Toward gene therapy of hypertension: Experimental study on hypertensive ISIAH rats

TiO₂-based nanocomposites were prepared to deliver oligonucleotides into cells. The nanocomposites were designed by the immobilization of polylysine-containing oligonucleotides on TiO₂-nanoparticles (TiO₂·PL-DNA). We showed for the first time the possibility of using the proposed nanocomposites for treatment of hypertensive disease by introducing them into hypertensive ISIAH rats developed as a model of stress-sensitive arterial hypertension. The mRNA of the gene encoding angiotensin I-converting enzyme (ACE1) involved in the synthesis of angiotensin II was chosen as a target. Administration (intraperitoneal injection and inhalation) of the nanocomposite showed a significant (by 20-30 mm Hg) decrease in systolic blood pressure when the nanocomposite contained the ACE1 gene-targeted oligonucleotide. When using the oligonucleotide with a random sequence, no effect was observed. Further development and improvement of the inhalation nanocomposite drug delivery to systemic hypertensive disease treatment promises new possibilities for clinical practice.     

Tashkent riffle minnow <Emphasis Type="Italic">Alburnoides oblongus</Emphasis> belongs to the genus <Emphasis Type="Italic">Alburnus</Emphasis> (Osteichthyes: Cyprinidae) as inferred from mitochondrial and nuclear DNA markers

Sequences of mitochondrial (cytochrome b) and nuclear (recombination activating gene 1–RAG1) DNA markers were obtained for two species of the genus Alburnoides, the Taskent riffle minnow A. oblongus Bulgakov 1923 and the Terek spirlin A. gmelini Bogutskaya and Coad 2009. Phylogenetic analysis revealed that A. oblongus belongs to the genus Alburnus.     

Keeping 53BP1 out of focus in mitosis

A recent study published in Science reveals the mechanism and biological importance of DNA damage response abrogation in mitotic cells.For many years, much research has focused on understanding how cells maintain genome integrity despite DNA being constantly challenged by factors of both endogenous and exogenous nature. DNA double-strand breaks (DSBs) are the most deleterious DNA lesions, and if left unrepaired or repaired incorrectly, a single DSB can trigger genome instability or even cell death¹. Therefore, any DSB has to be recognized and repaired by processes encompassed within the DNA damage response (DDR). Notably, while the ends of mammalian linear chromosomes naturally resemble DSBs, their structure and association with the so-called “Shelterin” complex normally makes them invisible to the DDR².As soon as a DSB is formed, it is sensed and directly bound by the Ku70-Ku80 and/or MRE11-NBS1-RAD50 protein complexes, which recruit and activate the DDR kinases DNA-PKcs and ATM, respectively. The first steps in the DDR to DSBs are followed by cascades of events involving protein post-translational modifications (PTMs) and formation of large protein assemblies at DSB sites known as ionizing radiation-induced foci (IRIF)³. Protein phosphorylation and ubiquitylation are at the heart of these signaling processes³. For example, following recruitment of the DDR mediator protein MDC1 to the phospho-epitope created by ATM and DNA-PKcs on variant histone H2AX, MDC1 is itself phosphorylated by ATM on multiple serines and threonines⁴. MDC1 phosphorylation on a group of threonines near its N-terminus and conforming to the consensus TQXF generates binding sites for the FHA domain of E3-ubiquitin ligase RNF8^5,6. Together with the E2-conjugating enzyme UBC13, RNF8, and another E3 ligase, RNF168, trigger formation of mainly lysine 63-linked ubiquitin adducts in DSB-proximal chromatin, promoting recruitment of downstream factors necessary for DNA repair, such as the RAP80-Abraxas-BRCA1 complex and 53BP1³.Significantly, the full DDR happens only in interphase cells, whereas if mitotic cells sustain DSBs, the process appears to be blocked at the stage of RNF8 recruitment, resulting in IRIF devoid of detectable ubiquitin conjugates⁷. Consequently, 53BP1 and BRCA1 are not recruited to IRIF during mitosis. Even more strikingly, although RNF8 and RNF168 are associated with mitotic IRIF in anaphase, hyperphosphorylated 53BP1 remains excluded from chromatin until cells progress into G1 phase⁷. Based on these findings, it was hypothesized that mitosis-specific PTMs on RNF8 and 53BP1 might preclude formation of repair-competent IRIF⁷. However, the precise mechanistic explanation of the “interrupted” DDR in mitosis remained to be unravelled.A recent study published in Science by the group of Daniel Durocher addressed the question of how full IRIF assembly and DSB repair are prevented in mitotic cells⁸. First, Orthwein et al. focused on the mechanism that abrogates RNF8 recruitment to DSBs during mitosis. They demonstrated that CDK1-dependent mitosis-specific phosphorylation of RNF8 on T198 abolished interaction between RNF8 and its target phospho-TQXF motifs in MDC1. This important finding was somewhat surprising, given that MDC1 binding is mediated by the RNF8 FHA domain^5,6 and T198 is located some distance away from this domain. It will thus be interesting to see how T198 phosphorylation abrogates MDC1 binding, for example via T198 being juxtaposed to the FHA domain in the RNF8 3D structure, through phosphorylated T198 docking with the phospho-binding region of the FHA domain, or via another mechanism. In this regard, we note that T198 is part of an STP motif, which upon modification by CDK1 could constitute a priming site for PLK1 kinase⁹. Thus, T198 phosphorylation might be followed by PLK1-mediated RNF8 phosphorylation. Interestingly, certain sites in RNF8 conform to the PLK1 consensus motif, with those at T39 and T316 being evolutionarily conserved in vertebrates. Moreover, T39 is located in the FHA domain, close to R42, mutation of which abolishes RNF8 interaction with MDC1^5,6. It would therefore be worthwhile mutating these potential PLK1 sites and establishing whether this affects mitotic control of RNF8 binding to MDC1.After identifying T198 as critical for preventing RNF8 recruitment to DSBs during mitosis, Orthwein et al. observed that, while mutating this residue to alanine restored recruitment of RNF8 (and BRCA1) to mitotic IRIF, 53BP1 still remained excluded from DSB sites. This prompted the authors to look for mitosis-specific PTMs of 53BP1 by mass spectrometry, leading to the discovery of two novel phosphosites mapped to the recently described ubiquitin-dependent recruitment (UDR) motif, which mediates binding to ubiquitylated H2A and is required for 53BP1 IRIF formation¹⁰. Notably, the same residues, T1609 and S1618, were also identified by Chowdhury and colleagues¹¹ as target sites for the PP4C/R3β phosphatase. This group showed that T1609 and S1618 must be dephosphorylated for 53BP1 to form IRIF. In accord with these findings, Orthwein et al. established that when T1609 and S1618 were mutated to alanines, the ensuing “53BP1-TASA” protein was recruited to sites of DNA damage during mitosis in cells expressing RNF8-T198A. Moreover, unlike normal cells, cells co-expressing RNF8-T198A and 53BP1-TASA carried out DSB joining reactions during mitosis and were extremely hypersensitive to ionizing radiation (IR). The authors also found that, following irradiation in mitosis, cells carrying these mutant RNF8 and 53BP1 proteins displayed increased rates of kinetochore-positive micronucleus formation, suggesting mis-segregation of full chromosomes. In addition, chromosomes in these cells were prone to sister telomere fusions, thereby helping to explain their elevated levels of aneuploidy and IR hypersensitivity.The research described above has not only revealed how DSB repair is suppressed in mitosis but has also established that this suppression is biologically important. Orthwein et al. propose that, as mitotic telomeres become “underprotected” when mitosis is prolonged upon stress¹², this could lead to telomere fusion if DNA end-joining pathway is active. The suppression of DSB signaling and repair mediated by RNF8 and 53BP1 mitotic phosphorylation therefore probably evolved as a mechanism to mitigate this threat to genome stability. A key question that still remains is why mitotic telomeres become underprotected in the first place? Also, what features in telomere structure or replication and segregation processes make it more beneficial for the cells to keep chromosome ends less protected at the cost of inhibiting the DDR during mitosis? Finally, given that cancers often harbor cell cycle and/or DDR defects¹, it will be of interest to see whether defective mitotic control of DSB repair might play a role in tumor evolution, or could provide opportunities for developing better anti-cancer therapies.     

Relation of the progestagenic activity and conformation of the 17beta-acetyl side chain in the D'6-pentarane series

The synthesis of 2' beta-methyl-16 alpha,17 alpha-cyclohexanoprogesterone and its MM2 conformational analysis have been performed. The acetyl side chain was shown to have an unusual conformation with the torsion angle C13-C17-C20-O20 being -32.1 degrees. This conformation is by 5.4 kJ.mol-1 more stable than the usual one with the torsion angle 130.3 degrees. 2' beta-Methyl-16 alpha,17 alpha-cyclohexanoprogesterone proved to be inactive as a progestogen (pregnancy maintenance and McPhail tests). The lack of the activity may be due to the additional methyl group in D'-ring causing a change of the conformation of the 17 beta-acetyl side chain, thus hindering the formation of the conformation necessary for binding to the progesterone receptor.     

DNA slows dissociation of progesterone receptor-steroid ligand complexes

Steroid ligands are known to affect the interactions of their respective receptors with DNA. In the present study, the possibility of DNA interference in progesterone receptor-ligand interactions was investigated. An oligonucleotide containing a hormone response element (HRE) was shown to decrease the dissociation rate of complexes of [3H]progesterone or [3H]16alpha,17alpha-cycloalkanoprogesterones with PRs from rabbit and rat uterine cytosol. The extent to which the oligonucleotide affected the dissociation constant varied from about 4- to 1.5-fold depending on the ligand structure and was ranked in the following order: progesterone>16alpha,17alpha-cyclopropanoprogesterone approximately 16alpha,17alpha-cyclopentanoprogesterone>/=16alpha,17alpha-cyclohex-2'-enoprogesterone approximately 6alpha-methyl-16alpha,17alpha-cyclohexanoprogesterone>/=16alpha,17alpha-cyclohexanoprogesterone. The control oligonucleotide lacking HRE had a weak effect, if any, on the dissociation kinetics. No influence of the HRE-containing oligonucleotide on the equilibrium binding of ligands to PR was observed. The results suggest that the DNA partner affects binding of PR to its ligand.     

Red blood of cosmonauts during missions aboard the international space station (ISS)

Morphological and biochemical investigations of red blood in cosmonauts on board the International Space Station (ISS) (from the 6th to the 12th expeditions) in the space experiment program “Hematology” were carried out 30 days before the space mission (SM), at the initial (days 6–10) and final (days 160–190) stages of SM, and after the SM (immediately after SM (day 0) and on the 7th and 15th days of the adaptation period to earth conditions). A reduction of the concentration of hemoglobin after a prolonged influence of SM factors has been found, which is probably related not only to the intensity of erythropoiesis but also with the possible early removal of a part of low-quality (probably, old) erythrocytes from the bloodstream, which is confirmed by the results on the metabolism of red blood cells and the state of the cell membrane. Stimulation of erythropoiesis (increase in erythropoietin, decreased level of iron in blood, removal of low-quality and old erythrocytes) in the period of readaptation to conditions on earth is aimed at maintenance of the optimal level of red blood cells required for increased oxygen demand in tissues under the conditions of earth gravitation and enhancement of muscular load.     

Structure-function analysis of mononucleotides and short oligonucleotides in the priming of enzymatic DNA synthesis

The reversed-phase chromatography technique was employed in the measurement of DNA synthesis at the primers d(pT)n, r(pU)n, d(pA)n, and r(pA)n (n = 1-16) in the presence of template poly(dA) or poly(dT). DNA synthesis was catalyzed by Escherichia coli DNA polymerase I Klenow fragment, Physarum polycephalum DNA polymerase beta-like, P. polycephalum DNA polymerase alpha, and human placenta DNA polymerase alpha. Values of Km and Vmax were measured as functions of the primer chain lengths. It was found that all mononucleotides and small oligonucleotides served as primers of DNA synthesis. Values of the logarithm of both Km and Vmax increased linearly until primers had attained a chain length of 9-12 nucleotides, where a break was observed. The incremental as well as the absolute values of Km were interpreted in terms of free binding energies. These together with other data indicate that the 3'-ultimate nucleotide of the primer contributes a decisive amount of free energy of binding to DNA polymerase both from the nucleoside and from the phosphate moiety. The incremental increase is due to a complementary interaction between bases of primer and template buried in the binding cleft of the polymerase. It is also the ultimate nucleotide that determines whether the ribonucleotide or the deoxyribonucleotide is an efficient primer. It is of interest that the major results seem preserved for all four DNA polymerases. An energetic model for the binding of the template-primer was proposed and compared with available crystallographic data.     

Preparation of DNA chips using polyamine-oligonucleotide conjugates

A convenient and efficient method for three-dimensional immobilizing oligonucleotides on glass was developed using oligonucleotide derivatives bearing a polyamine linker (PA-oligo conjugates). Polyamine (polylysine, poly(lysine, phenylalanine), polyethyleneimine) residues stipulate durable fixation of such conjugates to the glass surface with a high yield (90-95%). A DNA fragment (414-mer) is hybridized specifically to an immobilized oligonucleotide.     

3-O-methoxyimino group inhibits interactions between progestins and blood transcortin

The interactions between E- and Z-isomers of 3-O-methoxyimino-pregn-4-ene-20-one and its 17α-hydroxy derivative and transcortin from human blood were investigated. The substitution of the progesterone 3-oxo group for a 3-O-methoxyimino group was shown to diminish the affinity of the steroid for transcortin by approximately one order of magnitude irrespective of the substituent’s orientation. The data suggests that progesterone derivatives substituted thereby must have higher bioavailability compared to progesterone and must not significantly affect the biodynamics of glucocorticoid in vivo.