A class II phosphoinositide 3-kinase plays an indispensable role in hepatitis C virus replication

Phosphoinositides function as fundamental signaling molecules and play roles in diverse cellular processes. Certain types of viruses may employ host cell phosphoinositide signaling systems to facilitate their replication cycles. Here we demonstrate that the β isoform of class II PI3K (PI3K-C2β) plays an indispensable role in hepatitis C virus (HCV) propagation in human hepatocellular carcinoma cells. Knockdown of PI3K-C2β abrogated HCV propagation in the cell. Using an HCV replicon system, we found that knockdown of PI3K-C2β substantially repressed the full-genome replication, while showing relatively small reductions in sub-genome replication, in which structural proteins including core protein were deleted. We also found that HCV core protein showed the binding activity towards D4-phosphorylated phosphoinositides and overlapped localization with phosphatidylinositol 3,4-bisphosphate in the cell. These results suggest that the phosphoinositide generated by PI3K-C2β plays an indispensable role in the HCV replication cycle through the binding to HCV core protein.     

Modification of a novel angiogenic peptide, AG30, for the development of novel therapeutic agents

We previously identified a novel angiogenic peptide, AG30, with antibacterial effects that could serve as a foundation molecule for the design of wound-healing drugs. Toward clinical application, in this study we have developed a modified version of the AG30 peptide characterized by improved antibacterial and angiogenic action, thus establishing a lead compound for a feasibility study. Because AG30 has an α-helix structure with a number of hydrophobic and cationic amino acids, we designed a modified AG30 peptide by replacing several of the amino acids. The replacement of cationic amino acids (yielding a new molecule, AG30/5C), but not hydrophobic amino acids, increased both the angiogenic and the antimicrobial properties of the peptide. AG30/5C was also effective against methicillin-resistant Staphylococcus aureus (MRSA) and antibiotic-resistant Pseudomonas aeruginosa. In a diabetic mouse wound-healing model, the topical application of AG30/5C accelerated wound healing with increased angiogenesis and attenuated MRSA infection. To facilitate the eventual clinical investigation/application of these compounds, we developed a large-scale procedure for the synthesis of AG30/5C that employed the conventional solution method and met Good Manufacturing Practice guidelines. In the evaluation of stability of this peptide in saline solution, RP-HPLC analysis revealed that AG30/5C was fairly stable under 5°C for 12 months. Therefore, we propose the use of AG30/5C as a wound-healing drug with antibacterial and angiogenic actions.     

Activity dynamics and propagation of synchronous spiking in locally connected random networks

Random network models have been a popular tool for investigating cortical network dynamics. On the scale of roughly a cubic millimeter of cortex, containing about 100,000 neurons, cortical anatomy suggests a more realistic architecture. In this locally connected random network, the connection probability decreases in a Gaussian fashion with the distance between neurons. Here we present three main results from a simulation study of the activity dynamics in such networks. First, for a broad range of parameters these dynamics exhibit a stationary state of asynchronous network activity with irregular single-neuron spiking. This state can be used as a realistic model of ongoing network activity. Parametric dependence of this state and the nature of the network dynamics in other regimes are described. Second, a synchronous excitatory stimulus to a fraction of the neurons results in a strong activity response that easily dominates the network dynamics. And third, due to that activity response an embedding of a divergent-convergent feed-forward subnetwork (as in synfire chains) does not naturally lead to a stable propagation of synchronous activity in the subnetwork; this is in contrast to our earlier findings in isolated subnetworks of that type. Possible mechanisms for stabilizing the interplay of volleys of synchronous spikes and network dynamics by specific learning rules or generalizations of the subnetworks are discussed.     

Evaluation of characteristic aroma compounds of Citrus natsudaidai Hayata (Natsudaidai) cold-pressed peel oil

The characteristic aroma compounds of Citrus natsudaidai Hayata essential oil were evaluated by a combination of instrumental and sensory methods. Sixty compounds were identified and quantified, accounting for 94.08% of the total peel oil constituents. Limonene was the most abundant compound (80.68%), followed by gamma-terpinene (5.30%), myrcene (2.25%) and alpha-pinene (1.30%). Nineteen compounds which could not be identified in the original oil were identified in the oxygenated fraction. Myrcene, linalool, alpha-pinene, beta-pinene, limonene, nonanal, gamma-terpinene, germacrene D, and perillyl alcohol were the active aroma components (FD-factor > 3(6)), whereas beta-copaene, cis-sabinene hydrate and 1-octanol were suggested as characteristic aroma compounds, having a Natsudaidai-like aroma in the GC effluent. Three other compounds, heptyl acetate, (E)-limonene oxide and 2,3-butanediol, which each showed a high RFA value (>35) were considered to be important in the reconstruction of the original Natsudaidai oil from pure odor chemicals. The results indicate that 1-octanol was the aroma impact compound of C. natsudaidai Hayata peel oil.     

Tricin inhibits proliferation of human hepatic stellate cells in vitro by blocking tyrosine phosphorylation of PDGF receptor and its signaling pathways

4',5,7-Trihydroxy-3',5'-dimethoxyflavone (Tricin), a naturally occurring flavone, has anti-inflammatory potential and exhibits diverse biological activities including antigrowth activity in several human cancer cell lines and cancer chemopreventive effects in the gastrointestinal tract of mice. The present study aimed to investigate the biological actions of tricin on hepatic stellate cells (HSCs) in vitro, exploring its potential as a treatment of liver fibrosis, since HSC proliferation is closely related to the progression of hepatic fibrogenesis in chronic liver diseases leading to irreversible liver cirrhosis and hepatocellular carcinoma. Tricin inhibited platelet-derived growth factor (PDGF)-BB-induced cell proliferation by blocking cell cycle progression and cell migration in the human HSC line LI90 and culture-activated HSCs. It also reduced the phosphorylation of PDGF receptor β and the downstream signaling molecules ERK1/2 and Akt, which might be due to its tyrosine kinase inhibitor properties rather than inhibition of the direct binding between PDGF-BB and its receptor. Our findings suggest that tricin might be beneficial in HSC-targeting therapeutic or chemopreventive applications for hepatic fibrosis.     

Single-Molecule Analysis of the Rotation of F1-ATPase under High Hydrostatic Pressure

F₁-ATPase is the water-soluble part of ATP synthase and is an ATP-driven rotary molecular motor that rotates the rotary shaft against the surrounding stator ring, hydrolyzing ATP. Although the mechanochemical coupling mechanism of F₁-ATPase has been well studied, the molecular details of individual reaction steps remain unclear. In this study, we conducted a single-molecule rotation assay of F₁ from thermophilic bacteria under various pressures from 0.1 to 140 MPa. Even at 140 MPa, F₁ actively rotated with regular 120° steps in a counterclockwise direction, showing high conformational stability and retention of native properties. Rotational torque was also not affected. However, high hydrostatic pressure induced a distinct intervening pause at the ATP-binding angles during continuous rotation. The pause was observed under both ATP-limiting and ATP-saturating conditions, suggesting that F₁ has two pressure-sensitive reactions, one of which is evidently ATP binding. The rotation assay using a mutant F₁(βE190D) suggested that the other pressure-sensitive reaction occurs at the same angle at which ATP binding occurs. The activation volumes were determined from the pressure dependence of the rate constants to be +100 Å³ and +88 Å³ for ATP binding and the other pressure-sensitive reaction, respectively. These results are discussed in relation to recent single-molecule studies of F₁ and pressure-induced protein unfolding.     

Intra-strain biological and epidemiological characterization of plum pox virus

Plum pox virus (PPV) is one of the most important plant viruses causing serious economic losses. Thus far, strain typing based on the definition of 10 monophyletic strains with partially differentiable biological properties has been the sole approach used for epidemiological characterization of PPV. However, elucidating the genetic determinants underlying intra-strain biological variation among populations or isolates remains a relevant but unexamined aspect of the epidemiology of the virus. In this study, based on complete nucleotide sequence information of 210 Japanese and 47 non-Japanese isolates of the PPV-Dideron (D) strain, we identified five positively selected sites in the PPV-D genome. Among them, molecular studies showed that amino acid substitutions at position 2,635 in viral replicase correlate with viral titre and competitiveness at the systemic level, suggesting that amino acid position 2,635 is involved in aphid transmission efficiency and symptom severity. Estimation of ancestral genome sequences indicated that substitutions at amino acid position 2,635 were reversible and peculiar to one of two genetically distinct PPV-D populations in Japan. The reversible amino acid evolution probably contributes to the dissemination of the virus population. This study provides the first genomic insight into the evolutionary epidemiology of PPV based on intra-strain biological variation ascribed to positive selection.     

IL1RAPL1 Associated with Mental Retardation and Autism Regulates the Formation and Stabilization of Glutamatergic Synapses of Cortical Neurons through RhoA Signaling Pathway

Interleukin-1 receptor accessory protein-like 1 (IL1RAPL1) is associated with X-linked mental retardation and autism spectrum disorder. We found that IL1RAPL1 regulates synapse formation of cortical neurons. To investigate how IL1RAPL1 controls synapse formation, we here screened IL1RAPL1-interacting proteins by affinity chromatography and mass spectroscopy. IL1RAPL1 interacted with Mcf2-like (Mcf2l), a Rho guanine nucleotide exchange factor, through the cytoplasmic Toll/IL-1 receptor domain. Knockdown of endogenous Mcf2l and treatment with an inhibitor of Rho-associated protein kinase (ROCK), the downstream kinase of RhoA, suppressed IL1RAPL1-induced excitatory synapse formation of cortical neurons. Furthermore, we found that the expression of IL1RAPL1 affected the turnover of AMPA receptor subunits. Insertion of GluA1-containing AMPA receptors to the cell surface was decreased, whereas that of AMPA receptors composed of GluA2/3 was enhanced. Mcf2l knockdown and ROCK inhibitor treatment diminished the IL1RAPL1-induced changes of AMPA receptor subunit insertions. Our results suggest that Mcf2l-RhoA-ROCK signaling pathway mediates IL1RAPL1-dependent formation and stabilization of glutamatergic synapses of cortical neurons.     

Flavonoids isolated from the leaves of Melicope triphylla and their extracellular-superoxide dismutase-inducing activity

Two novel flavonoids, named meliflavones A (1) and B (2), were isolated from the leaves of Melicope triphylla (Lam.) Merr., along with thirteen known compounds (3–15). Four of the polymethoxyflavonoids bearing a prenyloxy (3-methylbut-2-enyloxy) function (1, 3–5) induced the expression of extracellular-superoxide dismutase (EC-SOD) in a human leukemic U937 cell-based assay.     

Lead optimization of 5-amino-6-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)-4-hydroxyhexanamides to reduce a cardiac safety issue: Discovery of DS-8108b,an orally active renin inhibitor

With the aim to address an undesired cardiac issue observed with our related compound in the recently disclosed novel series of renin inhibitors, further chemical modifications of this series were performed. Extensive structure–activity relationships studies as well as in vivo cardiac studies using the electrophysiology rat model led to the discovery of clinical candidate trans-adamantan-1-ol analogue 56 (DS-8108b) as a potent renin inhibitor with reduced potential cardiac risk. Oral administration of single doses of 3 and 10 mg/kg of 56 in cynomolgus monkeys pre-treated with furosemide led to significant reduction of mean arterial blood pressure for more than 12 h.