Noncoding RNAs have drawn significant attention in biology recently. Whereas the current research is highly inclined to microRNAs, research on other noncoding RNAs has lagged behind. Here, we investigated a novel noncoding RNA that has been known as precursor microRNA miR-886 (pre-miR-886). Pre-miR-886 has been proposed also as a vault RNA, a component of the vault complex implicated in cancer drug resistance. We identified pre-miR-886 as a 102-nucleotide-long, abundant cytoplasmic RNA that is neither a genuine pre-microRNA nor a vault RNA. Pre-miR-886 is physically associated with PKR (Protein Kinase RNA-activated), an interferon-inducible and double-stranded RNA dependent kinase. The suppression of pre-miR-886 activates PKR and its downstream pathways, eIF2a phosphorylation and the NF-kB pathway, leading to impaired cell proliferation. We also found that pre-miR-886 is suppressed in a wide-range of cancer cell lines and in clinical specimens. This study is the first intense characterization of pre-miR-886 as well as the initial report on its function as a PKR regulator, which suggests a critical role in tumorigenesis.

HDM2 regulates p53 by binding to its transactivation domain and promoting its ubiquitin‐dependent degradation. Crystallographic analysis of the HDM2/p53 complex revealed that three hydrophobic residues (F19, W23, L26) along one face of the p53 helical peptide are essential for binding (see picture). Terphenyl‐based antagonists mimic the α‐helical region of p53 and disrupt HDM2/p53 complexation.

The rational design of low-molecular weight ligands that disrupt protein-protein interactions is still a challenging goal in medicinal chemistry. Our approach to this problem involves the design of molecular scaffolds that mimic the surface functionality projected along one face of an alpha-helix. Using a terphenyl scaffold, which in a staggered conformation closely reproduces the projection of functionality on the surface of an alpha-helix, we designed mimics of the pro-apoptotic alpha-helical Bak-peptide as inhibitors of the Bak/Bcl-xL interaction. This led to the development of a potent Bcl-xL antagonist (KD = 114 nM), whose binding affinity for Bcl-xL was assessed by a fluorescence polarization assay. To determine the binding site of the developed inhibitor we used docking studies and an HSQC-NMR experiment with 15N-labeled Bcl-xL protein. These studies suggest that the inhibitor is binding in the same hydrophobic cleft as the Bak- and Bad-peptides.

We describe a general method for the mimicry of one face of an alpha-helix based on a terphenyl scaffold that spatially projects functionality in a manner similar to that of two turns of an alpha-helix. The synthetic scaffold reduces the flexibility and molecular weight of the mimicked protein secondary structure. We have applied this design to the development of antagonists of the alpha-helix binding protein Bcl-x(L). Using a sequential synthetic strategy, we have prepared a library of terphenyl derivatives to mimic the helical region of the Bak BH3 domain that binds Bcl-x(L). Fluorescence polarization assays were carried out to evaluate the ability of terphenyl derivatives to displace the Bcl-x(L)-bound Bak peptide. Terphenyl 14 exhibited good in vitro affinity with a K(i) value of 0.114 muM. These terphenyl derivatives were more selective at disrupting the Bcl-x(L)/Bak over the HDM2/p53 interaction, which involves binding of the N-terminal alpha-helix of p53 to HDM2. Structural studies using NMR spectroscopy and computer-aided docking simulations suggested that the helix binding area on the surface of Bcl-x(L) is the target for the synthetic ligands. Treatment of human embryonic kidney 293 (HEK293) cells with terphenyl derivatives resulted in the disruption of the binding of Bcl-x(L) to Bax in intact cells.

Accumulating evidence suggests cortical circuits may contribute to control of human locomotion. Here, noninvasive electroencephalography (EEG) recorded from able-bodied volunteers during a novel treadmill walking paradigm was used to assess neural correlates of walking. A systematic processing method, including a recently developed subspace reconstruction algorithm, reduced movement-related EEG artifact prior to independent component analysis and dipole source localization. We quantified cortical activity while participants tracked slow and fast target speeds across two treadmill conditions: an active mode that adjusted belt speed based on user movements and a passive mode reflecting a typical treadmill. Our results reveal frequency specific, multi-focal task related changes in cortical oscillations elicited by active walking. Low γ band power, localized to the prefrontal and posterior parietal cortices, was significantly increased during double support and early swing phases, critical points in the gait cycle since the active controller adjusted speed based on pelvis position and swing foot velocity. These phasic γ band synchronizations provide evidence that prefrontal and posterior parietal networks, previously implicated in visuo-spatial and somotosensory integration, are engaged to enhance lower limb control during gait. Sustained μ and β band desynchronization within sensorimotor cortex, a neural correlate for movement, was observed during walking thereby validating our methods for isolating cortical activity. Our results also demonstrate the utility of EEG recorded during locomotion for probing the multi-regional cortical networks which underpin its execution. For example, the cortical network engagement elicited by the active treadmill suggests that it may enhance neuroplasticity for more effective motor training.

The design of low-molecular-weight ligands (< 750 Da) that recognize protein surfaces and subsequently disrupt protein± protein interactions is an area of intense research. [1] Current strategies for identifying small-molecule protein-surface antagonists primarily involve the use of combinatorial methods.

reported that the adenine group in 5'-AMP forms a stronger stacking interaction to bipyridinecopper(1r) complexes than the uracil group in 5'-UMP. ['91 Such a stacking interaction between adenine and bipyridine may account for the high selectivity of 2 for 5a over 5b-d (Table I).Dinuclear complexes 1 and 2 are highly active in promoting hydrolytic cleavage of nucleoside 2',3'-cyclic monophosphates. Furthermore, 1 is highly regioselective and cleaves 5 c and 5 d predominantly to nucleoside 2'-monophosphates.On the other hand, 2 shows remarkable base selectivity and hydrolyzes 5a much faster than 5 b-d. Presumably, 1 achieves high regioselectivity by regioselectively binding to the bases in the substrates, while 2 attains base selectivity by a preferentialx--x stacking interaction between the bipyridine unit in 2 and the bases in the substrates. More experiments are in progress to probe the exact nature of interactions that give rise to the high reactivities and base and regioselectivities shown by 1 and 2. Experimental SectionThe preparation of 1 -4 has been described previously [12]. Cleavage of 5 by 1-4 was followed by HPLC (Ranin). The following procedure is typical: 1 (1.1 mM, 4.5 mL) in a buffer was mixed with 5a (2.0 mM, 0.5 mL) in the same buffer. Aliquots (ZOO pL) of the reaction mixture were quenched with ethylenediaminetetraacetate (EDTA, 20 mM, 200 pL). After any solid that formed was filtered, the quenched solution (20 pL) was injected onto a C-18 reversed-phase column and eluted for 10 min with 1-1070 CH,CN in H 2 0 containing 0.1 % CF,COOH (flow rate l.OmLmin-'). The eluent was monitored at Amax of the nucleobase (260 nm for adenine and uracil, 252 nm for guanine, and 268 nm for cytosine) by a Rainin UV-C detector. The first-order rate constants k were obtained as slopes of plots of In(AdA,) versus t, where AD and A , are integrations of the areas of the HPLC peaks for 5 at f = 0 and I, respectively. Selectivities for the formation of 3monophosphate (6) with respect to 2-monophosphate (7) were calculated from the ratios of integrations of the areas of the HPLC peaks for 6 and 7 The identities of 6 and 7 were established with authetic samples (Sigma). The two isomers have nearly identical extinction coefficients at A,,, of the nucleobase. Therefore, the ratio of the HPLC peaks for 6 and 7 is equal to the ratio of the amounts of 6 and 7 produced (i. e., the regioselectivity). In all cases the ratios remained constant throughout the entire course of hydrolysis. For slow reactions (k < 5 x lo-%'), appreciable amounts of 6 and 7 were further hydrolyzed to give inorganic phosphate and the correponding nucleosides before 5 was completely hydrolyzed. However, the ratio of 6 to 7 in the reaction mixture remained unchanged. Duplicate runs showed measurement errors of less than & 15 % for k and less than *3% for the regioselectivity.

A family of synthetic receptors for protein surface recognition has been prepared. The receptors are based on a design in which four peptide loops are arrayed around a central calilx[4]arene core. By varying the sequence of the loop regions a range of differently functionalized receptor surfaces approximately 450 Å2 in area can be prepared. From this family we have identified potent inhibitors of chymotrypsin that function by binding to the surface of the protein. The most potent of these (1) shows slow binding kinetics in an analogous manner to several of the natural protein proteinase inhibitors. Detailed kinetic analysis showed 1 to be a competitive inhibitor with K i and K i* values of 0.81 and 0.11 μM, respectively.