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Abstract
In the promoter of c-KIT proto-oncogene, whose deregulation has been implicated in many cancers, three G-rich regions (kit1, kit* and kit2) are able to fold into G-quadruplexes. While kit1 ...and kit2 have been studied in depth, little information is available on kit* folding behavior despite its key role in regulation of c-KIT transcription. Notably, kit* contains consensus sites for SP1 and AP2 transcription factors. Herein, a set of complementary spectroscopic and biophysical methods reveals that kit*, dGGCGAGGAGGGGCGTGGCCGGC, adopts a chair type antiparallel G-quadruplex with two G-quartets at physiological relevant concentrations of KCl. Heterogeneous ensemble of structures is observed in the presence of Na+ and NH4+ ions, which however stabilize pre-folded structure. In the presence of K+ ions stacking interactions of adenine and thymine residues on the top G-quartet contribute to structural stability together with a G10*C18 base pair and a fold-back motif of the five residues at the 3′-terminal under the bottom G-quartet. The 3′-tail enables formation of a bimolecular pre-folded structure that drives folding of kit* into a single G-quadruplex. Intriguingly, kinetics of kit* G-quadruplex formation matches timescale of transcriptional processes and might demonstrate interplay of kinetic and thermodynamic factors for understanding regulation of c-KIT proto-oncogene expression.
Ligands that bind to and stabilize guanine-quadruplex (G4) structures to regulate DNA replication have therapeutic potential for cancer and neurodegenerative diseases. Because there are several G4 ...topologies, ligands that bind to their specific types may have the ability to preferentially regulate the replication of only certain genes. Here, we demonstrated that binding ligands stalled the replication of template DNA at G4, depending on different topologies. For example, naphthalene diimide derivatives bound to the G-quartet of G4 with an additional interaction between the ligand and the loop region of a hybrid G4 type from human telomeres, which efficiently repressed the replication of the G4. Thus, these inhibitory effects were not only stability-dependent but also topology-selective based on the manner in which G4 structures interacted with G4 ligands. Our original method, referred to as a quantitative study of topology-dependent replication (QSTR), was developed to evaluate correlations between replication rate and G4 stability. QSTR enabled the systematic categorization of ligands based on topology-dependent binding. It also demonstrated accuracy in determining quantitatively how G4 ligands control the intermediate state of replication and the kinetics of G4 unwinding. Hence, the QSTR index would facilitate the design of new drugs capable of controlling the topology-dependent regulation of gene expression.
An NMR structural study of the interaction between a small‐molecule optical probe (DAOTA‐M2) and a G‐quadruplex from the promoter region of the c‐myc oncogene revealed that they interact at 1:2 ...binding stoichiometry. NMR‐restrained structural calculations show that binding of DAOTA‐M2 occurs mainly through π–π stacking between the polyaromatic core of the ligand and guanine residues of the outer G‐quartets. Interestingly, the binding affinities of DAOTA‐M2 differ by a factor of two for the outer G‐quartets of the unimolecular parallel G‐quadruplex under study. Unrestrained MD calculations indicate that DAOTA‐M2 displays significant dynamic behavior when stacked on a G‐quartet plane. These studies provide molecular guidelines for the design of triangulenium derivatives that can be used as optical probes for G‐quadruplexes.
Playing the triangle in a quartet: A small‐molecule optical probe (DAOTA‐M2) based on a triangulenium core binds to G‐quadruplexes in a 1:2 stoichiometry. Binding of DAOTA‐M2 occurs mainly through π–π stacking between the triangulenium core and the guanine residues of the outer G‐quartets. Interestingly, the binding affinities of DAOTA‐M2 for the two outer G‐quartets differ by a factor of two.
YES G‐rich oligonucleotide VK2 folds into an AGCGA‐quadruplex tetrahelical structure distinct and significantly different from G‐quadruplexes, even though it contains four G3 tracts. Herein, a ...bis‐quinolinium ligand 360A with high affinity for G‐quadruplex structures and selective telomerase inhibition is shown to strongly bind to VK2. Upon binding, 360A does not induce a conformational switch from VK2 to an expected G‐quadruplex. In contrast, NMR structural study revealed formation of a well‐defined VK2–360A complex with a 1:1 binding stoichiometry, in which 360A intercalates between GAGA‐ and GCGC‐quartets in the central cavity of VK2. This is the first high‐resolution structure of a G‐quadruplex ligand intercalating into a G‐rich tetrahelical fold. This unique mode of ligand binding into tetrahelical DNA architecture offers insights into the stabilization of an AGCGA‐quadruplex by a heterocyclic ligand and provides guidelines for rational design of novel VK2 binding molecules with selectivity for different DNA secondary structures.
Within a quadruplex structure: Ligand 360A, a strong G‐quadruplex binder, stabilizes the guanine‐rich VK2 AGCGA‐quadruplex. Interestingly, in this case the formation of a G‐quadruplex is not favored by 360A. Instead, the ligand intercalates between residues from the central cavity of the VK2 structure and is additionally stabilized by rare structural motifs.
EGFR is an oncogene which codifies for a tyrosine kinase receptor that represents an important target for anticancer therapy. Indeed, several human cancers showed an upregulation of the activity of ...this protein. The promoter of this gene contains some G-rich domains, thus representing a yet unexplored point of intervention to potentially silence this gene. Here, we explore the conformational equilibria of a 30-nt long sequence located at position -272 (EGFR-272). By merging spectroscopic and electrophoretic analysis performed on the wild-type sequence as well as on a wide panel of related mutants, we were able to prove that in potassium ion containing solution this sequence folds into two main G-quadruplex structures, one parallel and one hybrid. They show comparable thermal stabilities and affinities for the metal ion and, indeed, they are always co-present in solution. The folding process is driven by a hairpin occurring in the domain corresponding to the terminal loop which works as an important stabilizing element for both the identified G-quadruplex arrangements.
The lack of efficient methods to control the major diseases of crops most important to agriculture leads to huge economic losses and seriously threatens global food security. Many of the most ...important microbial plant pathogens, including bacteria, fungi, and oomycetes, secrete necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs), which critically contribute to the virulence and spread of the disease. NLPs are cytotoxic to eudicot plants, as they disturb the plant plasma membrane by binding to specific plant membrane sphingolipid receptors. Their pivotal role in plant infection and broad taxonomic distribution makes NLPs a promising target for the development of novel phytopharmaceutical compounds. To identify compounds that bind to NLPs from the oomycetes Pythium aphanidermatum and Phytophthora parasitica, a library of 587 small molecules, most of which are commercially unavailable, was screened by surface plasmon resonance. Importantly, compounds that exhibited the highest affinity to NLPs were also found to inhibit NLP-mediated necrosis in tobacco leaves and Phytophthora infestans growth on potato leaves. Saturation transfer difference-nuclear magnetic resonance and molecular modelling of the most promising compound, anthranilic acid derivative, confirmed stable binding to the NLP protein, which resulted in decreased necrotic activity and reduced ion leakage from tobacco leaves. We, therefore, confirmed that NLPs are an appealing target for the development of novel phytopharmaceutical agents and strategies, which aim to directly interfere with the function of these major microbial virulence factors. The compounds identified in this study represent lead structures for further optimization and antimicrobial product development.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
There have been increasing reports on the presence of Histoplasma capsulatum in some European countries. The study investigated the presence of Histoplasma in bat guanos, speleologists with records ...of visiting Histoplasma-endemic regions and patients with histoplasmosis. A commercial ALPHA Histoplasma Antigen enzyme immunoassay was tested as an alternative methodology to detect Histoplasma in environment and compared with polymerase chain reaction (PCR) assays. The presence of Histoplasma antigen in bat guanos was not confirmed by PCR. Among 14 healthy speleologists, two were indicated as having the Histoplasma antigen in urine, but expressed negative PCR-specific results for the Histoplasma antigen. Five unequivocal cases of imported acute pulmonary histoplasmosis in Slovenia between years 2005 and 2016 were confirmed in patients returning from North and South America after visiting hazardous localities e.g., caves with guano, and places with dust. Currently there is no evidence of autochthonous histoplasmosis in Slovenia, or that bat guano is a source of H. capsulatum. Involvement of histoplasmosis in travellers' and cavers' morbidity might be underestimated in non-endemic areas. It is crucial to ensure the use of appropriate protective equipment in Histoplasma hazardous localities, to spread information about this hazardous microbe to vulnerable populations and to monitor the health of the environment. A differential diagnosis for a febrile respiratory disease outbreak in patients returning from endemic regions should trigger routine consideration of possible histoplasmosis.
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