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1.  Crmp4 deletion promotes recovery from spinal cord injury by neuroprotection and limited scar formation 
Scientific Reports  2015;5:8269.
Axonal outgrowth inhibitors and scar formation are two major obstacles to central nervous system (CNS) repair. No target molecule that regulates both axonal growth and scarring has been identified. Here we identified collapsin response mediator protein 4 (CRMP4), a common mediator of inhibitory signals after neural injury, as a crucial factor that contributes to both axonal growth inhibition and scarring after spinal cord injury (SCI). We found increases in the inhibitory and toxic forms of CRMP4 in injured spinal cord. Notably, CRMP4 expression was evident in inflammatory cells as well as in neurons after spinal cord transection. Crmp4−/− mice displayed neuroprotection against SCI and reductions in inflammatory response and scar formation. This permissive environment for axonal growth due to CRMP4 deletion restored locomotor activity at an unusually early phase of healing. These results suggest that deletion of CRMP4 is a unique therapeutic strategy that overcomes two obstacles to CNS repair after SCI.
PMCID: PMC4317684  PMID: 25652774
2.  Residual Structure of Streptococcus mutans Biofilm following Complete Disinfection Favors Secondary Bacterial Adhesion and Biofilm Re-Development 
PLoS ONE  2015;10(1):e0116647.
Chemical disinfection of oral biofilms often leaves biofilm structures intact. This study aimed to examine whether the residual structure promotes secondary bacterial adhesion. Streptococcus mutans biofilms generated on resin-composite disks in a rotating disc reactor were disinfected completely with 70% isopropyl alcohol, and were again cultured in the same reactor after resupplying with the same bacterial solution. Specimens were subjected to fluorescence confocal laser scanning microscopy, viable cell counts and PCR-Invader assay in order to observe and quantify secondarily adhered cells. Fluorescence microscopic analysis, particularly after longitudinal cryosectioning, demonstrated stratified patterns of viable cells on the disinfected biofilm structure. Viable cell counts of test specimens were significantly higher than those of controls, and increased according to the amount of residual structure and culture period. Linear regression analysis exhibited a high correlation between viable and total cell counts. It was concluded that disinfected biofilm structures favored secondary bacterial adhesion.
PMCID: PMC4312048  PMID: 25635770
3.  Draft Genome Sequence of the Betaproteobacterial Endosymbiont Associated with the Fungus Mortierella elongata FMR23-6 
Genome Announcements  2014;2(6):e01272-14.
The fungus Mortierella elongata FMR23-6 harbors an endobacterium inside its mycelium. Attempts to isolate the endobacterium from the fungus were not yet successful, but a highly purified bacterial fraction was prepared. Here, we report the draft genome sequence of the endobacterium.
PMCID: PMC4263831  PMID: 25502669
4.  Prognostic implication of morphology, cyclinE2 and proliferation in EBV-associated T/NK lymphoproliferative disease in non-immunocompromised hosts 
EBV-associated T/NK-cell lymphoproliferative diseases (TNKLPD) is a rare spectrum of disease that occurs more commonly in Asia, and Central and South America. It commonly affects children and young adults and is an aggressive disease that is poorly understood with no known biologic markers that can predict prognosis. The systemic form of TNKLPD includes chronic active EBV infection of T/NK type, aggressive NK cell leukemia and systemic EBV + T-cell lymphoproliferative disease of childhood.
In this study, we analyse the clinicopathologic and genetic features of 22 cases of systemic TNKLPD in non-immunocompromised patients, including chronic active EBV infection of T/NK cell type and systemic EBV + T-cell lymphoproliferative disease of childhood. We also performed gene expression profiling in a subset of cases to identify markers that may be of prognostic relevance and validated our results using immunohistochemistry.
The median age is 14.9 years and two of our 22 cases occurring in patients older than 30 years. Fifteen of 17 cases (88%) with adequate data were of T-cell origin. Eleven of 22 cases revealed polymorphic cellular infiltrate (P-group) while the rest showed monomorphic lymphoid infiltrate (M-group). We found a significant difference in survival between P-group vs M-group patients with median survival not yet reached in P-group, and 1 month in M-group (p = 0.0001), suggesting a role for morphology in predicting patient outcome. We also performed gene expression profiling in a subset of patients and compared the genes differentially expressed between P-group and M-group cases to identify markers of prognostic value. We identified cyclin E2 gene and protein to be differentially expressed between patients with good outcome (P-group, median expression 8%) and poor outcome (M-group, median expression 42%) (p = 0.0005). In addition, the upregulation of cyclin E2 protein in M-group cases correlated with a higher Ki67 proliferation rate (Pearson correlation r = 0.73, p = 0.0006) detected by immunohistochemistry. High cyclin E2 expression was also significantly associated with shorter survival (p = 0.0002).
Our data suggests the potential role of monomorphic morphology, high cyclin E2 and Ki67 expression as adverse prognostic factors for TNKLPD.
Electronic supplementary material
The online version of this article (doi:10.1186/s13023-014-0165-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4263108  PMID: 25475054
EBV-associated T/NK lymphoproliferative disease; Molecular signature; Morphology; Cyclin E2; Proliferation; Prognosis
5.  Expression, purification, crystallization and preliminary X-ray analysis of NAD(P)H-dependent carbonyl reductase specifically expressed in thyroidectomized chicken fatty liver 
An NAD(P)H-dependent carbonyl reductase specifically expressed in thyroidectomized chicken fatty liver was successfully isolated and crystallized.
An NAD(P)H-dependent carbonyl reductase specifically expressed in thyroid­ectomized chicken fatty liver was crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol 300 as the precipitant. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 104.26, b = 81.32, c = 77.27 Å, β = 119.43°, and diffracted to 1.86 Å resolution on beamline NE3A at the Photon Factory. The overall R merge was 5.4% and the data completeness was 99.4%.
PMCID: PMC3509991  PMID: 23192050
thyroidectomized chicken fatty liver; NAD(P)H-dependent carbonyl reductases; short-chain dehydrogenase/reductase family
6.  Cdk5/p35 functions as a crucial regulator of spatial learning and memory 
Molecular Brain  2014;7(1):82.
Cyclin-dependent kinase 5 (Cdk5), which is activated by binding to p35 or p39, is involved in synaptic plasticity and affects learning and memory formation. In Cdk5 knockout (KO) mice and p35 KO mice, brain development is severely impaired because neuronal migration is impaired and lamination is disrupted. To avoid these developmental confounders, we generated inducible CreER-p35 conditional (cKO) mice to study the role of Cdk5/p35 in higher brain function.
CreER-p35 cKO mice exhibited spatial learning and memory impairments and reduced anxiety-like behavior. These phenotypes resulted from a decrease in the dendritic spine density of CA1 pyramidal neurons and defective long-term depression induction in the hippocampus.
Taken together, our findings reveal that Cdk5/p35 regulates spatial learning and memory, implicating Cdk5/p35 as a therapeutic target in neurological disorders.
Electronic supplementary material
The online version of this article (doi:10.1186/s13041-014-0082-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4239319  PMID: 25404232
Spatial learning; Memory; Kinase; Synaptic plasticity; Hippocampus
7.  Isolation, sequencing, and heterologous expression of the Paecilomyces variotii gene encoding S-hydroxymethylglutathione dehydrogenase (fldA) 
The filamentous fungus Paecilomyces variotii NBRC 109023 (teleomorph: Byssochlamys spectabilis NBRC 109023) degrades formaldehyde at concentrations as high as 2.4 % (w/v). In many prokaryotes and in all known eukaryotes, formaldehyde degradation is catalyzed by S-hydroxymethylglutathione (S-HMGSH) dehydrogenase. We report here the isolation and characterization of the gene encoding S-HMGSH dehydrogenase activity in P. variotii. The 1.6-kb fldA gene contained 5 introns and 6 exons, and the corresponding cDNA was 1143 bp, encoding a 40-kDa protein composed of 380 amino acids. FldA was predicted to have 74.3, 73.7, 68.5, and 67.4 % amino acid identity to the S-HMGSH dehydrogenases of Hansenula polymorpha, Candida boidinii, Saccharomyces cerevisiae, and Kluyveromyces lactis, respectively. The predicted protein also showed high amino acid similarity (84∼86 %) to the products of putative fldA genes from other filamentous fungi, including Aspergillus sp. and Penicillium sp. Notably, the P. variotii fldA gene was able to functionally complement a Saccharomyces cerevisiae strain (BY4741 ∆sfa1) lacking the gene for S-HMGSH dehydrogenase. The heterologous expression construct rendered BY4741 ∆sfa1 tolerant to exogenous formaldehyde. Although BY4741 (parental wild-type strain) was unable to degrade even low concentrations of formaldehyde, BY4741 ∆sfa1 harboring Paecilomyces fldA was able to degrade 4 mM formaldehyde within 30 h. The findings from this study confirm the essential role of S-HMGSH dehydrogenase in detoxifying formaldehyde.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-014-6203-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4322224  PMID: 25398285
Paecilomyces variotii; Formaldehyde degradation; S-hydroxymethylglutathione dehydrogenase; fldA; Heterologous expression
8.  Expansion of Natural Killer Cells in Peripheral Blood in a Japanese Elderly with Human T-Cell Lymphotropic Virus Type 1-Related Skin Lesions 
Natural killer (NK) cells were proposed to play an important role in the pathogenesis of human T-cell lymphotropic virus type 1- (HTLV-1-) associated neurologic disease. Our patient was a 77-year-old Japanese man, who had been treated for infective dermatitis associated with HTLV-1 for nearly 10 years. When referred to us, he had facial eczema/edema as well as extensive dermatitis at the neck/upper chest and nuchal area/upper back regions. Dermal lesions had CD3+CD4+ cells, but no NK cells. Flow cytometry of his peripheral blood showed a phenotype of CD2+ (97%), CD3+ (17%), CD4+ (12%), CD7+ (94%), CD8+ (6%), CD11c+ (70%), CD16+ (82%), CD19+ (0%), CD20+ (0%), CD56+ (67%), HLA-DR+ (68%), and NKp46+ (36%). Absolute numbers of CD56+NK cells in the peripheral blood were in a range of 986/μL–1,270/μL. The expanded NK cells in the peripheral blood are considered to be reactive, to maintain the confinement of the HTLV-1-positive CD4+ cells in the skin, and to prevent the progression of the disease.
PMCID: PMC4241692  PMID: 25431695
9.  An outbreak of blaOXA-51-like- and blaOXA-66-positive Acinetobacter baumannii ST208 in the emergency intensive care unit 
Journal of Medical Microbiology  2014;63(Pt 11):1517-1523.
A series of clinical isolates of drug-resistant (DR) Acinetobacter baumannii with diverse drug susceptibility was detected from eight patients in the emergency intensive care unit of Tokai University Hospital. The initial isolate was obtained in March 2010 (A. baumannii Tokai strain 1); subsequently, seven isolates were obtained from patients (A. baumannii Tokai strains 2–8) and one isolate was obtained from an air-fluidized bed used by five of the patients during the 3 months from August to November 2011. The isolates were classified into three types of antimicrobial drug resistance patterns (RRR, SRR and SSR) according to their susceptibility (S) or resistance (R) to imipenem, amikacin and ciprofloxacin, respectively. Genotyping of these isolates by multilocus sequence typing revealed one sequence type, ST208, whilst that by a DiversiLab analysis revealed two subtypes. All the isolates were positive for blaOXA-51-like and blaOXA-66, as assessed by PCR and DNA sequencing. A. baumannii Tokai strains 1–8 and 10 (RRR, SRR and SSR) had quinolone resistance-associated mutations in gyrA/parC, as revealed by DNA sequencing. The ISAba1 upstream of blaOXA-51-like and aminoglycoside resistance-associated gene, armA, were detected in A. baumannii Tokai strains 1–7 and 10 (RRR and SRR) as assessed by PCR. Among the genes encoding resistance–nodulation–division family pumps (adeB, adeG and adeJ) and outer-membrane porins (oprD and carO), overexpression of adeB and adeJ and suppression of oprD and carO were seen in isolates of A. baumannii Tokai strain 2 (RRR), as assessed by real-time PCR. Thus, the molecular characterization of a series of isolates of DR A. baumannii revealed the outbreak of ST208 and diverse antimicrobial drug susceptibilities, which almost correlated with differential gene alterations responsible for each type of drug resistance.
PMCID: PMC4209737  PMID: 25142965
10.  Regulation of Nuclear NF-κB Oscillation by a Diffusion Coefficient and Its Biological Implications 
PLoS ONE  2014;9(10):e109895.
The transcription factor NF-κB shuttles between the cytoplasm and the nucleus, and nuclear NF-κB is known to oscillate with a cycle of 1.5-2.5 h following the application of external stimuli. Oscillation pattern of NF-κB is implicated in regulation of the gene expression profile. In a previous report, we found that the oscillation pattern of nuclear NF-κB in a computational 3D spherical cell was regulated by spatial parameters such as nuclear to cytoplasmic volume ratio, nuclear transport, locus of protein synthesis, and diffusion coefficient. Here we report analyses and a biological implication for the regulation of oscillation pattern by diffusion coefficient. Our analyses show that the “reset” of nuclear NF-κB, defined as the return of nuclear NF-κB to the initial level or lower, was crucial for the oscillation; this was confirmed by the flux analysis. In addition, we found that the distant cytoplasmic location from the nucleus acted as a “reservoir” for storing newly synthesized IκBα. When the diffusion coefficient of proteins was large (≥10−11 m2/s), a larger amount of IκBα was stored in the “reservoir” with a large flux by diffusion. Subsequently, stored IκBα diffused back to the nucleus, where nuclear NF-κB was “reset” to the initial state. This initiated the next oscillation cycle. When the diffusion coefficient was small (≤10−13 m2/s), oscillation of nuclear NF-κB was not observed because a smaller amount of IκBα was stored in the “reservoir” and there was incomplete “reset” of nuclear NF-κB. If the diffusion coefficient for IκBα was increased to 10−11 m2/s keeping other proteins at 10−13 m2/s, the oscillation was rescued confirming the “reset” and “reservoir” hypothesis. Finally, we showed altered effective value of diffusion coefficient by diffusion obstacles. Thus, organelle crowding seen in stressed cells possibly changes the oscillation pattern by controlling the effective diffusion coefficient.
PMCID: PMC4193834  PMID: 25302804
11.  Association between serum γ-glutamyltranspeptidase and atherosclerosis: a population-based cross-sectional study 
BMJ Open  2014;4(10):e005413.
The purpose of this study was to determine the relationship between serum γ-glutamyltranspeptidase (GGT) and brachial-ankle pulse wave velocity (baPWV) as an indicator for atherosclerosis in Japanese men and women after adjusting for fatty liver.
A cross-sectional study.
A health check-up centre in Japan.
912 Japanese men and women aged 24–84 years recruited from people who received a medical health check-up programme with a standardised questionnaire and an automatic waveform analyser to measure baPWV.
Main outcome measures
We measured serum GGT concentrations and baPWV. Fatty liver was diagnosed by standardised criteria using abdominal ultrasonography. The postmenopausal state was defined as beginning 1 year after the cessation of menses.
In women, log2 GGT was positively associated with baPWV (β=0.11, 95% CI 0.02 to 0.19, p<0.05), independent of age, body mass index, systolic blood pressure, fasting plasma glucose, triglycerides, estimated glomerular filtration rate, fatty liver, menopausal state and parameters of lifestyles. However, in men, the positive association of log2 GGT with baPWV was not significant (β=−0.04, 95% CI −0.10 to 0.03, p=0.28) in multivariable linear regression analyses.
The serum GGT level was associated with baPWV, independently of covariates including fatty liver or menopausal state just in women, but not in men.
PMCID: PMC4187458  PMID: 25280803
12.  Development of New Multilocus Variable Number of Tandem Repeat Analysis (MLVA) for Listeria innocua and Its Application in a Food Processing Plant 
PLoS ONE  2014;9(9):e105803.
Listeria innocua is an important hygiene indicator bacterium in food industries because it behaves similar to Listeria monocytogenes, which is pathogenic to humans. PFGE is often used to characterize bacterial strains and to track contamination source. However, because PFGE is an expensive, complicated, time-consuming protocol, and poses difficulty in data sharing, development of a new typing method is necessary. MLVA is a technique that identifies bacterial strains on the basis of the number of tandem repeats present in the genome varies depending on the strains. MLVA has gained attention due to its high reproducibility and ease of data sharing. In this study, we developed a MLVA protocol to assess L. innocua and evaluated it by tracking the contamination source of L. innocua in an actual food manufacturing factory by typing the bacterial strains isolated from the factory. Three VNTR regions of the L. innocua genome were chosen for use in the MLVA. The number of repeat units in each VNTR region was calculated based on the results of PCR product analysis using capillary electrophoresis (CE). The calculated number of repetitions was compared with the results of the gene sequence analysis to demonstrate the accuracy of the CE repeat number analysis. The developed technique was evaluated using 60 L. innocua strains isolated from a food factory. These 60 strains were classified into 11 patterns using MLVA. Many of the strains were classified into ST-6, revealing that this MLVA strain type can contaminate each manufacturing process in the factory. The MLVA protocol developed in this study for L. innocua allowed rapid and easy analysis through the use of CE. This technique was found to be very useful in hygiene control in factories because it allowed us to track contamination sources and provided information regarding whether the bacteria were present in the factories.
PMCID: PMC4157758  PMID: 25198191
13.  Phylogeny and Functions of Bacterial Communities Associated with Field-Grown Rice Shoots 
Microbes and Environments  2014;29(3):329-332.
Metagenomic analysis was applied to bacterial communities associated with the shoots of two field-grown rice cultivars, Nipponbare and Kasalath. In both cultivars, shoot microbiomes were dominated by Alphaproteobacteria (51–52%), Actinobacteria (11–15%), Gammaproteobacteria (9–10%), and Betaproteobacteria (4–10%). Compared with other rice microbiomes (root, rhizosphere, and phyllosphere) in public databases, the shoot microbiomes harbored abundant genes for C1 compound metabolism and 1-aminocyclopropane-1-carboxylate catabolism, but fewer genes for indole-3-acetic acid production and nitrogen fixation. Salicylate hydroxylase was detected in all microbiomes, except the rhizosphere. These genomic features facilitate understanding of plant–microbe interactions and biogeochemical metabolism in rice shoots.
PMCID: PMC4159046  PMID: 25130883
metagenomics; methanol; plant hormone; rice; shoot
14.  Structure of a UDP-glucose dehydrogenase from the hyperthermophilic archaeon Pyrobaculum islandicum  
Acta Crystallographica Section F  2012;68(Pt 9):1003-1007.
The crystal structure of an extremely thermostable UDP-glucose dehydrogenase from a hyperthermophile was determined.
The crystal structure of an extremely thermostable UDP-glucose dehydrogenase (UDP-GDH) from the hyperthermophilic archaeon Pyrobaculum islandicum was determined at a resolution of 2.0 Å. The overall fold was comprised of an N-­terminal NAD+ dinucleotide binding domain and a C-terminal UDP-sugar binding domain connected by a long α-helix, and the main-chain coordinates of the enzyme were similar to those of previously studied UDP-GDHs, including the enzymes from Burkholderia cepacia, Streptococcus pyogenes and Klebsiella pneumoniae. However, the sizes of several surface loops in P. islandicum UDP-GDH were much smaller than the corresponding loops in B. cepacia UDP-GDH but were comparable to those of the S. pyogenes and K. pneumoniae enzymes. Structural comparison revealed that the presence of extensive intersubunit hydrophobic interactions, as well as the formation of an intersubunit aromatic pair network, is likely to be the main factor contributing to the hyperthermo­stability of P. islandicum UDP-GDH.
PMCID: PMC3433186  PMID: 22949183
hyperthermophiles; Pyrobaculum islandicum; UDP-glucose dehydrogenases; archaea; thermostability
15.  Differential knockdown of TGF-β ligands in a three-dimensional co-culture tumor- stromal interaction model of lung cancer 
BMC Cancer  2014;14(1):580.
Transforming growth factor (TGF)-β plays a pivotal role in cancer progression through regulating cancer cell proliferation, invasion, and remodeling of the tumor microenvironment. Cancer-associated fibroblasts (CAFs) are the predominant type of stromal cell, in which TGF-β signaling is activated. Among the strategies for TGF-β signaling inhibition, RNA interference (RNAi) targeting of TGF-β ligands is emerging as a promising tool. Although preclinical studies support the efficacy of this therapeutic strategy, its effect on the tumor microenvironment in vivo remains unknown. In addition, differential effects due to knockdown of various TGF-β ligand isoforms have not been examined. Therefore, an experimental model that recapitulates tumor–stromal interaction is required for validation of therapeutic agents.
We have previously established a three-dimensional co-culture model of lung cancer, and demonstrated the functional role of co-cultured fibroblasts in enhancing cancer cell invasion and differentiation. Here, we employed this model to examine how knockdown of TGF-β ligands affects the behavior of different cell types. We developed lentivirus vectors carrying artificial microRNAs against human TGF-β1 and TGF-β2, and tested their effects in lung cancer cells and fibroblasts.
Lentiviral vectors potently and selectively suppressed the expression of TGF-β ligands, and showed anti-proliferative effects on these cells. Furthermore, knockdown of TGF-β ligands attenuated fibroblast-mediated collagen gel contraction, and diminished lung cancer cell invasion in three-dimensional co-culture. We also observed differential effects by targeting different TGF-β isoforms in lung cancer cells and fibroblasts.
Our findings support the notion that RNAi-mediated targeting of TGF-β ligands may be beneficial for lung cancer treatment via its action on both cancer and stromal cells. This study further demonstrates the usefulness of this three-dimensional co-culture model to examine the effect of therapeutic agents on tumor–stromal interaction.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-580) contains supplementary material, which is available to authorized users.
PMCID: PMC4132906  PMID: 25107280
RNA interference; MicroRNA; Lentivirus vector; TGF-β; Three-dimensional co-culture; Gel contraction assay
16.  An autopsy case of unicentric Castleman’s disease associated with bronchiolitis obliterans 
Respirology Case Reports  2014;2(3):105-107.
A 34-year-old woman visited the hospital suffering from enanthema of the tongue, hair loss, and nonproductive cough. Corticosteroid administration slightly resolved the enanthema and hair loss, but not the nonproductive cough. She was transferred to another hospital for the resection of a retroperitoneal mass, which was histopathologically diagnosed as unicentric, hyaline vascular type Castleman’s disease. She was then referred to our hospital due to progressive dyspnea and was diagnosed as having bronchiolitis obliterans based on computed tomography scan findings and a lung function test, while paraneoplastic pemphigus was clinically considered for her enanthema. After her death from respiratory failure in spite of corticosteroid administration, autopsy confirmed constrictive bronchiolitis in the lung. Mucocutaneous lesions, clinically considered as paraneoplastic pemphigus, were histologically different from pemphigus. The constrictive bronchiolitis and other pathological findings, including nonviral chronic hepatitis and interstitial nephritis, confirmed the diagnosis of paraneoplastic autoimmune multiorgan syndrome associated with the Castleman’s disease.
PMCID: PMC4184740  PMID: 25473583
Bronchiolitis obliterans; paraneoplastic autoimmune multiorgan syndrome; paraneoplastic pemphigus; unicentric Castleman’s disease
17.  Conserved Neutralizing Epitope at Globular Head of Hemagglutinin in H3N2 Influenza Viruses 
Journal of Virology  2014;88(13):7130-7144.
Neutralizing antibodies that target the hemagglutinin of influenza virus either inhibit binding of hemagglutinin to cellular receptors or prevent the low-pH-induced conformational change in hemagglutinin required for membrane fusion. In general, the former type of antibody binds to the globular head formed by HA1 and has narrow strain specificity, while the latter type binds to the stem mainly formed by HA2 and has broad strain specificity. In the present study, we analyzed the epitope and function of a broadly neutralizing human antibody against H3N2 viruses, F005-126. The crystal structure of F005-126 Fab in complex with hemagglutinin revealed that the antibody binds to the globular head, spans a cleft formed by two hemagglutinin monomers in a hemagglutinin trimer, and cross-links them. It recognizes two peptide portions (sites L and R) and a glycan linked to asparagine at residue 285 using three complementarity-determining regions and framework 3 in the heavy chain. Binding of the antibody to sites L (residues 171 to 173, 239, and 240) and R (residues 91, 92, 270 to 273, 284, and 285) is mediated mainly by van der Waals contacts with the main chains of the peptides in these sites and secondarily by hydrogen bonds with a few side chains of conserved sequences in HA1. Furthermore, the glycan recognized by F005-126 is conserved among H3N2 viruses. F005-126 has the ability to prevent low-pH-induced conformational changes in hemagglutinin. The newly identified conserved epitope, including the glycan, should be immunogenic in humans and may induce production of broadly neutralizing antibodies against H3 viruses.
IMPORTANCE Antibodies play an important role in protection against influenza virus, and hemagglutinin is the major target for virus neutralizing antibodies. It has long been believed that all effective neutralizing antibodies bind to the surrounding regions of the sialic acid-binding pocket and inhibit the binding of hemagglutinin to the cellular receptor. Since mutations are readily introduced into such epitopes, this type of antibody shows narrow strain specificity. Recently, however, broadly neutralizing antibodies have been isolated. Most of these bind either to conserved sites in the stem region or to the sialic acid-binding pocket itself. In the present study, we identified a new neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against H3N2. This epitope may be useful for design of vaccines.
PMCID: PMC4054433  PMID: 24719430
18.  Comprehensive enzymatic analysis of the amylolytic system in the digestive fluid of the sea hare, Aplysia kurodai: Unique properties of two α-amylases and two α-glucosidases 
FEBS Open Bio  2014;4:560-570.
•The amylolytic system of the digestive fluid of sea hare (Aplysia kurodai) was studied.•Two α-amylases and two α-glucosidases were purified from the digestive fluid.•Sea hare efficiently digests sea lettuce to glucose by a combination of these enzymes.•Starch in sea lettuce is a predominant glucose source for sea hare.
Sea lettuce (Ulva pertusa) is a nuisance species of green algae that is found all over the world. East-Asian species of the marine gastropod, the sea hare Aplysia kurodai, shows a clear feeding preference for sea lettuce. Compared with cellulose, sea lettuce contains a higher amount of starch as a storage polysaccharide. However, the entire amylolytic system in the digestive fluid of A. kurodai has not been studied in detail. We purified α-amylases and α-glucosidases from the digestive fluid of A. kurodai and investigated the synergistic action of these enzymes on sea lettuce. A. kurodai contain two α-amylases (59 and 80 kDa) and two α-glucosidases (74 and 86 kDa). The 59-kDa α-amylase, but not the 80-kDa α-amylase, was markedly activated by Ca2+ or Cl−. Both α-amylases degraded starch and maltoheptaose, producing maltotriose, maltose, and glucose. Glucose production from starch was higher with 80-kDa α-amylase than with 59-kDa α-amylase. Kinetic analysis indicated that 74-kDa α-glucosidase prefers short α-1,4-linked oligosaccharide, whereas 86-kDa α-glucosidase prefers large α-1,6 and α-1,4-linked polysaccharides such as glycogen. When sea lettuce was used as a substrate, a 2-fold greater amount of glucose was released by treatment with 59-kDa α-amylase and 74-kDa α-glucosidase than by treatment with 45-kDa cellulase and 210-kDa β-glucosidase of A. kurodai. Unlike mammals, sea hares efficiently digest sea lettuce to glucose by a combination of two α-amylases and two α-glucosidases in the digestive fluids without membrane-bound maltase–glucoamylase and sucrase–isomaltase complexes.
PMCID: PMC4141080  PMID: 25161866
ApAmy59, Aplysia 59-kDa α-amylase; ApAmy80, Aplysia 80-kDa α-amylase; ApAGL74, Aplysia 74-kDa α-glucosidase; ApAGL86, Aplysia 86-kDa α-glucosidase; CMC, carboxymethylcellulose; 4MU-α-Glu, 4-methylumbelliferyl α-glucoside; GHF, glycoside hydrolase family; PVDF, polyvinylidene fluoride; TLC, thin layer chromatography; α-Amylase; α-Glucosidase; Sea hare; Sea lettuce
19.  Establishment of a Simple and Rapid Identification Method for Listeria spp. by Using High-Resolution Melting Analysis, and Its Application in Food Industry 
PLoS ONE  2014;9(6):e99223.
Listeria monocytogenes is the causative bacteria of listeriosis, which has a higher mortality rate than that of other causes of food poisoning. Listeria spp., of which L. monocytogenes is a member, have been isolated from food and manufacturing environments. Several methods have been published for identifying Listeria spp.; however, many of the methods cannot identify newly categorized Listeria spp. Additionally, they are often not suitable for the food industry, owing to their complexity, cost, or time consumption. Recently, high-resolution melting analysis (HRMA), which exploits DNA-sequence differences, has received attention as a simple and quick genomic typing method. In the present study, a new method for the simple, rapid, and low-cost identification of Listeria spp. has been presented using the genes rarA and ldh as targets for HRMA. DNA sequences of 9 Listeria species were first compared, and polymorphisms were identified for each species for primer design. Species specificity of each HRM curve pattern was estimated using type strains of all the species. Among the 9 species, 7 were identified by HRMA using rarA gene, including 3 new species. The remaining 2 species were identified by HRMA of ldh gene. The newly developed HRMA method was then used to assess Listeria isolates from the food industry, and the method efficiency was compared to that of identification by 16S rDNA sequence analysis. The 2 methods were in coherence for 92.6% of the samples, demonstrating the high accuracy of HRMA. The time required for identifying Listeria spp. was substantially low, and the process was considerably simplified, providing a useful and precise method for processing multiple samples per day. Our newly developed method for identifying Listeria spp. is highly valuable; its use is not limited to the food industry, and it can be used for the isolates from the natural environment.
PMCID: PMC4053416  PMID: 24918440
20.  Identification, Purification, and Characterization of a Novel Amino Acid Racemase, Isoleucine 2-Epimerase, from Lactobacillus Species 
Journal of Bacteriology  2013;195(22):5207-5215.
Accumulation of d-leucine, d-allo-isoleucine, and d-valine was observed in the growth medium of a lactic acid bacterium, Lactobacillus otakiensis JCM 15040, and the racemase responsible was purified from the cells and identified. The N-terminal amino acid sequence of the purified enzyme was GKLDKASKLI, which is consistent with that of a putative γ-aminobutyrate aminotransferase from Lactobacillus buchneri. The putative γ-aminobutyrate aminotransferase gene from L. buchneri JCM 1115 was expressed in recombinant Escherichia coli and then purified to homogeneity. The enzyme catalyzed the racemization of a broad spectrum of nonpolar amino acids. In particular, it catalyzed at high rates the epimerization of l-isoleucine to d-allo-isoleucine and d-allo-isoleucine to l-isoleucine. In contrast, the enzyme showed no γ-aminobutyrate aminotransferase activity. The relative molecular masses of the subunit and native enzyme were estimated to be about 49 kDa and 200 kDa, respectively, indicating that the enzyme was composed of four subunits of equal molecular masses. The Km and Vmax values of the enzyme for l-isoleucine were 5.00 mM and 153 μmol·min−1·mg−1, respectively, and those for d-allo-isoleucine were 13.2 mM and 286 μmol·min−1·mg−1, respectively. Hydroxylamine and other inhibitors of pyridoxal 5′-phosphate-dependent enzymes completely blocked the enzyme activity, indicating the enzyme requires pyridoxal 5′-phosphate as a coenzyme. This is the first evidence of an amino acid racemase that specifically catalyzes racemization of nonpolar amino acids at the C-2 position.
PMCID: PMC3811583  PMID: 24039265
21.  Hyphema is a risk factor for failure of trabeculectomy in neovascular glaucoma: a retrospective analysis 
BMC Ophthalmology  2014;14:55.
Several retinal ischemic diseases can cause neovascular glaucoma (NVG). Trabeculectomy with mitomycin C (MMC) is a relatively better treatment modality in the management of eyes with NVG than other glaucoma surgeries. The aim of this study was to investigate the factors that may influence the outcome of trabeculectomy with MMC for NVG.
Forty-nine NVG eyes from 43 patients (26 males and 17 females) underwent primary trabeculectomy with MMC. The mean follow-up period was 16.8 ± 8.1 months (range, 6 to 34 months). Twenty-one eyes of 21 patients received intravitreal bevacizumab (IVB) 3.6 ± 1.8 days before trabeculectomy with MMC. A Kaplan-Meier survival-curve analysis was used to summarize the cumulative probability of success. We examined the relationship between the surgical outcome and the following surgical factors: gender, age, history of panretinal photocoagulation, history of cataract surgery, history of vitrectomy, preoperative IVB, NVG in the fellow eye, and postoperative complications (hyphema, choroidal detachment, and formation of fibrin) by multivariate analysis.
The survival rate was 83.7% after 6 months, 70.9% after 12 months, and 60.8% after 24 months. The Kaplan-Meier survival curves showed no significant difference in the survival rate between the eyes with preoperative IVB (n = 21) and the eyes without preoperative IVB (n = 28) (p = 0.14). The multiple logistic regression analysis showed that postoperative hyphema (odds ratio, 6.54; 95% confidence interval, 1.41 to 35.97) was significantly associated with the surgical outcome (p = 0.02).
Postoperative hyphema was significantly correlated with the outcome of trabeculectomy for NVG. There was no significant association between preoperative IVB and postoperative hyphema or the results of trabeculectomy.
PMCID: PMC4026882  PMID: 24766841
Neovascular glaucoma; Trabeculectomy; Hyphema; Mitomycin C; Intravitreal bevacizumab
22.  IL-33 Enhanced the Proliferation and Constitutive Production of IL-13 and IL-5 by Fibrocytes 
BioMed Research International  2014;2014:738625.
Interleukin-33 appears to play important roles in the induction of allergic airway inflammation. However, whether IL-33 is involved in airway remodeling remains unclear. Because fibrocytes contribute to tissue remodeling in the setting of chronic inflammation, we examined the effects of IL-33 on fibrocyte functions. Fibrocytes were generated in vitro from peripheral blood mononuclear cells by culturing in the presence of platelet derived growth factors and the cells were stimulated with IL-33. IL-33 enhanced cell proliferation, α-SMA expression, and pro-MMP-9 activity by the fibrocytes without increasing endogenous transforming growth factor-β1 production. Fibrocytes constitutively expressed IL-13 and IL-5, and their production was augmented by stimulation with IL-33. Dexamethasone inhibited the functions of fibrocytes, but IL-33 made fibrocytes slightly refractory to the inhibitory effect of dexamethasone in terms of IL-13 production. Montelukast suppressed IL-13 production by nonstimulated fibrocytes but not those stimulated by IL-33. These findings suggest that IL-33 is involved in the airway remodeling process through its modulation of fibrocyte function independent of antigen stimulation. IL-33 might partially reduce the therapeutic effects of glucocorticoid and cysteinyl leukotriene receptor antagonist on fibrocyte-mediated Th2 responses.
PMCID: PMC4005215  PMID: 24822215
23.  In vivo imaging and tracking of individual nanodiamonds in drosophila melanogaster embryos 
Biomedical Optics Express  2014;5(4):1250-1261.
In this work, we incorporate and image individual fluorescent nanodiamonds in the powerful genetic model system Drosophila melanogaster. Fluorescence correlation spectroscopy and wide-field imaging techniques are applied to individual fluorescent nanodiamonds in blastoderm cells during stage 5 of development, up to a depth of 40 µm. The majority of nanodiamonds in the blastoderm cells during cellularization exhibit free diffusion with an average diffusion coefficient of (6 ± 3) × 10−3 µm2/s, (mean ± SD). Driven motion in the blastoderm cells was also observed with an average velocity of 0.13 ± 0.10 µm/s (mean ± SD) µm/s and an average applied force of 0.07 ± 0.05 pN (mean ± SD). Nanodiamonds in the periplasm between the nuclei and yolk were also found to undergo free diffusion with a significantly larger diffusion coefficient of (63 ± 35) × 10−3 µm2/s (mean ± SD). Driven motion in this region exhibited similar average velocities and applied forces compared to the blastoderm cells indicating the transport dynamics in the two cytoplasmic regions are analogous.
PMCID: PMC3985983  PMID: 24761304
(160.4236) Nanomaterials; (160.2540) Fluorescent and luminescent materials; (180.2520) Fluorescence microscopy
24.  Phosphorylation of Drebrin by Cyclin-Dependent Kinase 5 and Its Role in Neuronal Migration 
PLoS ONE  2014;9(3):e92291.
Cyclin-dependent kinase 5 (Cdk5)-p35 is a proline-directed Ser/Thr kinase which plays a key role in neuronal migration, neurite outgrowth, and spine formation during brain development. Dynamic remodeling of cytoskeletons is required for all of these processes. Cdk5-p35 phosphorylates many cytoskeletal proteins, but it is not fully understood how Cdk5-p35 regulates cytoskeletal reorganization associated with neuronal migration. Since actin filaments are critical for the neuronal movement and process formation, we aimed to find Cdk5 substrates among actin-binding proteins. In this study, we isolated actin gels from mouse brain extracts, which contain many actin-binding proteins, and phosphorylated them by Cdk5-p35 in vitro. Drebrin, a side binding protein of actin filaments and well known for spine formation, was identified as a phosphorylated protein. Drebrin has two isoforms, an embryonic form drebrin E and an adult type long isoform drebrin A. Ser142 was identified as a common phosphorylation site to drebrin E and A and Ser342 as a drebrin A-specific site. Phosphorylated drebrin is localized at the distal area of total drebrin in the growth cone of cultured primary neurons. By expressing nonphosphorylatable or phosphorylation mimicking mutants in developing neurons in utero, the reversible phosphorylation/dephosphorylation reaction of drebrin was shown to be involved in radial migration of cortical neurons. These results suggest that Cdk5-p35 regulates neuronal migration through phosphorylation of drebrin in growth cone processes.
PMCID: PMC3956921  PMID: 24637538
25.  Expression, purification, crystallization and preliminary X-ray diffraction analysis of a galactose 1-phosphate uridylyltransferase from the hyperthermophilic archaeon Pyrobaculum aerophilum  
A galactose 1-phosphate uridylyltransferase from a hyperthermophilic archaeon was successfully isolated and crystallized.
A galactose 1-phosphate uridylyltransferase from the hyperthermophilic archaeon Pyrobaculum aerophilum was crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol 8000 as the precipitant. The crystals belonged to the tetragonal space group P41, with unit-cell parameters a = b = 73.3, c = 126.1 Å, and diffracted to 2.73 Å resolution on beamline BL5A at the Photon Factory. The overall R merge was 7.3% and the data completeness was 99.8%.
PMCID: PMC3310544  PMID: 22442236
hyperthermophiles; Pyrobaculum aerophilum; galactose 1-phosphate uridylyltransferase

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