on a Gi protein-coupled receptor, most likely the putative guanosine receptor. The protection by guanosine was also abolished by the selective inhibitor of the enzyme PI-3-K/Akt/PKB (LY294002), confirming that this pathway plays a decisive role in this effect of guanosine. Neither MPP⁺ nor guanosine had any significant effect on α-synuclein expression. Thus, guanosine antagonizes and reverses MPP⁺-induced cytotoxicity of neuroblastoma cells via activation of the cell survival pathway, PI-3-K/Akt/PKB. Our results suggest that guanosine may be an effective pharmacological intervention in PD.     

Pulmonary Inflammation Is Regulated by the Levels of the Vesicular Acetylcholine Transporter

Acetylcholine (ACh) plays a crucial role in physiological responses of both the central and the peripheral nervous system. Moreover, ACh was described as an anti-inflammatory mediator involved in the suppression of exacerbated innate response and cytokine release in various organs. However, the specific contributions of endogenous release ACh for inflammatory responses in the lung are not well understood. To address this question we have used mice with reduced levels of the vesicular acetylcholine transporter (VAChT), a protein required for ACh storage in secretory vesicles. VAChT deficiency induced airway inflammation with enhanced TNF-α and IL-4 content, but not IL-6, IL-13 and IL-10 quantified by ELISA. Mice with decreased levels of VAChT presented increased collagen and elastic fibers deposition in airway walls which was consistent with an increase in inflammatory cells positive to MMP-9 and TIMP-1 in the lung. In vivo lung function evaluation showed airway hyperresponsiveness to methacholine in mutant mice. The expression of nuclear factor-kappa B (p65-NF-kB) in lung of VAChT-deficient mice were higher than in wild-type mice, whereas a decreased expression of janus-kinase 2 (JAK2) was observed in the lung of mutant animals. Our findings show the first evidence that cholinergic deficiency impaired lung function and produce local inflammation. Our data supports the notion that cholinergic system modulates airway inflammation by modulation of JAK2 and NF-kB pathway. We proposed that intact cholinergic pathway is necessary to maintain the lung homeostasis.     

Genetic polymorphism (ABO, Rh, Kell) in Kabul (Afghanistan)

ABO, rhesus and Kell blood group data on 1327 donors in Kabul are analysed by ethnic affinity and compared with existing data on Afghanistan peoples. Blood group frequencies are very similar in Pushtu and Tadjik, despite their different historical, linguistic, and cultural backgrounds. Inclusion of the small sample of Hazara in the analysis shows overall heterogeneity in rhesus D, E, and e frequencies, suggesting the existence of a broader pattern of genetic variation among the peoples of Afghanistan.     

Distribution of rhesus blood group system in the French basques: a reappraisal using the allele-specific primers PCR method

OBJECTIVES: To determine for the first time using PCR the distribution of Rhesus (Rh) blood group in French Basques and compare these results with those obtained by serology in the same sample and in the historical series from various Basque subgroups. METHODS: Rh polymorphism was determined in a sample of 127 autochthonous French Basques using allele-specific primers (ASP) PCR and traditional serological technique. Statistical comparisons were performed between both techniques and with the data published from various Basque subpopulations. RESULTS: No intra-sample discrepancies were detected between ASP-PCR and serology. A high frequency of RH Ddel exceeding 0.50, as typically described from several decades, was found here (0.511), as the peculiar frequency of cde (0.456) and cDE (0.073) haplotypes. This profile, obtained by molecular analysis, was within the range of previous historical studies in various Basque subpopulations using serological approach. The Rh polymorphism among the reviewed autochthonous Basque samples indicates a heterogeneous pattern of distribution, with individuals from the Alava province demonstrating the most atypical profile. CONCLUSIONS: Molecular biology approach using PCR confirms the peculiar pattern of Rh polymorphism which was previously defined by serology among Basques. Nevertheless, this distribution profile is not homogeneous within the Basque area.     

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.     

Lateral root emission in woody taproots of Fraxinus ornus L.

Abstract

We applied environmental stresses, namely dehydration, pruning and bending, to woody taproots of Fraxinus ornus L. in order to: (i) identify a method that could be applied in routine studies of lateral root development from a secondary structure; and (ii) carry out anatomical investigations to identify the tissue involved in the recruitment of lateral root mother cells (LRMC). We found that all three methods induce the formation of new lateral roots from a woody parental root. However, bending stress considerably reduced the zone of the woody parental root (the curvature) for analysis when studying the process of emission of a new lateral root. The trace left by a new lateral root in the taproot secondary xylem forms a V-shaped insertion zone that starts in contact with a growth ring and enlarges toward the periphery. This type of insertion zone suggests that the vascular cambium is the tissue-source of initials that produce the root primordium of a new lateral root. In the case of root bending, the emission of a new lateral root occurs also in the convex side of the curvature and is preceded by the formation, at the same site, of a small amount of reaction wood. Thus, reaction wood and lateral root emission are two aspects of the same response mechanism to bending. Consequently, anatomical and cytological studies of lateral root formation should focus on this part of the woody taproot. By peeling off the bark at this site, one has direct access to the underlying living tissues and can thus investigate lateral root formation also at a biochemical and molecular level.     

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.