Induction of a senescent-like phenotype does not confer the ability of bovine immortal cells to support the development of nuclear transfer embryos

Previously, we reported that cloned embryos derived from an immortalized bovine mammary epithelial cell line (MECL) failed to develop beyond 12- to 16-cell stage. To analyze whether induction of a senescent-like phenotype in MECL can improve their ability to support the development after transfer into enucleated oocytes, we treated MECL with DNA methylation inhibitor 5-aza-2-deoxycytidine (Aza-C), histone deacetylase inhibitors trichostatin A (TSA), sodium butyrate (NaBu), or 5-bromodeoxyuridine and used those cells for nuclear transfer. Primary bovine fetal fibroblasts (BFF) were used as control. All agents were capable to induce features of senescence including reduced cell proliferation, enlarged cell size with a considerable proportion of cells stained positive for acidic senescence-associated beta-galactosidase and G1/S cell cycle boundary arrest in MECL. Aza-C treatment induced genome demethylation. Acetylation of H3 and H4 was increased after TSA treatment in both MECL and BFF, whereas no obvious changes in global H3 or H4 acetylation were detected after NaBu treatment. Nuclear transfer experiments following diverse treatments demonstrated that the induced senescent-like phenotype of MECL did not confer their ability to support embryonic development, although 7.3% of reconstructed embryos derived from NaBu-treated cells developed to morula stage. Intriguingly, a much higher proportion of cloned embryos developed to blastocysts when using NaBu-treated BFF, compared with using untreated BFF (59% versus 26%). Our results suggest that the developmental failure of donor nuclei from bovine immortal cells could not be reversed by induction of senescent-like phenotype. The beneficial effect of NaBu on the developmental potential of cloned embryos reconstructed from BFF merits further studies.     

Identification of the primate papovavirus HD as the stump-tailed macaque virus.

The recently isolated primate papovavirus HD is shown to be indistinguishable from the stump-tailed macaque virus by immunofluorescent reactivity, by restriction endonuclease analysis, and by nucleic acid hybridization assay.     

Establishment and Growth Kinetics of the Mutant Ehrlich-Lettré Ascites Cell Strain Hd33 In Permanent Suspension Culture1,2

Cells of a mutant in vivo subline of the Ehrlich-Lettré mouse ascites tumour (ELAT) were converted to growth in suspension culture. Kinetic analysis revealed the selective character of the conversion process; without a detectable adaptation period, a fraction of about 2 × 10^-5 of the explanted cells continued to grow in vitro. the resulting, mutant Ehrlich-Lettré ascites cell strain was designated HD33 and propagated uninterruptedly from 1974 on. the corresponding in vivo ELAT subline HD33 was derived from the HD33 ascites cell strain by intraperitoneal retransplantation. In HD33 cell suspension cultures, the population doubling time, the average intermitotic interval, as determined by videomonitoring, and the average duration of the cell cycle, as determined from percentage of labelled mitoses (PLM) data, were all measured at 15 hr. Cell loss and quiescent compartments were insignificant. the duration of the G₁ phase was effectively zero. Both PLM data and [³H]/[¹⁴C] thymidine double-labetling measurements revealed an S-phase duration of between 11 and 12 hr. the G₂ phase lasted 3–5 hr. The HD33 strain differs from comparable suspension strains of wild-type Ehrlich ascites cells in the insignificant role of density-dependent inhibition in growth, and the striking prolongation of the S phase which is associated with an excessive, cytoplasmic storage of glycogen by the mutant cells.     

Dual 5-HT1A agonists and 5-HT re-uptake inhibitors by combination of indole-butyl-amine and chromenonyl-piperazine structural elements in a single molecular entity

The dual serotonin (5-HT) re-uptake inhibitor and 5-HT(1A) receptor agonist vilazodone was found to increase central serotonin levels in rat brain. In the course of structural modifications of vilazodone 3-[4-[4-(2-oxo-2H-1-benzopyran-6-yl)-1-piperazinyl]-butyl]-1H-indole-5-carbonitrile 8i and its fluorine analogue 6-[4-[4-(5-fluor-3-indolyl)-butyl]-1-piperazinyl]-2H-1-benzopyran-2-one have been identified. These unsubstituted chromenones are equally potent at the 5-HT(1A) receptor and 5-HT transporter. The implementation of nitrogen functionalities in position 3 of the chromenones resulted in compounds acting as agonists at the 5-HT(1A) receptor and as 5-HT re-uptake inhibitors like vilazodone. Ex vivo 5-HT re-uptake inhibition and in vitro 5-HT agonism were determined in the PCA- and GTPgammaS-assay, respectively. The potential of these chromenones to increase central 5-HT levels was measured in microdialysis studies and especially the derivatives 3-[4-[4-(3-amino-2-oxo-2H-chromen-6-yl)-piperazin-1-yl]-butyl]-1H-indole-5-carbonitrile 8f, ethyl (6-[4-[4-(5-cyano-1H-indol-3-yl)-butyl]-piperazin-1-yl]-2-oxo-2H-chromen-3-yl)-carbamate 8h and N-(6-[4-[4-(5-cyano-1H-indol-3-yl)-butyl]-piperazin-1-yl]-2-oxo-2H-chromen-3-yl)-acetamide 8k give rise to rapid development of increased serotonin levels in rat brain cortex, lasting longer than 3h.     

Beiträge zur Vitalfärbung von Pilzmyzelien II

Ohne ZusammenfassungBeitr. z. Vitalf. v. Pilzmyzelien I; Flora134, S. 58, 1939.     

Binding of histamine to the H1 receptor—a molecular dynamics study

Binding of histamine to the G-protein coupled histamine H₁ receptor plays an important role in the context of allergic reactions; however, no crystal structure of the resulting complex is available yet. To deduce the histamine binding site, we performed unbiased molecular dynamics (MD) simulations on a microsecond time scale, which allowed to monitor one binding event, in which particularly the residues of the extracellular loop 2 were involved in the initial recognition process. The final histamine binding pose in the orthosteric pocket is characterized by interactions with Asp107^3.32, Tyr108^3.33, Thr194^5.43, Asn198^5.46, Trp428^6.48, Tyr431^6.51, Phe432^6.52, and Phe435^6.55, which is in agreement with existing mutational data. The conformational stability of the obtained complex structure was subsequently confirmed in 2 μs equilibrium MD simulations, and a metadynamics simulation proved that the detected binding site represents an energy minimum. A complementary investigation of a D107A mutant, which has experimentally been shown to abolish ligand binding, revealed that this exchange results in a significantly weaker interaction and enhanced ligand dynamics. This finding underlines the importance of the electrostatic interaction between the histamine ammonium group and the side chain of Asp107^3.32 for histamine binding.     

Synthesis and evaluation of [¹⁸F]fluororasagiline, a novel positron emission tomography (PET) radioligand for monoamine oxidase B (MAO-B)

The aim of this study was to synthesize and evaluate a novel fluorine-18 labeled analogue of rasagiline (6) as a PET radioligand for monoamine oxidase B (MAO-B). The corresponding non-radioactive fluorine-19 ligand, (1S,2S)-2-fluoro-N-(prop-2-yn-1-yl)indan-1-amine (4), was characterized in in vitro assays. The precursor compound (3aS,8aR)-3-(prop-2-yn-1-yl)-3,3a,8,8a-tetrahydroindeno[1,2-d][1,2,3]oxathiazole 2,2-dioxide (3) and reference standard 4 were synthesized in multi-step syntheses. Recombinant human MAO-B and MAO-A enzyme preparations were used in order to determine IC(50) values for compound 4 by use of an enzymatic assay employing kynuramine as substrate. Radiolabeling was accomplished by a two-step synthesis, compromising a nucleophilic substitution followed by hydrolysis of the sulphamidate group. Human whole hemisphere autoradiography (ARG) was performed with [(18)F]fluororasagiline. Blocking experiments with pirlindole (MAO-A), L-deprenyl and rasagiline (MAO-B) were conducted to demonstrate the specificity of the binding. A positron emission tomography (PET) study was carried out in a cynomolgus monkey where time activity curves for whole brain and regions with high and low MAO-B activity were recorded. Radiometabolites were measured in monkey plasma using gradient HPLC. Compound 4 inhibited MAO-B with an IC(50) of 27 nM and MAO-A with an IC(50) of 2.3 μM. Radiolabeling of precursor 3 and subsequent hydrolysis of the protecting group towards (1S,2S)-2-[(18)F]fluoro-N-(prop-2-yn-1-yl)indan-1-amine (6) was successfully accomplished with an radiochemical yield of 40-70%, a radiochemical purity higher than 99% and a specific radioactivity higher than 200GBq/μmol. ARG demonstrated selective binding for [(18)F]fluororasagiline (6) to MAO-B containing brain regions, for example, striatum. The initial uptake in the monkey brain was 250% SUV at 4 min post injection. The highest amounts of radioactivity were observed in the striatum and thalamus as expected whereas in the cortex and cerebellum lower levels were observed. Metabolite studies demonstrated 30% unchanged radioligand at 90 min post injection. Our investigations demonstrated that the new ligand [(18)F]fluororasagiline (6) binds specifically to MAO-B in vitro and has a MAO-B specific binding pattern in vivo. Thus, it could serve as a novel potential candidate for human PET studies.