Yhh1p/Cft1p directly links poly(A) site recognition and RNA polymerase II transcription termination

RNA polymerase II (pol II) transcription termination requires co-transcriptional recognition of a functional polyadenylation signal, but the molecular mechanisms that transduce this signal to pol II remain unclear. We show that Yhh1p/Cft1p, the yeast homologue of the mammalian AAUAAA interacting protein CPSF 160, is an RNA-binding protein and provide evidence that it participates in poly(A) site recognition. Interestingly, RNA binding is mediated by a central domain composed of predicted -propeller-forming repeats, which occurs in proteins of diverse cellular functions. We also found that Yhh1p/Cft1p bound specifically to the phosphorylated C-terminal domain (CTD) of pol II in vitro and in a two-hybrid test in vivo. Furthermore, transcriptional run-on analysis demonstrated that yhh1 mutants were defective in transcription termination, suggesting that Yhh1p/Cft1p functions in the coupling of transcription and 3'-end formation. We propose that direct interactions of Yhh1p/Cft1p with both the RNA transcript and the CTD are required to communicate poly(A) site recognition to elongating pol II to initiate transcription termination.

Microphase separation in double crystalline poly (ethylene oxide)-block-poly (caprolactone)/poly (4-vinyl phenol) blends

We report microphase separation induced by competitive hydrogen bonding interactions in double crystalline diblock copolymer/homopolymer blends of poly(ethylene oxide)-block-poly(ɛ-caprolactone) (PEO-b-PCL) and poly(4-vinyl phenol) (PVPh). The diblock copolymer PEO-b-PCL consists of two immiscible crystallizable blocks wherein both PEO and PCL blocks can form hydrogen bonds with PVPh. In these A-b-B/C diblock copolymer/homopolymer blends, microphase separation takes place due to the disparity in intermolecular interactions; specifically PVPh and PEO block interact strongly whereas PVPh and PCL block interact weakly. The TEM and SAXS results show that the cubic PEO-b-PCL diblock copolymer changes into ordered hexagonal cylindrical morphology upon addition of 20 wt % PVPh followed by disordered bicontinuous phase in the blend with 40 wt % PVPh and then to homogenous phase at 60 wt% PVPh and above. Up to 40 wt % PVPh there is only weak interaction between PVPh and PCL due to the selective hydrogen bonding between PVPh and PEO. However, with higher PVPh concentration, the blends become homogeneous since a sufficient amount of PVPh is available to form hydrogen bonds with both PEO and PCL. A structural model was proposed to explain the self-assembly and morphology of these blends based on the experimental results obtained. The formation of nanostructures and changes in morphologies depend on the relative strength of hydrogen bonding interaction between each block of the block copolymer and the homopolymer (1-3).

New drug targets in depression : inflammatory, cell-mediated immune, oxidative and nitrosative stress, mitochondrial, antioxidant, and neuroprogressive pathways. And new drug candidates—Nrf2 activators and GSK-3 inhibitors

This paper reviews new drug targets in the treatment of depression and new drug candidates to treat depression. Depression is characterized by aberrations in six intertwined pathways: (1) inflammatory pathways as indicated by increased levels of proinflammatory cytokines, e.g. interleukin-1 (IL-1), IL-6, and tumour necrosis factor α. (2) Activation of cell-mediated immune pathways as indicated by an increased production of interferon γ and neopterin. (3) Increased reactive oxygen and nitrogen species and damage by oxidative and nitrosative stress (O&NS), including lipid peroxidation, damage to DNA, proteins and mitochondria. (4) Lowered levels of key antioxidants, such as coenzyme Q10, zinc, vitamin E, glutathione, and glutathione peroxidase. (5) Damage to mitochondria and mitochondrial DNA and reduced activity of respiratory chain enzymes and adenosine triphosphate production. (6) Neuroprogression, which is the progressive process of neurodegeneration, apoptosis, and reduced neurogenesis and neuronal plasticity, phenomena that are probably caused by inflammation and O&NS. Antidepressants tend to normalize the above six pathways. Targeting these pathways has the potential to yield antidepressant effects, e.g. using cytokine antagonists, minocycline, Cox-2 inhibitors, statins, acetylsalicylic acid, ketamine, ω3 poly-unsaturated fatty acids, antioxidants, and neurotrophic factors. These six pathways offer new, pathophysiologically guided drug targets suggesting that novel therapies could be developed that target these six pathways simultaneously. Both nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activators and glycogen synthase kinase-3 (GSK-3) inhibitors target the six above-mentioned pathways. GSK-3 inhibitors have antidepressant effects in animal models of depression. Nrf2 activators and GSK-3 inhibitors have the potential to be advanced to phase-2 clinical trials to examine whether they augment the efficacy of antidepressants or are useful as monotherapy.

Coupled RNA polymerase II transcription and 3′ end formation with yeast whole-cell extracts

 RNA polymerase II (RNAP II) transcription and pre-mRNA 3' end formation are linked through physical and functional interactions. We describe here a highly efficient yeast in vitro system that reproduces both transcription and 3' end formation in a single reaction. The system is based on simple whole-cell extracts that were supplemented with a hybrid Gal4-VP16 transcriptional activator and supercoiled plasmid DNA templates encoding G-less cassette reporters. We found that the coupling of transcription and processing in vitro enhanced pre-mRNA 3' end formation and reproduced requirements for poly(A) signals and polyadenylation factors. Unexpectedly, however, we show that in vitro transcripts lacked m⁷G-caps. Reconstitution experiments with CF IA factor assembled entirely from heterologous components suggested that the CTD interaction domain of the Pcf11 subunit was required for proper RNAP II termination but not 3' end formation. Moreover, we observed reduced termination activity associated with extracts prepared from cells carrying a mutation in the 5'-3' exonuclease Rat1 or following chemical inhibition of exonuclease activity. Thus, in vitro transcription coupled to pre-mRNA processing recapitulates hallmarks of poly(A)-dependent RNAP II termination. The in vitro transcription/processing system presented here should provide a useful tool to further define the role of factors involved in coupling.

Δ-6 desaturase substrate competition : dietary linoleic acid (18∶2n-6) has only trivial effects on α-linolenic acid (18∶3n-3) bioconversion in the teleost rainbow trout

It is generally accepted that, in vertebrates, omega-3 (n-3) and omega-6 (n-6) poly-unsaturated fatty acids (PUFA) compete for ?-6 desaturase enzyme in order to be bioconverted into long-chain PUFA (LC-PUFA). However, recent studies into teleost fatty acid metabolism suggest that these metabolic processes may not conform entirely to what has been previously observed in mammals and other animal models. Recent work on rainbow trout has led us to question specifically if linoleic acid (LA, 18:2n-6) and ?-linolenic acid (ALA, 18:3n-3) (?-6 desaturase substrates) are in direct competition for access to ?-6 desaturase. Two experimental diets were formulated with fixed levels of ALA, while LA levels were varied (high and low) to examine if increased availability of LA would result in decreased bioconversion of ALA to its LC-PUFA products through substrate competition. No significant difference in ALA metabolism towards n-3 LC-PUFA was exhibited between diets while significant differences were observed in LA metabolism towards n-6 LC-PUFA. These results are evidence for minor if any competition between substrates for ?-6 desaturase, suggesting that, paradoxically, the activity of ?-6 desaturase on n-3 and n-6 substrates is independent. These results call for a paradigm shift in the way we approach teleost fatty acid metabolism. The findings are also important with regard to diet formulation in the aquaculture industry as they indicate that there should be no concern for possible substrate competition between 18:3n-3 and 18:2n-6, when aiming at increased n-3 LC-PUFA bioconversion in vivo.

Preparation and dielectric properties of sulfonated poly(aryl ether ketone)/acidified graphite nanosheet composites

Percolative dielectric composites of sulfonated poly(aryl ether ketone) (SPAEK) and acidified graphite nanosheets (AGSs) were fabricated by a solution method. The dielectric constant of the as-prepared composite with 4.01 vol % AGSs was found to be 330 at 1000 Hz; this was a significant increase compared to that of pure SPAEK. Through the calculation, a low percolation threshold of the AGS/SPAEK composite was confirmed at 3.18 vol % (0.0318 volume fraction) AGSs; this was attributed to the large surface area and high conductivity of the AGSs. Additionally, our percolative dielectric composites also exhibited good mechanical performances and good thermostability, with a tensile strength of 71.7 MPa, a tensile modulus of 1.91 GPa, a breaking elongation of 16.4%, and a mass loss temperature at 5% of 336°C.

Understanding water and ion transport behaviour and permeability through poly(amide) thin film composite membrane

Molecular dynamics (MD) together with the adaptive biasing force (ABF) and metadynamics free energy calculation methods was used to investigate the permeation properties of salt water through poly(amide) thin film composite reverse osmosis membranes. The thin films were generated by annealing an amorphous cell of poly(amide) chains through an MD method. The MD results showed they have typical structural properties of the active layer of thin film composite membranes and comparable water diffusivity (2.13×10^-5cm²/s for the film with a density of 1.06g/cm³) and permeability (9.27×10^-15cm³cm/cm²sPa) to experimental data. The simulations of water permeation through the films under different transmembrane pressures revealed the behaviours of water molecules in the thin films and the dynamic regimes of water permeation, including Brownian diffusion, flush and jump diffusion regimes. The intermolecular interactions of water and ions with poly(amide) chains showed a strong dependence on the local structure of films. The attraction between water and ploy(amide) molecules can be up to 8.5kcal/mol in dense polymer regions and 5kcal/mol in the pores of about 3nm. The ABF and metadynamics simulations produced the profiles of free energy potential of water and ions along the depth of the thin films, which provided important information for quantitatively determining the barrier energy required for water permeation and rejection of ions. The thin film with a density of 1.06g/cm³ and a thickness of 6nm offers a rejection to Na⁺ but a slight absorption of Cl^- (0.25kcal/mol) at 0.3-0.4nm distance to its surface. Water molecules must overcome 63kcal/mol energy to move to the centre of the film. The dependences of the barrier energy and the water-polymer interaction energy on the local free volume size in the thin film were analysed. The simulations of water permeation under high transmembrane pressures showed a nonlinear response of the concentration and distribution of water molecules in the film to the imposed pressure. Compaction of the film segments close to the porous substrate and water congestion in dense regions significantly influenced the water permeation when the membrane was operated under pressures of more than 3.0MPa.

NDRG2 promotes myoblast proliferation and caspase 3/7 activities during differentiation, and attenuates hydrogen peroxide - but not palmitate-induced toxicity

The function of the stress-responsive N-myc downstream-regulated gene 2 (NDRG2) in the control of myoblast growth, and the amino acids contributing to its function, are not well characterized. Here, we investigated the effect of increased NDRG2 levels on the proliferation, differentiation and apoptosis in skeletal muscle cells under basal and stress conditions. NDRG2 overexpression increased C2C12 myoblast proliferation and the expression of positive cell cycle regulators, cdk2, cyclin B and cyclin D, and phosphorylation of Rb, while the serine/threonine-deficient NDRG2, 3A-NDRG2, had less effect. The onset of differentiation was enhanced by NDRG2 as determined through the myogenic regulatory factor expression profiles and myocyte fusion index. However, the overall level of differentiation in myotubes was not different. While NDRG2 up-regulated caspase 3/7 activities during differentiation, no increase in apoptosis was measured by TUNEL assay or through cleavage of caspase 3 and PARP proteins. During H2O2 treatment to induce oxidative stress, NDRG2 helped protect against the loss of proliferation and ER stress as measured by GRP78 expression with 3A-NDRG2 displaying less protection. NDRG2 also attenuated apoptosis by reducing cleavage of PARP and caspase 3 and expression of pro-apoptotic Bax while enhancing the pro-survival Bcl-2 and Bcl-xL levels. In contrast, Mcl-1 was not altered, and NDRG2 did not protect against palmitate-induced lipotoxicity. Our findings show that NDRG2 overexpression increases myoblast proliferation and caspase 3/7 activities without increasing overall differentiation. Furthermore, NDRG2 attenuates H2O2-induced oxidative stress and specific serine and threonine amino acid residues appear to contribute to its function in muscle cells.