Solubility properties of a 65-kDa peptide prepared by restricted digestion of myosin with astacin-like squid metalloprotease

Substructure of the myosin rod and its correlation to filament formation is largely based on studies of proteolytic digests and expressed proteins. However, tryptic digestion of myosin always produces polymorphous peptides. Consequently, it is difficult to determine the relation between myosin substructure and filament formation. Similarly, filament formation with recombinant myosin protein is also difficult to interpret because it is never clear whether the recombinant protein folds like the native protein. We recently reported a novel metal protease isolated from squid liver, astacin-like squid metalloprotease (ALSM), which can specifically hydrolyze in vitro myosin heavy chain. In the present study, we examined the solubility properties of the 65-kDa peptide and light meromyosin (LMM) prepared by ALSM isoform II and trypsin digestion, respectively. The 65-kDa peptide is much less soluble than LMM under physiological conditions, even though the length of 65-kDa peptide is shorter than that of LMM. These results suggest that a novel substructure of myosin drives filament assembly.     

Improvement of cognitive function in Alzheimer's disease model mice by genetic and pharmacological inhibition of the EP(4) receptor

Amyloid-β peptide (Aβ), which is generated by the β- and γ-secretase-mediated proteolysis of β-amyloid precursor protein (APP), plays an important role in the pathogenesis of Alzheimer's disease (AD). We recently reported that prostaglandin E(2) (PGE(2) ) stimulates the production of Aβ through both EP(2) and EP(4) receptors and that activation of the EP(4) receptor stimulates Aβ production through endocytosis and activation of γ-secretase. We here found that transgenic mice expressing mutant APP (APP23) mice showed a greater or lesser apparent cognitive deficit when they were crossed with mice lacking EP(2) or EP(4) receptors, respectively. Mice lacking the EP(4) receptor also displayed lower levels of Aβ plaque deposition and less neuronal and synaptic loss than control mice. Oral administration of a specific EP(4) receptor antagonist, AE3-208 to APP23 mice, improved their cognitive performance, as well as decreasing brain levels of Aβ and suppressing endocytosis and activation of γ-secretase. Taken together, these results suggest that inhibition of the EP(4) receptor improves the cognitive function of APP23 mice by suppressing Aβ production and reducing neuronal and synaptic loss. We therefore propose that EP(4) receptor antagonists, such as AE3-208, could be therapeutically beneficial for the prevention and treatment of AD.     

Synthesis and biological evaluation of sulfonylhydrazone-substituted imidazo[1,2-a]pyridines as novel PI3 kinase p110alpha inhibitors

We have previously reported the imidazo[1,2-a]pyridine derivative 4 as a novel p110alpha inhibitor; however, although 4 is a potent inhibitor of p110alpha enzymatic activity and tumor cell proliferation in vitro, it is unstable in solution and ineffective in vivo. To increase stability the pyrazole of 4 was replaced with a hydrazone and a moderately potent p110alpha inhibitor 7a was obtained. Subsequent optimization of 7a afforded exceptionally potent p110alpha inhibitors, including 8c and 8h, with IC(50) values of 0.30 nM and 0.26 nM, respectively; to the best of our knowledge, these compounds are the most potent PI3K p110alpha inhibitors reported to date. Compound 8c was also stable in solution and exhibited significant anti-tumor effectiveness in vivo.     

A single-headed recombinant fragment of Dictyostelium cytoplasmic dynein can drive the robust sliding of microtubules

A cytoplasmic dynein is a microtubule-based motor protein involved in diverse cellular functions, such as organelle transport and chromosome segregation. The dynein has two ring-shaped heads that contain six repeats of the AAA domain responsible for ATP hydrolysis. It has been proposed that the ATPase-dependent swing of a stalk and a stem emerging from each of the heads generates the power stroke (Burgess, S.A. (2003) Nature 421, 715-718). To understand the molecular mechanism of the dynein power stroke, it is essential to establish an easy and reproducible method to express and purify the recombinant dynein with full motor activities. Here we report the expression and purification of the C-terminal 380-kDa fragment of the Dictyostelium cytoplasmic dynein heavy-chain fused with an affinity tag and green fluorescent protein. The purified single-headed recombinant protein drove the robust minus-end-directed sliding of microtubules at a velocity of 1.2 microm/s. This recombinant protein had a high basal ATPase activity (approximately 4s(-1)), which was further activated by >15-fold on the addition of 40 microM microtubules. These results show that the 380-kDa recombinant fragment retains all the structures required for motor functions, i.e. the ATPase activity highly stimulated by microtubules and the robust motility.     

The role of CCR7 in allergic airway inflammation induced by house dust mite exposure

House dust mite (HDM), the most common allergen, activate both the IgE-associated and innate immune responses. To clarify the process of sensitization, we investigated the role of the CCL21, CCL19, and CCR7 axis in a mouse model of HDM-induced allergic asthma. HDM inhalation without systemic immunization resulted in a HDM-specific IgE response. CCR7-knockout (CCR7KO) mice exhibited greater airway inflammation and IgE responses compared to wild-type mice. We examined FoxP3 expression in these mice to clarify the contribution of regulatory cells to the responses. FoxP3 expression was higher in the lungs but not in the lymph nodes of CCR7KO mice compared to wild-type mice. In CCR7KO mice, FoxP3-positive cells were found in lung, but we observed higher release of IL-13, IL-5, TGF-β, IL-17, and HMGB1 in bronchoalveolar lavage fluid. We demonstrate here that immuno-regulation through CCR7 expression in T cells plays a role in HDM-specific sensitization in the airway.     

Influence of Municipality-Level Mean Income on Access to Aortic Valve Surgery: A Cross-Sectional Observational Study under Japan's Universal Health-Care Coverage

Background

Universal health-care coverage has attracted the interest of policy makers as a way of achieving health equity. However, previous reports have shown that despite universal coverage, socioeconomic disparity persists in access to high-tech invasive care, such as cardiac treatment. In this study, we aimed to investigate the association between socioeconomic status and care of aortic stenosis in the context of Japan''s health-care system, which is mainly publicly funded.

Methods

We chose aortic stenosis in older people as a target because such patients are likely to be affected by socioeconomic disparity. Using a large Japanese claim-based inpatient database, we identified 12,893 isolated aortic stenosis patients aged over 65 years who were hospitalized between July 2010 and March 2012. Municipality socioeconomic status was represented by the mean household income of the patients'' residential municipality, categorized into quartiles. The likelihood of undergoing aortic valve surgery and in-hospital mortality was regressed against socioeconomic status level with adjustments for hospital volume, regional number of cardiac surgeons per 1 million population, and patients'' clinical status.

Results

We found no significant differences between the highest and lowest quartile groups in surgical indication (odds ratio, 0.84; 95% confidence interval, 0.69–1.03) or in-hospital mortality (1.00; 0.68–1.48). Hospital volume was significantly associated with lower postoperative mortality (odds ratio of the highest volume tertile to the lowest, 0.49; 0.34–0.71).

Conclusions

Under Japan''s current universal health-care coverage, municipality socioeconomic status did not appear to have a systematic relationship with either treatment decision for surgical intervention or postoperative survival following aortic valve surgery among older patients. Our results imply that universal health-care coverage with high publicly funded coverage offers equal access to high-tech cardiovascular care.     

Randomized Phase III Trial of Adjuvant Chemotherapy with S-1 after Curative Treatment in Patients with Squamous-Cell Carcinoma of the Head and Neck (ACTS-HNC)

BackgroundWe conducted a phase III study to evaluate S-1 as compared with UFT as control in patients after curative therapy for stage III, IVA, or IVB squamous-cell carcinoma of the head and neck (SCCHN).ResultsA total of 526 patients were enrolled, and 505 were eligible for analysis. The 3-year DFS rate was 60.0% in the UFT group and 64.1% in the S-1 group (HR, 0.87; 95%CI, 0.66-1.16; p = 0.34). The 3-year OS rate was 75.8% and 82.9%, respectively (HR, 0.64; 95% CI, 0.44-0.94; p = 0.022). Among grade 3 or higher adverse events, the incidences of leukopenia (5.2%), neutropenia (3.6%), thrombocytopenia (2.0%), and mucositis/stomatitis (2.4%) were significantly higher in the S-1 group.ConclusionsAlthough DFS did not differ significantly between the groups, OS was significantly better in the S-1 group than in the UFT group. S-1 is considered a treatment option after curative therapy for stage III, IVA, IVB SCCHN.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00336947","term_id":"NCT00336947"}}NCT00336947 http://clinicaltrials.gov/show/{"type":"clinical-trial","attrs":{"text":"NCT00336947","term_id":"NCT00336947"}}NCT00336947     

GM1 Ganglioside is Involved in Epigenetic Activation Loci of Neuronal Cells

Gangliosides are sialic acid-containing glycosphingolipids that are most abundant in the nerve tissues. The quantity and expression pattern of gangliosides in brain change drastically throughout development and are mainly regulated through stage-specific expression of glycosyltransferase (ganglioside synthase) genes. We previously demonstrated that acetylation of histones H3 and H4 on the N-acetylgalactosaminyltransferase I (GalNAcT, GA2/GM2/GD2/GT2-synthase) gene promoter resulted in recruitment of trans-activation factors. In addition, we reported that epigenetic activation of the GalNAcT gene was also detected as accompanied by an apparent induction of neuronal differentiation in neural stem cells responding to an exogenous supplement of ganglioside GM1. Here, we present evidence supporting the concept that nuclear GM1 is associated with gene regulation in neuronal cells. We found that nuclear GM1 binds acetylated histones on the promoters of the GalNAcT and NeuroD1 genes in differentiated neurons. Our study demonstrates for the first time that GM1 interacts with chromatin via acetylated histones at the nuclear periphery of neuronal cells.     

Rab12 regulates mTORC1 activity and autophagy through controlling the degradation of amino‐acid transporter PAT4

Autophagy is an evolutionarily conserved catabolic mechanism that targets intracellular molecules and damaged organelles to lysosomes. Autophagy is achieved by a series of membrane trafficking events, but their regulatory mechanisms are poorly understood. Here, we report small GTPase Rab12 as a new type of autophagic regulator that controls the degradation of an amino‐acid transporter. Knockdown of Rab12 results in inhibition of autophagy and in increased activity of mTORC1 (mammalian/mechanistic target of rapamycin complex 1), an upstream regulator of autophagy. We also found that Rab12 promotes constitutive degradation of PAT4 (proton‐coupled amino‐acid transporter 4), whose accumulation in Rab12‐knockdown cells modulates mTORC1 activity and autophagy. Our findings reveal a new mechanism of regulation of mTORC1 signalling and autophagy, that is, quality control of PAT4 by Rab12.