TRF2 dysfunction elicits DNA damage responses associated with senescence in proliferating neural cells and differentiation of neurons

Telomeres are specialized structures at the ends of chromosomes that consist of tandem repeats of the DNA sequence TTAGGG and several proteins that protect the DNA and regulate the plasticity of the telomeres. The telomere-associated protein TRF2 (telomeric repeat binding factor 2) is critical for the control of telomere structure and function; TRF2 dysfunction results in the exposure of the telomere ends and activation of ATM (ataxia telangiectasin mutated)-mediated DNA damage response. Recent findings suggest that telomere attrition can cause senescence or apoptosis of mitotic cells, but the function of telomeres in differentiated neurons is unknown. Here, we examined the impact of telomere dysfunction via TRF2 inhibition in neurons (primary embryonic hippocampal neurons) and mitotic neural cells (astrocytes and neuroblastoma cells). We demonstrate that telomere dysfunction induced by adenovirus-mediated expression of dominant-negative TRF2 (DN-TRF2) triggers a DNA damage response involving the formation of nuclear foci containing phosphorylated histone H2AX and activated ATM in each cell type. In mitotic neural cells DN-TRF2 induced activation of both p53 and p21 and senescence (as indicated by an up-regulation of beta-galactosidase). In contrast, in neurons DN-TRF2 increased p21, but neither p53 nor beta-galactosidase was induced. In addition, TRF2 inhibition enhanced the morphological, molecular and biophysical differentiation of hippocampal neurons. These findings demonstrate divergent molecular and physiological responses to telomere dysfunction in mitotic neural cells and neurons, indicate a role for TRF2 in regulating neuronal differentiation, and suggest a potential therapeutic application of inhibition of TRF2 function in the treatment of neural tumors.     

Prolonging bovine sperm-oocyte incubation in modified medium 199 improves embryo development rate and the viability of vitrified blastocysts

This study was conducted to compare the efficacy of four in vitro fertilization (IVF) media: Bracket and Oliphant's medium (BO), modified medium 199 (IVF-M199), modified Tyrode's medium (MTM), and modified KSOM (m-KSOM) on fertilization efficiency and blastocyst formation rate. In addition, we wanted to investigate the benefit of prolonging the IVF period (from 6 to 18 h) using the two most effective IVF media determined in our initial experiment; subsequently, blastocyst viability was assessed following vitrification. A higher incidence of polyspermic fertilization was observed in the MTM (6%) and in BO, in both the 6 and 18 h (7% and 11%, respectively) groups, than in the m-KSOM (1%) or in the IVF-M199 6 or 18 h (1 and 3%, respectively) groups. Cleavage rates were similar in BO, IVF-M199, and MTM 48 h post-fertilization; however, the lowest cleavage rate was observed for m-KSOM. A greater proportion of zygotes developed into 8-cell embryos in IVF-M199 than in other IVF media. Subsequently, a greater proportion of blastocyst formation and hatching was achieved in IVF-M199 (40% and 79%, respectively) or BO (35% and 74%, respectively) than in m-KSOM (18% and 58%, respectively) or MTM (22% and 66%, respectively). Prolonging IVF to 18 h did not alter cleavage rates; however, the highest rate of overall blastocyst formation was achieved in the IVF-M199 18 h (49%), rather than in the BO 18 h (20%) group. Vitrified/thawed blastocysts from IVF-M199 groups re-expanded and developed better, as compared to the BO 18 h group, and hatching rate and total cell number in IVF-M199 18 h group was comparable to the control groups (non-vitrified). Vitrification reduced survival compared to controls. In conclusion, IVF-M199 was successfully used for IVF, compared favorably to BO medium, and offered the advantage of an extended IVF period for up to 18 h that requires only one-half a dose of semen, and resulted in better quality blastocysts that endured vitrification with a hatching rate comparable to that of control groups.     

Preferential localization of abscisic acid in primordial and nursing cells of reproductive organs of Arabidopsis and cucumber

Abscisic acid (ABA) is known to function in plant stress responses and seed dormancy, and much is known about its detailed mechanisms of signal transduction. Recent studies suggest that this hormone may also play important roles in sugar signaling and assimilate distribution during fruit development. However, little is known about the role of ABA in actively growing or differentiating fruits and other plant organs or tissues. To explore whether ABA functions during the early development of reproductive organs, we carried out ABA immunolocalization using monoclonal antibodies. The specific ABA accumulation pattern was verified by gas chromatography-mass spectrometry (GC-MS). ABA was not only detected in primordial cells of flower organs, but was also detected in nursing cells (e.g. tapetum and integuments), which function in supplying nutrition for germ cell development. These findings suggest that, in addition to its well-known function as a 'negative hormone', ABA may play some 'positive' roles during plant development, including possible involvement in the regulation of assimilate distribution.     

Microbial desulfurization of gasoline by free whole-cells of Rhodococcus erythropolis XP

Rhodococcus erythropolis XP could grow well with condensed thiophenes, mono-thiophenic compounds and mercaptans present in gasoline. Rhodococcus erythropolis XP was also capable of efficiently degrading the condensed thiophenes in resting cell as well as biphasic reactions in which n-octane served as a model oil phase. Free whole cells of R. erythropolis XP were adopted to desulfurize fluid catalytic cracking (FCC) and straight-run (SR) gasoline oils. About 30% of the sulfur content of FCC gasoline and 85% of sulfur in SR gasoline were reduced, respectively. Gas chromatography analysis with atomic emission detection also showed depletion of sulfur compounds in SR gasoline. Rhodococcus erythropolis XP could partly resist the toxicity of gasoline and had an application potential to biodesulfurization of gasoline.     

Influence of rhamnose substituents on the potency of SL0101, an inhibitor of the Ser/Thr kinase, RSK

We have previously reported the isolation of kaempferol 3-O-(3',4'-di-O-acetyl-alpha-l-rhamnopyranoside) from Forsteronia refracta [Xu, Y.-M.; Smith, J. A.; Lannigan, D. A.; Hecht, S. M. Biorg. Med. Chem.2006, 14, 3974-3977.]. This flavonoid glycoside, termed SL0101, is a specific inhibitor of p90 ribosomal S6 kinase (RSK) with a dissociation constant of 1 microM. In intact cells, however, the EC50 for inhibition of RSK activity is 50 microM, which suggests that the efficacy of SL0101 could be limited by cellular uptake. Therefore, we investigated the possibility of developing a more potent RSK inhibitor by synthesizing SL0101 analogs with increased hydrophobic character. The total syntheses of kaempferol 3-O-(3',4'-di-O-butyryl-alpha-L-rhamnopyranoside) (Bu-SL0101) and kaempferol 3-O-(2',3',4'-tri-O-acetyl-alpha-L-rhamnopyranoside) (3Ac-SL0101) were performed. The IC50 for inhibition of RSK activity in in vitro kinase assays for the analogs was similar to that obtained for SL0101. 3Ac-SL0101 demonstrated the same remarkable specificity for inhibiting RSK activity in intact cells as SL0101; however, Bu-SL0101 was not completely specific. 3Ac-SL0101 was approximately 2-fold more potent at inhibiting MCF-7 cell proliferation compared to SL0101 and preferentially decreased MCF-7 cell growth, as compared to the growth of the normal human breast line, MCF-10A. Thus the discovery of 3Ac-SL0101 as a more potent RSK-specific inhibitor than SL0101 should facilitate the development of RSK inhibitors as anti-cancer chemotherapeutic agents.     

Scaffold compound L971 exhibits anti‐inflammatory activities through inhibition of JAK/STAT and NFκB signalling pathways

JAK/STAT and NFκB signalling pathways play essential roles in regulating inflammatory responses, which are important pathogenic factors of various serious immune‐related diseases, and function individually or synergistically. To find prodrugs that can treat inflammation, we performed a preliminary high‐throughput screening of 18 840 small molecular compounds and identified scaffold compound L971 which significantly inhibited JAK/STAT and NFκB driven luciferase activities. L971 could inhibit the constitutive and stimuli‐dependent activation of STAT1, STAT3 and IκBα and could significantly down‐regulate the proinflammatory gene expression in mouse peritoneal macrophages stimulated by LPS. Gene expression profiles upon L971 treatment were determined using high‐throughput RNA sequencing, and significant differentially up‐regulated and down‐regulated genes were identified by DESeq analysis. The bioinformatic studies confirmed the anti‐inflammatory effects of L971. Finally, L971 anti‐inflammatory character was further verified in LPS‐induced sepsis shock mouse model in vivo. Taken together, these data indicated that L971 could down‐regulate both JAK/STAT and NFκB signalling activities and has the potential to treat inflammatory diseases such as sepsis shock.     

Insights into xanthine riboswitch structure and metal ion-mediated ligand recognition

Riboswitches are conserved functional domains in mRNA that mostly exist in bacteria. They regulate gene expression in response to varying concentrations of metabolites or metal ions. Recently, the NMT1 RNA motif has been identified to selectively bind xanthine and uric acid, respectively, both are involved in the metabolic pathway of purine degradation. Here, we report a crystal structure of this RNA bound to xanthine. Overall, the riboswitch exhibits a rod-like, continuously stacked fold composed of three stems and two internal junctions. The binding-pocket is determined by the highly conserved junctional sequence J1 between stem P1 and P2a, and engages a long-distance Watson–Crick base pair to junction J2. Xanthine inserts between a G–U pair from the major groove side and is sandwiched between base triples. Strikingly, a Mg²⁺ ion is inner-sphere coordinated to O6 of xanthine and a non-bridging oxygen of a backbone phosphate. Two further hydrated Mg²⁺ ions participate in extensive interactions between xanthine and the pocket. Our structure model is verified by ligand binding analysis to selected riboswitch mutants using isothermal titration calorimetry, and by fluorescence spectroscopic analysis of RNA folding using 2-aminopurine-modified variants. Together, our study highlights the principles of metal ion-mediated ligand recognition by the xanthine riboswitch.     

Trichomonas vaginalis infection impairs anion secretion in vaginal epithelium

Trichomonas vaginalis is a common protozoan parasite, which causes trichomoniasis associated with severe adverse reproductive outcomes. However, the underlying pathogenesis has not been fully understood. As the first line of defense against invading pathogens, the vaginal epithelial cells are highly responsive to environmental stimuli and contribute to the formation of the optimal luminal fluid microenvironment. The cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel widely distributed at the apical membrane of epithelial cells, plays a crucial role in mediating the secretion of Cl⁻ and HCO₃⁻. In this study, we investigated the effect of T. vaginalis on vaginal epithelial ion transport elicited by prostaglandin E₂ (PGE₂), a major prostaglandin in the semen. Luminal administration of PGE₂ triggered a remarkable and sustained increase of short-circuit current (I_SC) in rat vaginal epithelium, which was mainly due to Cl⁻ and HCO₃⁻ secretion mediated by the cAMP-activated CFTR. However, T. vaginalis infection significantly abrogated the I_SC response evoked by PGE₂, indicating impaired transepithelial anion transport via CFTR. Using a primary cell culture system of rat vaginal epithelium and a human vaginal epithelial cell line, we demonstrated that the expression of CFTR was significantly down-regulated after T. vaginalis infection. In addition, defective Cl⁻ transport function of CFTR was observed in T. vaginalis-infected cells by measuring intracellular Cl⁻ signals. Conclusively, T. vaginalis restrained exogenous PGE₂-induced anion secretion through down-regulation of CFTR in vaginal epithelium. These results provide novel insights into the intervention of reproductive complications associated with T. vaginalis infection such as infertility and disequilibrium in vaginal fluid microenvironment.