35-10-9.pdf

The microtubule organizational changes in the isolated generative cells of Allemanda schottii were followed using immunofluorescence and confocal laser scanning microscopy. Due to the improved resolution and the lack of out-of-focus flares, the microtubule cytoskeleton of the generative cells could be visualized more clearly than using conventional epifluorescence systems. Immediately after isolation the microtubule cytoskeleton of the generative cells was cage-like composed of longitudinally oriented microtubule bundles. Later, some bundles began to depolymerize and at the same time some smaller bundles appearred. The smaller bundles unlike the longitudinal bundles crisscrossed throughout the cell. Later still, the cells became spherical. Both the longitudinal and the smaller bundles disappearred. At the same time some of the microtubules began to aggregate around the nucleus. These perinuclear microtubules were apparently not very stable, because soon afterwards,they started to disintegrate. By the time the cells became completely spherical,the cytoplasm became filled with diffuse fluorescence indicating that the tubulin was no longer existing in a polymerized form but in a monomeric form inside the cell. After the fuberlin had completely depolymerized the microtubules started to reform. The sequence of events leading to the reformation of the microtubule cytoskeleton in the spherical cells was as follow: A few nucleating centres began to form first. Then the nucleating centres gave rise to microtubule bundles. The bundles extended and aggregated to form a reticulate network. This cytoskeletal network appearred stable and well organized. It also had a lot of microtubule-bundle junctions. The network persisted after Triton X-l00 extraction.     

Dial 9-1-1 for DNA damage: the Rad9-Hus1-Rad1 (9-1-1) clamp complex

Genotoxic stress activates checkpoint signaling pathways that block cell cycle progression, trigger apoptosis, and regulate DNA repair. Studies in yeast and humans have shown that Rad9, Hus1, Rad1, and Rad17 play key roles in checkpoint activation. Three of these proteins-Rad9, Hus1, and Rad1-interact in a heterotrimeric complex (dubbed the 9-1-1 complex), which resembles a PCNA-like sliding clamp, whereas Rad17 is part of a clamp-loading complex that is related to the PCNA clamp loader, replication factor-C (RFC). In response to genotoxic damage, the 9-1-1 complex is loaded around DNA by the Rad17-containing clamp loader. The DNA-bound 9-1-1 complex then facilitates ATR-mediated phosphorylation and activation of Chk1, a protein kinase that regulates S-phase progression, G2/M arrest, and replication fork stabilization. In addition to its role in checkpoint activation, accumulating evidence suggests that the 9-1-1 complex also participates in DNA repair. Taken together, these findings suggest that the 9-1-1 clamp is a multifunctional complex that is loaded onto DNA at sites of damage, where it coordinates checkpoint activation and DNA repair.     

9-1-1: PCNA's specialized cousin

All living organisms are vulnerable to DNA damage. Cells respond to this hazard by activating a complex network of checkpoint and repair proteins to preserve genomic integrity. The DNA-encircling, ring-shaped heterotrimeric 9-1-1 complex, a relative of the replication protein PCNA, is a central coordinator of these events. 9-1-1 is loaded to damaged sites where it serves as a platform for the selective recruitment of checkpoint and repair proteins. In this Opinion article, 9-1-1 and proliferating cell nuclear antigen (PCNA) are compared and discussed in light of their respective structures and functions. We propose that the interaction partners of 9-1-1 possess specific 9-1-1-interaction boxes, which discriminate between 9-1-1 and PCNA thereby enabling specific interactions with individual 9-1-1 subunits.     

BDKRB2 GENE -9/+9 POLYMORPHISM AND SWIMMING PERFORMANCE

The aim of the study was to evaluate the association between swimming performance and the -9/+9 (rs5810761) polymorphism within the BDKRB2 gene in successful competitive swimmers. Best individual swimming results expressed in FINA points achieved at short, middle and long distance events of 157 well-trained Polish swimmers were incorporated into an analysis. Athletes’ genotype and allele distributions were analysed in comparison to 230 unrelated sedentary subjects who served as controls with the χ² test. All samples were genotyped for the BDKRB2 -9/+9 polymorphism using the polymerase chain reaction (PCR). The effects of genotype on swimming performance were analysed with two-way (3 x 2; genotype x gender) analysis of variance with metrical age as a covariate for each distance specialization. No statistical differences in the genotype and allele frequencies were found in long distance swimmers when compared with the total group of swimmers or controls. The BDKRB2 +9/-9 genotype had no significant effect on swimming performance at short, middle or long distance, regardless of gender. The results of this study do not support the hypothesis that the BDKRB2 -9/+9 polymorphism is associated with swimming performance in Polish swimmers.     

Kinetic mechanism of RGS9-1 potentiation by R9AP

The duration of the photoreceptor's response to a light stimulus determines the speed at which an animal adjusts to ever-changing conditions of the visual environment. One critical component which regulates the photoresponse duration on the molecular level is the complex between the ninth member of the regulators of G protein signaling family (RGS9-1) and its partner, type 5 G protein beta-subunit (Gbeta5L). RGS9-1.Gbeta5L is responsible for the activation of the GTPase activity of the photoreceptor-specific G protein, transducin. Importantly, this function of RGS9-1.Gbeta5L is regulated by its membrane anchor, R9AP, which drastically potentiates the ability of RGS9-1.Gbeta5L to activate transducin GTPase. In this study, we address the kinetic mechanism of R9AP action and find that it consists primarily of a direct increase in the RGS9-1.Gbeta5L activity. We further showed that the binding site for RGS9-1.Gbeta5L is located within the N-terminal putative trihelical domain of R9AP, and even though this domain is sufficient for binding, it takes the entire R9AP molecule to potentiate the activity of RGS9-1.Gbeta5L. The mechanism revealed in this study is different from and complements another well-established mechanism of regulation of RGS9-1.Gbeta5L by the effector enzyme, cGMP phosphodiesterase, which is based entirely on the enhancement in the affinity between RGS9-1.Gbeta5L and transducin. Together, these mechanisms ensure timely transducin inactivation in the course of the photoresponse, a requisite for normal vision.     

1-(2,6-Dibenzyloxybenzoyl)-3-(9H-fluoren-9-yl)-urea: A novel cyclophilin A allosteric activator

Cyclophilin A (CypA) plays an important role in many physiology processes and its overexpression has been involved in many diseases including immune disease, viral infection, neuro-degenerative disease, and cancer. However, the actual role of CypA in the diseases is still far from clear, and a complete understanding of CypA is necessary in order to direct more specific and effective therapeutic strategies. Based on the screening of our in-house library through the isomer-specific proteolysis method, we find a CypA activator (1-(2,6-Dibenzyloxybenzoyl)-3-(9H-fluoren-9-yl)-urea), compound 1a, which can increase CypA’s PPIase activity and give allosteric behavior. The binding affinity of compound 1a to CypA has been confirmed by Fortebio’s Octet RED system and the increased phosphorylation of ERK in H446 cells is observed by treatment with both compound 1a and CsA. In order to further evaluate the binding mode between the activator and CypA, the allosteric binding site and allosteric mechanism of CypA are investigated by molecular dynamics (MD) simulations in combination with mutagenesis experiments. The results show that the allosteric binding site of CypA is 7 Å away from its catalytic site and is composed of Cys52, His70, His54, Lys151, Thr152 and Lys155. Compound 1a binds to the allosteric site of CypA, stabilizing the active conformation of catalytic residues, and finally promotes the catalytic efficiency of CypA. We believe our finding of the CypA allosteric activator will be used as an effective chemical tool for further studies of CypA mechanisms in diseases.     

9-[(10-(aden-9-yl)-4,8-diazadecyl)amino]-6-chloro-2-methoxy-acridine incises DNA at apurinic sites.

The incision of DNA at apurinic/apyrimidinic sites (AP-sites) by chloro-6-methoxy-2 [(adenyl-9)-11)-4,8 diazadecyl]amino-9 acridine (Ade-Z-Acr), a 9-aminoacridine linked to an adenine, at nanomolar concentrations is described. Moreover, this drug, Ade-Z-Acr, is one of the most efficient drugs which cleaves DNA at AP-sites. The high activity is the result of the composition of the drug, since the individual components have no incising activity in the concentration range studied. The termini left by the Ade-Z-Acr molecule are a 3'deoxyribose and a 5'nucleotide. The termini and the inability of the Ade-Z-Acr to incise DNA with reduced AP-sites suggest that the mechanism of cleavage is beta-elimination.     

Synthesis of 9-[(Phosphonomethoxy)methyl]guanine and 9-[2-Hydroxy-1-(phosphonomethoxy)ethyl]guanine

Abstract

The synthesis of 9-[(phosphonomethoxy)methyl]guanine (3) and 9-[2-hydroxy-1-(phosphonomethoxy)ethyl]guanine (4) is described.     

Immunogenicity of Outer Membrane Proteins VirB9-1 and VirB9-2, a Novel Nanovaccine against Anaplasma marginale

Anaplasma marginale is the most prevalent tick-borne livestock pathogen and poses a significant threat to cattle industry. In contrast to currently available live blood-derived vaccines against A. marginale, alternative safer and better-defined subunit vaccines will be of great significance. Two proteins (VirB9-1 and VirB9-2) from the Type IV secretion system of A. marginale have been shown to induce humoral and cellular immunity. In this study, Escherichia coli were used to express VirB9-1 and VirB9-2 proteins. Silica vesicles having a thin wall of 6 nm and pore size of 5.8 nm were used as the carrier and adjuvant to deliver these two antigens both as individual or mixed nano-formulations. High loading capacity was achieved for both proteins, and the mouse immunisation trial with individual as well as mixed nano-formulations showed high levels of antibody titres over 10⁷ and strong T-cell responses. The mixed nano-formulation also stimulated high-level recall responses in bovine T-cell proliferation assays. These results open a promising path towards the development of efficient A. marginale vaccines and provide better understanding on the role of silica vesicles to deliver multivalent vaccines as mixed nano-formulations able to activate both B-cell and T-cell immunity, for improved animal health.