Identification of annexin A1 as a novel substrate for E6AP‐mediated ubiquitylation

E6‐associated protein (E6AP) is a cellular ubiquitin protein ligase that mediates ubiquitylation and degradation of p53 in conjunction with the high‐risk human papillomavirus E6 proteins. However, the physiological functions of E6AP are poorly understood. To identify a novel biological function of E6AP, we screened for binding partners of E6AP using GST pull‐down and mass spectrometry. Here we identified annexin A1, a member of the annexin superfamily, as an E6AP‐binding protein. Ectopic expression of E6AP enhanced the degradation of annexin A1 in vivo. RNAi‐mediated downregulation of endogenous E6AP increased the levels of endogenous annexin A1 protein. E6AP interacted with annexin A1 and induced its ubiquitylation in a Ca²⁺‐dependent manner. GST pull‐down assay revealed that the annexin repeat domain III of annexin A1 is important for the E6AP binding. Taken together, our data suggest that annexin A1 is a novel substrate for E6AP‐mediated ubiquitylation. Our findings raise the possibility that E6AP may play a role in controlling the diverse functions of annexin A1 through the ubiquitin‐proteasome pathway. J. Cell. Biochem. 106: 1123–1135, 2009. © 2009 Wiley‐Liss, Inc.     

RNA Polymerase Activity and Specific RNA Structure Are Required for Efficient HCV Replication in Cultured Cells

We have previously reported that the NS3 helicase (N3H) and NS5B-to-3′X (N5BX) regions are important for the efficient replication of hepatitis C virus (HCV) strain JFH-1 and viral production in HuH-7 cells. In the current study, we investigated the relationships between HCV genome replication, virus production, and the structure of N5BX. We found that the Q377R, A450S, S455N, R517K, and Y561F mutations in the NS5B region resulted in up-regulation of J6CF NS5B polymerase activity in vitro. However, the activation effects of these mutations on viral RNA replication and virus production with JFH-1 N3H appeared to differ. In the presence of the N3H region and 3′ untranslated region (UTR) of JFH-1, A450S, R517K, and Y561F together were sufficient to confer HCV genome replication activity and virus production ability to J6CF in cultured cells. Y561F was also involved in the kissing-loop interaction between SL3.2 in the NS5B region and SL2 in the 3′X region. We next analyzed the 3′ structure of HCV genome RNA. The shorter polyU/UC tracts of JFH-1 resulted in more efficient RNA replication than J6CF. Furthermore, 9458G in the JFH-1 variable region (VR) was responsible for RNA replication activity because of its RNA structures. In conclusion, N3H, high polymerase activity, enhanced kissing-loop interactions, and optimal viral RNA structure in the 3′UTR were required for J6CF replication in cultured cells.     

Decalcification for electron microscopy with L-ascorbic acid

L-Ascorbic acid decalcification was used for electron microscopy of mammalian tooth germs and bone after fixation in a glutaraldehyde-paraformaldehyde mixture. The recommended decalcifying solution is 2% with respect to L-ascorbic acid and 0.9% with respect to sodium chloride. The method has the advantage that decalcification is complete within a quarter of the time required with EDTA. The fine structure of ameloblasts and hard tissue is preserved as well as with EDTA.     

The extract of Japanese soybean, Kurosengoku activates the production of IL-12 and IFN-γ by DC or NK1.1(+) cells in a TLR4- and TLR2-dependent manner

During the search for immuno-improving foods, we found that a variety of the Japanese soybean, Glycine max cv. Kurosengoku (Kurosengoku), which activated Type-1 immunity in a Toll-like receptor (TLR)4- and TLR2-dependent manner. Namely, the extract of Kurosengoku first caused production of IL-12 from DC and sequentially induced IFN-γ production by NK1.1⁺ NK cells and NKT cells. The IFN-γ production was significantly blocked by neutralizing mAb against IL-12 or TLR4- and TLR2-deficient condition, indicating that TLR4- and TLR2-dependent activation of DC to produce IL-12 was essential for the production of IFN-γ from spleen cells by Kurosengoku. Moreover, the extract of Kurosengoku also enhanced production of IFN-γ from human PBMC by co-stimulation with anti-CD3 mAb in a TLR2- and TLR4-dependent manner. Thus, our findings strongly suggest that Kurosengoku might a novel immuno-improving food, which would be a useful tool for preventing the tip of immune balance in developed countries.     

Complications associated with endobronchial ultrasound-guided transbronchial needle aspiration: a nationwide survey by the Japan Society for Respiratory Endoscopy

Background

With the recent widespread use of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA), there have been occasional reports on complications associated with its use. Previous reviews on EBUS-TBNA have been limited to studies by skilled operators, thus the results may not always be applicable to recent clinical practice. To assess the safety of EBUS-TBNA for the staging and diagnosis of lung cancer in Japan, a nationwide survey on its current usage status and complications associated with its use was conducted by the Japan Society for Respiratory Endoscopy (JSRE).

Methods

A questionnaire about EBUS-TBNA performed between January 2011 and June 2012 was mailed to 520 JSRE-accredited facilities.

Results

Responses were obtained from 455 facilities (87.5%). During the study period, EBUS-TBNA was performed in 7,345 cases in 210 facilities (46.2%) using a convex probe ultrasound bronchoscope, for 6,836 mediastinal and hilar lesions and 275 lung parenchymal lesions. Ninety complications occurred in 32 facilities. The complication rate was 1.23% (95% confidence interval, 0.97%-1.48%), with hemorrhage being the most frequent complication (50 cases, 0.68%). Infectious complications developed in 14 cases (0.19%) (Mediastinitis, 7; pneumonia, 4; pericarditis, 1; cyst infection, 1; and sepsis, 1). Pneumothorax developed in 2 cases (0.03%), one of which required tube drainage. Regarding the outcome of the cases with complications, prolonged hospitalization was observed in 14 cases, life-threatening conditions in 4, and death in 1 (severe cerebral infarction) (mortality rate, 0.01%). Breakage of the ultrasound bronchoscope occurred in 98 cases (1.33%) in 67 facilities (31.9%), and that of the puncture needle in 15 cases (0.20%) in 8 facilities (3.8%).

Conclusions

Although the complication rate associated with EBUS-TBNA was found to be low, severe complications, including infectious complications, were observed, and the incidence of device breakage was high. Since the use of EBUS-TBNA is rapidly expanding in Japan, an educational program for its safe performance should be immediately established.     

A method for detection of 4-hydroxy-2-nonenal adducts in proteins

We developed a procedure to measure 4-hydroxy-2-nonenal (HNE)-amino acid adducts using the fluorescent probe 2-aminopyridine (2-AP). The method is based on the fact that HNE forms Michael addition-type amino acid adducts possessing an aldehyde functionality, which upon reaction with 2-AP in the presence of NaBH₃CN can be converted to their pyridylaminated derivatives. The HNE-amino acid adducts, namely Michael addition-type HNE-cysteine, HNE-histidine, and HNE-lysine adducts, after pyridylamination were resistant to conventional acid-hydrolysis conditions for protein (6 N HCl/110 °C/24 h) and could be detected by HPLC with a fluorescence detector. The reductive amination-based fluorescent labeling of HNE adducts is a simple and accurate technique that may be widely used to reveal increased levels of covalently modified proteins with HNE and its related aldehydes during aging and disease.     

PML tumor suppressor protein is required for HCV production

PML tumor suppressor protein, which forms discrete nuclear structures termed PML-nuclear bodies, has been associated with several cellular functions, including cell proliferation, apoptosis and antiviral defense. Recently, it was reported that the HCV core protein colocalizes with PML in PML-NBs and abrogates the PML function through interaction with PML. However, role(s) of PML in HCV life cycle is unknown. To test whether or not PML affects HCV life cycle, we examined the level of secreted HCV core and the infectivity of HCV in the culture supernatants as well as the level of HCV RNA in HuH-7-derived RSc cells, in which HCV-JFH1 can infect and efficiently replicate, stably expressing short hairpin RNA targeted to PML. In this context, the level of secreted HCV core and the infectivity in the supernatants from PML knockdown cells was remarkably reduced, whereas the level of HCV RNA in the PML knockdown cells was not significantly affected in spite of very effective knockdown of PML. In fact, we showed that PML is unrelated to HCV RNA replication using the subgenomic HCV-JFH1 replicon RNA, JRN/3-5B. Furthermore, the infectivity of HCV-like particle in the culture supernatants was significantly reduced in PML knockdown JRN/3-5B cells expressing core to NS2 coding region of HCV-JFH1 genome using the trans-packaging system. Finally, we also demonstrated that INI1 and DDX5, the PML-related proteins, are involved in HCV production. Taken together, these findings suggest that PML is required for HCV production.     

Optical effects on the surrounding structure during quantitative analysis using indocyanine green videoangiography: A phantom vessel study

Various reports have been published regarding quantitative evaluations of intraoperative fluorescent intensity studies using indocyanine green (ICG) with videoangiography (VAG). The effects of scattering and point‐spread functions (PSF) on quantitative ICG‐VAG evaluations have not been investigated. Clinically, when ICG is administered through the peripheral vein, it reaches the tissue intra‐arterially. To achieve more reliable intraoperative quantitative intensity evaluations, we examined the impact of high‐intensity structures on close areas. The study was conducted using a phantom model and surgical fluorescent microscope. A region of interest (ROI) was created for the vessel model and another ROI was created within 3 cm of that. With an ROI of 6.8 mm in the vessel phantom model, 10% intensity was confirmed, even though there was no fluorescent structure. Intensity decreased gradually as the ROI moved further from the vessel model. Our study results suggest that the presence of a high‐intensity structure and the size of the ROI may affect quantitative intensity evaluations using ICG‐VAG. Results of linear regression analysis indicate that the relationship of intensity (Y) and distance (X) is as follows: Y(real/A) = 29 Exp(?0.062X) + 164.3 Exp(?1.81X). The optical effect should be considered when performing an intraoperative intensity study with a surgical microscope.      

Deficiency of Tenascin-C Alleviates Neuronal Apoptosis and Neuroinflammation After Experimental Subarachnoid Hemorrhage in Mice

Tenascin-C (TNC), a matricellular protein, is upregulated in brain parenchyma after experimental subarachnoid hemorrhage (SAH). Recent studies emphasize that early brain injury (EBI) should be overcome to improve post-SAH outcomes. The aim of this study was to investigate effects of TNC knockout (TNKO) on neuronal apoptosis and neuroinflammation, both of which are important constituents of EBI after SAH. C57BL/6 wild-type (WT) mice or TNKO mice underwent sham or filament perforation SAH modeling. Twenty-five WT mice and 25 TNKO mice were randomly divided into sham+WT (n?=?10), sham+TNKO (n?=?8), SAH+WT (n?=?15), and SAH+TNKO (n?=?17) groups. Beam balance test, neurological score, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, immunostaining of Toll-like receptor 4 (TLR4), and Western blotting were performed to evaluate neurobehavioral impairments, neuronal apoptosis, and neuroinflammation at 24 h post-SAH. Deficiency of TNC significantly alleviated post-SAH neurobehavioral impairments and neuronal apoptosis. The protective effects of TNKO on neurons were associated with the inhibition of a caspase-dependent apoptotic pathway, which was at least partly mediated by TLR4/nuclear factor-κB/interleukin-1β and interleukin-6 signaling cascades. This study first provided the direct evidence that TNC causes post-SAH neuronal apoptosis and neuroinflammation, potentially leading to the development of a new molecular targeted therapy against EBI.     

Homocysteine, another risk factor for Alzheimer disease, impairs apolipoprotein E3 function

Apolipoprotein E (apoE) ε4 and hyperhomocysteinemia are risk factors for Alzheimer disease (AD). The dimerization of apoE3 by disulfide bonds between cysteine residues enhances apoE3 function to generate HDL. Because homocysteine (Hcy) harbors a thiol group, we examined whether Hcy interferes with the dimerization of apoE3 and thereby impairs apoE3 function. We found that Hcy inhibits the dimerization of apoE3 and reduces apoE3-mediated HDL generation to a level similar to that by apoE4, whereas Hcy does not affect apoE4 function. Western blot analysis of cerebrospinal fluid showed that the ratio of apoE3 dimers was significantly lower in the samples from the patients with hyperhomocysteinemia than in those that from control subjects. Hyperhomocysteinemia induced by subcutaneous injection of Hcy to apoE3 knock-in mice decreased the level of the apoE3 dimer in the brain homogenate. Because apoE-HDL plays a role in amyloid β-protein clearance, these results suggest that two different risk factors, apoE4 and hyperhomocysteinemia, may share a common mechanism that accelerates the pathogenesis of AD in terms of reduced HDL generation.