Inhibition of PrPSc formation in scrapie infected N2a cells by 5,7,8-trimethyl-3,4-dihydro-2H-1,4-benzoxazine derivatives

Prion diseases are fatal, neurodegenerative diseases characterized by the structural conversion of the normal, cellular prion protein, PrP^C into an abnormally structured, aggregated and partially protease-resistant isoform, termed PrP^Sc. Although substantial research has been directed toward development of therapeutics targeting prions, there is still no curative treatment for the disease. Benzoxazines are bicyclic heterocyclic compounds possessing several pharmaceutically important properties, including neuroprotection and reactive oxygen species scavenging. In an effort to identify novel inhibitors of prion formation, several 5,7,8-trimethyl-1,4-benzoxazine derivatives were evaluated in vitro for their effectiveness on the expression levels of normal PrP^C and its conversion to the abnormal isoforms of PrP^Sc in a scrapie-infected cell culture model. The most potent compound was 2-(4-methoxyphenyl)-5,7,8-trimethyl-3,4-dihydro-2H-1,4-benzoxazine, with a diminishing effect on the formation of PrP^Sc, thus establishing a class of compounds with a promising therapeutic use against prion diseases.     

Disease phenotype in sheep after infection with cloned murine scrapie strains

Prion diseases exhibit different disease phenotypes in their natural hosts and when transmitted to rodents, and this variability is regarded as indicative of prion strain diversity. Phenotypic characterization of scrapie strains in sheep can be attempted by histological, immunohistochemical and biochemical approaches, but it is widely considered that strain confirmation and characterization requires rodent bioassay. Examples of scrapie strains obtained from original sheep isolates by serial passage in mice include ME7, 79A, 22A and 87V. In order to address aspects of prion strain stability across the species barrier, we transmitted the above murine strains to sheep of different breeds and susceptible Prnp genotypes. The experiment included 40 sheep dosed by the oral route alone and 36 sheep challenged by combined subcutaneous and intracerebral routes. Overall, the combined route produced higher attack rates (~100%) than the oral route (~50%) and 2–4 times shorter incubation periods. Uniquely, 87V given orally was unable to infect any sheep. Overall, scrapie strains adapted and cloned in mice produce distinct but variable disease phenotypes in sheep depending on breed or Prnp genotype. Further re-isolation experiments in mice are in progress in order to determine whether the original cloned murine disease phenotype will reemerge.     

Modulation of distinct isoforms of L-type calcium channels by Gq-coupled receptors in Xenopus oocytes

L-type voltage dependent Ca²⁺ channels (L-VDCCs; Ca_v1.2) are crucial in cardiovascular physiology. In heart and smooth muscle, hormones and transmitters operating via G_q enhance L-VDCC currents via essential protein kinase C (PKC) involvement. Heterologous reconstitution studies in Xenopus oocytes suggested that PKC and G_q-coupled receptors increased L-VDCC currents only in cardiac long N-terminus (NT) isoforms of α_1C, whereas known smooth muscle short-NT isoforms were inhibited by PKC and G_q activators. We report a novel regulation of the long-NT α_1C isoform by Gβγ. Gβγ inhibited whereas a Gβγ scavenger protein augmented the G_q- but not phorbol ester-mediated enhancement of channel activity, suggesting that Gβγ acts upstream from PKC. In vitro binding experiments reveal binding of both Gβγ and PKC to α_1C-NT. However, PKC modulation was not altered by mutations of multiple potential phosphorylation sites in the NT, and was attenuated by a mutation of C-terminally located serine S1928. The insertion of exon 9a in intracellular loop 1 rendered the short-NT α_1C sensitive to PKC stimulation and to Gβγ scavenging. Our results suggest a complex antagonistic interplay between G_q-activated PKC and Gβγ in regulation of L-VDCC, in which multiple cytosolic segments of α_1C are involved.     

SRSF1-dependent alternative splicing attenuates BIN1 expression in non–small cell lung cancer

Decreased bridging integrator 1 (BIN1) expression has great significance in promoting the progression of malignant tumors. Reduced messenger RNA expression is partly due to aberrant alternative splicing (AS). However, the AS status of BIN1 and its correlation with BIN1 inactivation in non–small cell lung cancer (NSCLC) remains poorly defined. Here we reported that BIN1 inactivation was not related to DNA methylation in NSCLC. Importantly, BIN1 with exon 12A inclusion (BIN1+12A isoform), the most frequent aberrant splicing variant in tumors was also observed in NSCLC, and might be accounted for BIN1 inactivation. Furthermore, we showed that the aberrant splicing of BIN1 was under the control of serine and arginine-rich factor 1 (SRSF1) in NSCLC. In addition, colony formation assay showed that BIN1+12A isoform could abolish the tumor-inhibiting ability of BIN1 in NSCLC cells. Meanwhile, transwell, wound healing and apoptosis experiments demonstrated that the occurrence of BIN1+12A could abrogate the invasion suppressing activity and proapoptotic property of BIN1 in NSCLC. Significantly, we also found that BIN1+12A isoform neutralized the tumor-suppressing functions of BIN1 via affecting its subcellular localization. Altogether, these data revealed an aberrant splicing phenomenon which abated the expression and tumor-inhibiting activity of BIN1 in NSCLC, and the related mechanisms were associated with SRSF1.     

Contribution of genetic and epigenetic mechanisms to Wnt pathway activity in prevalent skeletal disorders

We reported previously that the expression of Wnt-related genes is lower in osteoporotic hip fractures than in osteoarthritis. We aimed to confirm those results by analyzing β-catenin levels and explored potential genetic and epigenetic mechanisms involved.     

Dominant-negative effects in prion diseases: insights from molecular dynamics simulations on mouse prion protein chimeras

Mutations in the prion protein (PrP) can cause spontaneous prion diseases in humans (Hu) and animals. In transgenic mice, mutations can determine the susceptibility to the infection of different prion strains. Some of these mutations also show a dominant-negative effect, thus halting the replication process by which wild type mouse (Mo) PrP is converted into Mo scrapie. Using all-atom molecular dynamics (MD) simulations, here we studied the structure of HuPrP, MoPrP, 10?Hu/MoPrP chimeras, and 1 Mo/sheepPrP chimera in explicit solvent. Overall, ～2?μs of MD were collected. Our findings suggest that the interactions between α1 helix and N-terminal of α3 helix are critical in prion propagation, whereas the β2–α2 loop conformation plays a role in the dominant-negative effect.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:4.     

Sequence,Packing and Nanometer Scale Structure in STM Images of Nucleic Acids Under Water

Abstract

Scanning tunneling microscope (STM) images of random-sequence nucleic acid polymers under water show internal structure which depends strongly on the packing density of the polymer. Images of dense aggregates have a semicrystalline order with the individual polymers adopting simple periodic structures. Loose aggregates (or isolated molecules) show structural variability with considerable local bending and curving on a nanometer scale. It is not clear to what extent this structure is induced by the operation of the microscope. In order to investigate the possibility that the structure is sequence directed, we have imaged various DNA and RNA polymers at low packing densities. We present results here for random sequence DNA, poly(dAT) · poly(dAT), poly(dA) · poly(dT), poly(dCG) · poly(dCG) and for random sequence RNA and poly(U). In contrast to loose aggregates of the random sequence material, the homopolymers show few sharp bends. Furthermore, the homopolymers appear to yield characteristic backbone patterns, usually at resolutions in excess of that obtained with random sequence polymers. The random sequence polymers show much more evidence of image distortion due to tip-molecule interactions, suggesting that they are, on average, mechanically less stable in the STM tunnel-gap than the homopolymers. Thus, while some of the structure observed in STM images is a consequence of tip-molecule interactions, it is related to sequence-directed properties of the polymer.