AMP-dependent Kinase Inhibits Oxidative Stress-induced Caveolin-1 Phosphorylation and Endocytosis by Suppressing the Dissociation between c-Abl and Prdx1 Proteins in Endothelial Cells

Caveolin-1 is the primary structural component of endothelial caveolae that is essential for transcellular trafficking of albumin and is also a critical scaffolding protein that regulates the activity of signaling molecules in caveolae. Phosphorylation of caveolin-1 plays a fundamental role in the mechanism of oxidant-induced vascular hyper permeability. However, the regulatory mechanism of caveolin-1 phosphorylation remains unclear. Here we identify a previously unexpected role for AMPK in inhibition of caveolin-1 phosphorylation under oxidative stress. A pharmacological activator of AMPK, 5-amino-4-imidazole carboxamide riboside (AICAR), inhibited oxidative stress-induced phosphorylation of both caveolin-1 and c-Abl, which is the major kinase of caveolin-1, and endocytosis of albumin in human umbilical vein endothelial cell. These effects were abolished by treatment with two specific inhibitors of AICAR, dipyridamole, and 5-iodotubericidin. Consistently, knockdown of the catalytic AMPKα subunit by siRNA abolished the inhibitory effect of AICAR on oxidant-induced phosphorylation of both caveolin-1 and c-Abl. Pretreatment with specific c-Abl inhibitor, imatinib mesylate, and knock down of c-Abl significantly decreased the caveolin-1 phosphorylation after H₂O₂ exposure and abolished the inhibitory effect of AICAR on the caveolin-1 phosphorylation. Interestingly, knockdown of Prdx-1, an antioxidant enzyme associated with c-Abl, increased phosphorylation of both caveolin-1 and c-Abl and abolished the inhibitory effect of AICAR on the caveolin-1 phosphorylation. Furthermore, co-immunoprecipitation experiment showed that AICAR suppressed the oxidant-induced dissociation between c-Abl and Prdx1. Overall, our results suggest that activation of AMPK inhibits oxidative stress-induced caveolin-1 phosphorylation and endocytosis, and this effect is mediated in part by stabilizing the interaction between c-Abl and Prdx-1.     

A Novel Bivalent Vaccine Based on a PB2-Knockout Influenza Virus Protects Mice from Pandemic H1N1 and Highly Pathogenic H5N1 Virus Challenges

Vaccination is an effective means to protect against influenza virus. Although inactivated and live-attenuated vaccines are currently available, each vaccine has disadvantages (e.g., immunogenicity and safety issues). To overcome these problems, we previously developed a replication-incompetent PB2-knockout (PB2-KO) influenza virus that replicates only in PB2 protein-expressing cells. Here, we generated two PB2-KO viruses whose PB2-coding regions were replaced with the HA genes of either A/California/04/2009 (H1N1pdm09) or A/Vietnam/1203/2004 (H5N1). The resultant viruses comparably, or in some cases more efficiently, induced virus-specific antibodies in the serum, nasal wash, and bronchoalveolar lavage fluid of mice relative to a conventional formalin-inactivated vaccine. Furthermore, mice immunized with these PB2-KO viruses were protected from lethal challenges with not only the backbone virus strain but also strains from which their foreign HAs originated, indicating that PB2-KO viruses with antigenically different HAs could serve as bivalent influenza vaccines.     

Synthesis and biological evaluation of pyrrolidine derivatives as novel and potent sodium channel blockers for the treatment of ischemic stroke

A novel series of pyrrolidine derivatives as Na⁺ channel blockers was synthesized and evaluated for their inhibitory effects on neuronal Na⁺ channels. Structure–activity relationship (SAR) studies of a pyrrolidine analogue 2 led to the discovery of 5e as a potent Na⁺ channel blocker with a low inhibitory action against human ether-a-go-go-related gene (hERG) channels. Compound 5e showed remarkably neuroprotective activity in a rat transient middle cerebral artery occlusion (MCAO) model, suggesting that 5e would act as a neuroprotectant for ischemic stroke.     

Isolation of Protein Inhibitors of Papain,Trypsin, and α-Amylase in the Grain of Foxtail Millet

The amino acid sequence of Indian peafowl egg-white lysozyme has been identified. The reduced and carboxymethylated lysozyme was digested with trypsin followed by purification of the resulting peptides by reverse-phase HPLC. The tryptic peptides obtained were sequenced using the DABITC/PITC double coupling manual sequencing method. The alignment of the tryptic peptides were deduced by comparison with corresponding peptides of hen egg-white lysozyme. This protein proved to consist of 129 amino acid residues, and a relative molecular mass of 14423 Da was calculated. Amino acid sequence comparison of peafowl lysozyme and other phasianoid bird lysozymes revealed a maximum homology ratio of 98% with turkey lysozyme.     

Aminoimidazole Carboxamide Ribonucleotide (AICAR) Inhibits the Growth of Retinoblastoma In Vivo by Decreasing Angiogenesis and Inducing Apoptosis

5-Aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR), an analog of AMP is widely used as an activator of AMP-kinase (AMPK), a protein that regulates the responses of the cell to energy change. Recently, we showed that AICAR-induced AMPK activation inhibits the growth of retinoblastoma cells in vitro by decreasing cyclins and by inducing apoptosis and S-phase arrest. In this study, we investigated the effects of AMPK activator AICAR on the growth of retinoblastoma in vivo. Intraperitoneal injection of AICAR resulted in 48% growth inhibition of Y79 retinoblastoma cell tumors in mice. Tumors isolated from mice treated with AICAR had decreased expression of Ki67 and increased apoptotic cells (TUNEL positive) compared with the control. In addition, AICAR treatment suppressed significantly tumor vessel density and macrophage infiltration. We also showed that AICAR administration resulted in AMPK activation and mTOR pathway inhibition. Paradoxically observed down-regulation of p21, which indicates that p21 may have a novel function of an oncogene in retinoblastoma tumor. Our results indicate that AICAR treatment inhibited the growth of retinoblastoma tumor in vivo via AMPK/mTORC1 pathway and by apoptogenic, anti-proliferative, anti-angiogenesis mechanism. AICAR is a promising novel non-chemotherapeutic drug that may be effective as an adjuvant in treating Retinoblastoma.     

Analysis of Factors That Determine Weight Gain during Smoking Cessation Therapy

Cigarette smokers are generally known to gain weight after quitting smoking, and such weight gain is thought to contribute to the worsening of glucose tolerance. While smoking cessation therapy such as nicotine replacement is useful to minimize post-cessation weight gain, substantial gain occurs even during the therapy. The purpose of the present study was to identify factors associated with weight gain during smoking cessation therapy. We evaluated 186 patients(132 males and 54 females)who visited our outpatient clinic for smoking cessation, and successfully achieved smoking abstinence. We performed gender-adjusted regression analysis for the rate of BMI increase from the beginning of cessation to 3 months after initiation. Furthermore, we performed multivariate analysis to investigate factors that determine the BMI increase after smoking cessation. The mean BMI significantly (p<0.0001) increased from 23.5±3.6 kg/m² at the initial consultation to 23.9±3.8 kg/m² at 3 months after the start of therapy. There was no significant difference in the extent of BMI increase between nicotine patch and varenicline therapy groups. Factors significantly correlated with the %BMI increase at 3 months after the start of therapy were triglyceride (p = 0.0006, βa = 0.260), high-density lipoprotein cholesterol (p = 0.0386, βa = −0.168), daily cigarette consumption (p = 0.0385, βa = 0.154), and the Fagerström Test for Nicotine Dependence (FTND) score (p = 0.0060, βa = 0.203). Stepwise multivariate analysis demonstrated that triglyceride and the FTND score were the factors determining the post-cessation BMI increase and that the FTND score was the strongest one. The present study demonstrated that smokers with a high FTND score are more likely to gain weight during smoking cessation therapy. Thus, smokers with a high nicotine dependency may require intervention against weight gain in the cessation clinic.     

Adaptation changes in dynamic postural control and contingent negative variation during repeated transient forward translation in the elderly

Background

Adaptation changes in postural control and contingent negative variation (CNV) for the elderly were investigated during repeated forward floor translation.

Methods

Fifteen healthy elderly persons, living in the suburban area of Kanazawa City, Japan, underwent backward postural disturbance by a forward-floor translation (S2) 2 s after an auditory warning signal (S1). A set with 20 trials was repeated until a negative peak of late CNV was recognized in the 600-ms period before S2, and the last set was defined as the final set. Electroencephalograms, center of foot pressure in the anteroposterior direction (CoPap), and electromyograms of postural muscles were analyzed.

Results

CoPap displacement generated by the floor translation was significantly decreased until the twelfth trial in the first set, and mean CoPap displacement was smaller in the second and final sets than in the first set. The mean displacement was significantly smaller in the final set than the previous set. A late CNV with a negative peak was not recognized in the first and second sets. However, most subjects (13/15) showed a negative peak by the fourth set, when the late CNV started to increase negatively from about 1,000 ms after S1 and peaked at about 300 ms before S2. At about 160 ms before the CNV peak, the CoPap forward shift started. The increase in timing of the gastrocnemius activity related to the CoPap shift was significantly correlated with the CNV peak timing (r = 0.64). After S2, peak amplitudes of the anterior postural muscles were significantly decreased in the final set compared to the first set.

Conclusions

It was demonstrated that even for the elderly, with so many repetitions of postural disturbance, a late CNV with a negative peak was recognized, leading to accurate postural preparation. This suggests the improvement of frontal lobe function (e.g., anticipatory attention and motor preparation) in the elderly.     

Endothelial Protein C Receptor Function in Murine and Human Breast Cancer Development

Several markers identify cancer stem cell-like populations, but little is known about the functional roles of stem cell surface receptors in tumor progression. Here, we show that the endothelial protein C receptor (EPCR), a stem cell marker in hematopoietic, neuronal and epithelial cells, is crucial for breast cancer growth in the orthotopic microenvironment of the mammary gland. Mice with a hypomorphic allele of EPCR show reduced tumor growth in the PyMT-model of spontaneous breast cancer development and deletion of EPCR in established PyMT tumor cells significantly attenuates transplanted tumor take and growth. We find expansion of EPCR⁺ cancer stem cell-like populations in aggressive, mammary fat pad-enhanced human triple negative breast cancer cells. In this model, EPCR-expressing cells have markedly increased mammosphere- and tumor-cell initiating activity compared to another stable progenitor-like subpopulation present at comparable frequency. We show that receptor blocking antibodies to EPCR specifically attenuate in vivo tumor growth initiated by either EPCR⁺ cells or the heterogenous mixture of EPCR⁺ and EPCR^- cells. Furthermore, we have identified tumor associated macrophages as a major source for recognized ligands of EPCR, suggesting a novel mechanism by which cancer stem cell-like populations are regulated by innate immune cells in the tumor microenvironment.