Synthesis of Macrocyclic Hexaoxazole (6OTD) Dimers, Containing Guanidine and Amine Functionalized Side Chains, and an Evaluation of Their Telomeric G4 Stabilizing Properties

Structure-activity relationship studies were carried out on macrocyclic hexaoxazole (6OTD) dimers, whose core structure stabilizes telomeric G-quadruplexes (G4). Two new 6OTD dimers having side chain amine and guanidine functional groups were synthesized and evaluated for their stabilizing ability against a telomeric G4 DNA sequence. The results show that the 6OTD dimers interact with the DNA to form 1:1 complexes and stabilize the antiparallel G4 structure of DNA in the presence of potassium cation. The guanidine functionalized dimer displays a potent stabilizing ability of the G4 structure, as determined by using a FRET melting assay (ΔTm = 14°C)

Stereochemical assignment of the protein-protein interaction inhibitor JBIR-22 by total synthesis

The authors acknowledge the EPSRC and Cancer Research UK (CRUK Grant No. C21383/A6950) for funding this research.Recent reports have highlighted the biological activity associated with a sub-family of the tetramic acid class of natural products. Despite the fact that members of this sub-family act as protein-protein interaction inhibitors of relevance to proteasome assembly, no synthetic work has been reported. This may be because this sub-family contains an unnatural 4,4-disubstitued glutamic acid, the synthesis of which provides a key challenge. Here we describe a highly stereoselective route to a masked form of this unnatural amino acid. This enabled the synthesis of two of the possible diastereomers of JBIR-22 and allowed its relative and absolute stereochemistry to be assigned.Publisher PDFPeer reviewe

Cation Involvement in Telomestatin Binding to G-Quadruplex DNA

The binding mode of telomestatin to G-quadruplex DNA has been investigated using electrospray mass spectrometry, by detecting the intact complexes formed in ammonium acetate. The mass measurements show the incorporation of one extra ammonium ion in the telomestatin complexes. Experiments on telomestatin alone also show that the telomestatin alone is able to coordinate cations in a similar way as a crown ether. Finally, density functional theory calculations suggest that in the G-quadruplex-telomestatin complex, potassium or ammonium cations are located between the telomestatin and a G-quartet. This study underlines that monovalent cation coordination capabilities should be integrated in the rational design of G-quadruplex binding ligands

A gene expression test for depression

Purpose: Recently, we could distinguished patients with major depressive disorder (MDD) from nonpsychiatric controls with high accuracy using a panel of five gene expression markers (ARHGAP24, HDAC5, PDGFC, PRNP, and SLC6A4) in leukocyte. In the present study, we examined whether this biological test is able to discriminate patients with MDD from those without MDD, including those with schizophrenia and bipolar disorder. Patients and methods: We measured messenger ribonucleic acid expression levels of the aforementioned five genes in peripheral leukocytes in 17 patients with schizophrenia and 36 patients with bipolar disorder using quantitative real-time polymerase chain reaction (PCR), and we combined these expression data with our previous expression data of 25 patients with MDD and 25 controls. Subsequently, a linear discriminant function was developed for use in discriminating between patients with MDD and without MDD. Results: This expression panel was able to segregate patients with MDD from those without MDD with a sensitivity and specificity of 64% and 67.9%, respectively. Conclusion: Further research to identify MDD-specific markers is needed to improve the performance of this biological test

Novel macrolactam compound produced by the heterologous expression of a large cryptic biosynthetic gene cluster of Streptomyces rochei IFO12908

In the course of our studies on the heterologous expression of giant biosynthetic genes, we discovered a novel cryptic biosynthetic gene cluster in Streptomyces rochei IFO12908. During our efforts to express biosynthetic genes using the host SUKA strain derived from Streptomyces avermitilis, a novel polyene macrolactam compound designated as JBIR-156 was produced. We report herein the cloning and heterologous expression of the JBIR-156 biosynthetic gene cluster, and the isolation, structure determination, and cytotoxic activity of this novel compound

Mammalian DNA2 helicase/nuclease cleaves G-quadruplex DNA and is required for telomere integrity

Efficient and faithful replication of telomeric DNA is critical for maintaining genome integrity. The G-quadruplex (G4) structure arising in the repetitive TTAGGG sequence is thought to stall replication forks, impairing efficient telomere replication and leading to telomere instabilities. However, pathways modulating telomeric G4 are poorly understood, and it is unclear whether defects in these pathways contribute to genome instabilities in vivo. Here, we report that mammalian DNA2 helicase/nuclease recognizes and cleaves telomeric G4 in vitro. Consistent with DNA2’s role in removing G4, DNA2 deficiency in mouse cells leads to telomere replication defects, elevating the levels of fragile telomeres (FTs) and sister telomere associations (STAs). Such telomere defects are enhanced by stabilizers of G4. Moreover, DNA2 deficiency induces telomere DNA damage and chromosome segregation errors, resulting in tetraploidy and aneuploidy. Consequently, DNA2-deficient mice develop aneuploidy-associated cancers containing dysfunctional telomeres. Collectively, our genetic, cytological, and biochemical results suggest that mammalian DNA2 reduces replication stress at telomeres, thereby preserving genome stability and suppressing cancer development, and that this may involve, at least in part, nucleolytic processing of telomeric G4

Identification, cloning and heterologous expression of biosynthetic gene cluster for desertomycin

From our in-house microbial genome database of secondary metabolite producers, we identified a candidate biosynthetic gene cluster for desertomycin from Streptomyces nobilis JCM4274. We report herein the cloning of the 127-kb entire gene cluster for desertomycin biosynthesis using bacterial artificial chromosome vector. The entire biosynthetic gene cluster for desertomycin was introduced in the heterologous host, Streptomyces lividans TK23, with an average yield of more than 130 mg l(-1)

Spectroscopic Observations of the WZ Sge-Type Dwarf Nova GW Librae during the 2007 Superoutburst

We carried out an international spectroscopic observation campaign of the dwarf nova GW Librae (GW Lib) during the 2007 superoutburst. Our observation period covered the rising phase of the superoutburst, maximum, slowly decaying phase (plateau), and long fading tail after the rapid decline from the plateau. The spectral features dramatically changed during the observations. In the rising phase, only absorption lines of H$\alpha$, H$\beta$, and H$\gamma$ were present. Around the maximum, the spectrum showed singly-peaked emission lines of H$\alpha$, He I 5876, He I 6678, He II 4686, and C III/N III as well as absorption lines of Balmer components and He I. These emission lines significantly weakened in the latter part of the plateau phase. In the fading tail, all the Balmer lines and He I 6678 were in emission, as observed in quiescence. We find that the center of the H$\alpha$ emission component was mostly stable over the whole orbital phase, being consistent with the low inclination of the system. Comparing with the observational results of WZ Sge during the 2001 superoutburst, the same type of stars as GW Lib seen with a high inclination angle, we interpret that the change of the H$\alpha$ profile before the fading tail phase is attributed to a photoionized region formed at the outer edge of the accretion disk, irradiated from the white dwarf and inner disk.Comment: 10 pages, 9 figures, accepted for publication in PAS

Novel Plant-Associated Acidobacteria Promotes Growth of Common Floating Aquatic Plants, Duckweeds

Duckweeds are small, fast growing, and starch- and protein-rich aquatic plants expected to be a next generation energy crop and an excellent biomaterial for phytoremediation. Despite such an importance, very little is known about duckweed–microbe interactions that would be a key biological factor for efficient industrial utilization of duckweeds. Here we first report the duckweed growth promoting ability of bacterial strains belonging to the phylum Acidobacteria, the members of which are known to inhabit soils and terrestrial plants, but their ecological roles and plant–microbe interactions remain largely unclear. Two novel Acidobacteria strains, F-183 and TBR-22, were successfully isolated from wild duckweeds and phylogenetically affiliated with subdivision 3 and 6 of the phylum, respectively, based on 16S rRNA gene sequence analysis. In the co-culture experiments with aseptic host plants, the F-183 and TBR-22 strains visibly enhanced growth (frond number) of six duckweed species (subfamily Lemnoideae) up to 1.8–5.1 times and 1.6–3.9 times, respectively, compared with uninoculated controls. Intriguingly, both strains also increased the chlorophyll content of the duckweed (Lemna aequinoctialis) up to 2.4–2.5 times. Under SEM observation, the F-183 and TBR-22 strains were epiphytic and attached to the surface of duckweed. Taken together, our findings suggest that indigenous plant associated Acidobacteria contribute to a healthy growth of their host aquatic plants

The G-Quadruplex Ligand Telomestatin Impairs Binding of Topoisomerase IIIα to G-Quadruplex-Forming Oligonucleotides and Uncaps Telomeres in ALT Cells

In Alternative Lengthening of Telomeres (ALT) cell lines, specific nuclear bodies called APBs (ALT-associated PML bodies) concentrate telomeric DNA, shelterin components and recombination factors associated with telomere recombination. Topoisomerase IIIα (Topo III) is an essential telomeric-associated factor in ALT cells. We show here that the binding of Topo III to telomeric G-overhang is modulated by G-quadruplex formation. Topo III binding to G-quadruplex-forming oligonucleotides was strongly inhibited by telomestatin, a potent and specific G-quadruplex ligand. In ALT cells, telomestatin treatment resulted in the depletion of the Topo III/BLM/TRF2 complex and the disruption of APBs and led to the segregation of PML, shelterin components and Topo III. Interestingly, a DNA damage response was observed at telomeres in telomestatin-treated cells. These data indicate the importance of G-quadruplex stabilization during telomere maintenance in ALT cells. The function of TRF2/Topo III/BLM in the resolution of replication intermediates at telomeres is discussed