Publications by authors named "Sachiko Takahashi"

39 Publications

Semi-empirical model to estimate ideal conditions for the growth of large protein crystals.

Acta Crystallogr D Struct Biol 2020 Dec 26;76(Pt 12):1174-1183. Epub 2020 Nov 26.

Confocal Science Inc., Hayakawa 2nd Building 7F, 2-12-2 Iwamoto-cho, Chiyoda-ku, Tokyo 101-0032, Japan.

A large high-quality crystal is required to specify the positions of H atoms in neutron structural analysis. Consequently, several methods have been proposed for obtaining such large crystals, and theoretical considerations for growing them have been presented. However, further investigation is required to obtain a numerical model that can provide quantitative experimental conditions for obtaining a single large crystal. In the case of protein crystallization experiments, the amount of sample is often limited. Therefore, it is more realistic to make a rough estimation from a small number of experiments. This paper proposes a method of estimating the optimum experimental conditions for the growth of large protein crystals by performing a small number of experiments using a micro-batch method and reporting a numerical model based on nucleation theory and a linear approximation of the crystal-growth rate. Specifically, micro-batch experiments are performed to provide the empirical parameters for the model and to help to estimate the conditions for the growth of a crystal of a predetermined size using a certain sample concentration and volume. This method is offered as a step on the path towards efficiently and rationally producing large crystals that can be subjected to neutron diffraction without depending on luck or on performing many experiments. It is expected to contribute to drug design and the elucidation of protein molecular functions and mechanisms by obtaining positional information on H atoms in the protein molecule, which is an advantage of neutron diffraction.
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http://dx.doi.org/10.1107/S205979832001445XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7709197PMC
December 2020

Association Between Autistic Symptoms and Self-Stigma in Patients with Schizophrenia Spectrum Disorders.

Neuropsychiatr Dis Treat 2020 29;16:2553-2561. Epub 2020 Oct 29.

Department of Psychiatry, Miyagi Psychiatric Center, Natori, Miyagi, Japan.

Purpose: Self-stigma negatively influences self-esteem, quality of life, self-efficacy, treatment adherence, and recovery in psychiatric patients. By revealing personality traits that influence self-stigma, we can gain useful knowledge for the management of self-stigma. A previous meta-analysis indicated that patients with schizophrenia have higher scores on the Autism-Spectrum Quotient (AQ) than healthy controls. However, the relationship between autistic symptoms and self-stigma in patients with schizophrenia spectrum disorders remains unclear. Therefore, the present study aimed to reveal the association between autistic symptoms and self-stigma in patients with schizophrenia spectrum disorders.

Patients And Methods: We recruited 127 patients with schizophrenia spectrum disorders (schizophrenia, schizoaffective disorder, and delusional disorder). We assessed participants' self-stigma and autistic symptoms using the Internalized Stigma for Mental Illness (ISMI) scale and the Autism-Spectrum Quotient (AQ), respectively. The differences in the scores of ISMI and AQ according to patient characteristics were investigated. Multiple regression analysis controlling for age and gender was performed to determine the relationship between the total scores on the AQ and IMSI scale.

Results: Female patients showed a higher level of self-stigma than males. Unmarried patients showed a significantly higher score on the AQ than married patients. Multiple regression analysis adjusted for age and gender indicated that the total score on AQ might be a predictor of the overall rating on ISMI in patients with schizophrenia spectrum disorders.

Conclusion: This study is the first to reveal the association between autistic symptoms and self-stigma in patients with schizophrenia spectrum disorders. Our results highlight the importance of considering autistic symptoms in the assessment and management of self-stigma in patients with schizophrenia spectrum disorders.
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http://dx.doi.org/10.2147/NDT.S280485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605940PMC
October 2020

Improved preparation of group-specific component (Gc) protein to derive macrophage activating factor.

Protein Expr Purif 2020 11 29;175:105714. Epub 2020 Jul 29.

Laboratory of Basic Science on Healthy Longevity, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan. Electronic address:

Cancer immunotherapy has recently attracted attention as an approach for cancer treatment through the activation of the immune system. Group-specific component (Gc) protein is a precursor for macrophage activating factor (GcMAF), which has a promising immunomodulatory effect on the suppression of tumor growth and angiogenesis. In this study, we successfully purified Gc protein from human serum using anion-exchange chromatography combined with affinity chromatography using a 25-OH-D-immobilized column. The purity of Gc protein reached 95.0% after anion-exchange chromatography. The known allelic variants of Gc protein are classified into three subtypes-Gc1F, Gc1S and Gc2. The fragment sequence of residues 412-424 determined according to their MS/MS spectra is available to evaluate the subtypes of Gc protein. The data showed that the Gc protein purified in this study consisted of the Gc1F and Gc2 subtypes. Our method improved the purity of Gc protein, which was not affected by the treatment to convert it into GcMAF using β-galactosidase- or neuraminidase-immobilized resin, and will be useful for biological studies and/or advanced clinical uses of GcMAF, such as cancer immunotherapy.
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http://dx.doi.org/10.1016/j.pep.2020.105714DOI Listing
November 2020

Orally administered heat-killed Lactobacillus paracasei MCC1849 enhances antigen-specific IgA secretion and induces follicular helper T cells in mice.

PLoS One 2018 13;13(6):e0199018. Epub 2018 Jun 13.

Research Center for Food Safety, Graduate School of Agricultural and Life Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan.

Antigen-specific immunoglobulin (Ig) A plays a major role in host defense against infections in gut mucosal tissue. Follicular helper T (Tfh) cells are located in germinal centers and promote IgA production via interactions with germinal center B cells. Several studies have demonstrated that some lactic acid bacteria (LAB) strains activate the host's acquired immune system, inducing IgA secretion in the intestine. However, the precise molecular mechanisms underlying the effects of LAB on IgA production and Tfh cells are not fully resolved. Lactobacillus paracasei MCC1849 is a probiotic strain isolated from the intestine of a healthy adult. In this study, we investigated the effects of orally administered heat-killed MCC1849 on IgA production in the intestine and on Tfh cell induction in vivo. We found that orally administered MCC1849 induced antigen-specific IgA production in the small intestine, serum and lungs. We also observed that MCC1849 increased the proportion of IgA+ B cells and Tfh cells in Peyer's patches (PPs). In addition, MCC1849 increased the gene expression of IL-12p40, IL-10, IL-21, STAT4 and Bcl-6 associated with Tfh cell differentiation. These results suggest that orally administered MCC1849 enhances antigen-specific IgA production and likely affects Tfh cell differentiation in PPs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0199018PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5999281PMC
January 2019

Electrodiagnostic Characterization of Hereditary Neuropathy With Liability to Pressure Palsies.

J Clin Neuromuscul Dis 2017 Mar;18(3):119-124

*Department of Clinical Neurological Sciences, London Health Sciences Centre, University Hospital, London, ON, Canada; and †Schulich School of Medicine and Dentistry, Western University, London, ON, Canada.

Objectives: The study objective was electrodiagnostic characterization of a large cohort of patients with genetically confirmed hereditary neuropathy with liability to pressure palsies (HNPP).

Methods: A retrospective review was conducted on all patients with HNPP seen at the neuromuscular clinic (London, Canada) from 1977 to 2015. Clinical data obtained included patient characteristics, examination findings, and nerve conduction study results.

Results: A total of 46 patients were analyzed. The mean age and median disease duration were 42.6 and 5.0 years, respectively. Most patients had abnormalities on sural nerve conduction studies. The most common focal neuropathies at compressive sites were ulnar nerve at the elbow (85.7%), distal median nerve at the wrist (84.4%), and fibular nerve at the fibular head (36.7%). Distal median neuropathy was associated with a mean terminal latency of 6.64 milliseconds.

Conclusions: The presence of polyneuropathy, median terminal motor latency prolongation, and multiple compressive neuropathies are the most common findings associated with HNPP.
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http://dx.doi.org/10.1097/CND.0000000000000152DOI Listing
March 2017

Lifestyle Modifications Versus Antihypertensive Medications in Reducing Cardiovascular Events in an Aging Society: A Success Rate-oriented Simulation.

Intern Med 2016 15;55(16):2185-95. Epub 2016 Aug 15.

Community Health Science Center, Saitama Medical University, Japan.

Objective It is difficult to compare directly the practical effects of lifestyle modifications and antihypertensive medications on reducing cardiovascular disease (CVD). The purpose of this study was to compare the hypothetical potential of lifestyle modifications with that of antihypertensive medications in reducing CVD in an aging society using a success rate-oriented simulation. Methods We constructed a simulation model for virtual Japanese subpopulations according to sex and age at 10-year intervals from 40 years of age as an example of an aging society. The fractional incidence rate of CVD was calculated as the product of the incidence rate at each systolic blood pressure (SBP) level and the proportion of the SBP frequency distribution in the fractional subpopulations of each SBP. The total incidence rate was calculated by the definite integral of the fractional incidence rate at each SBP level in the sex- and age-specific subpopulations. Results If we consider the effects of lifestyle modifications on metabolic factors and transfer them onto SBP, the reductions in the total incidence rate of CVD were competitive between lifestyle modifications and antihypertensive medications in realistic scenarios. In middle-aged women, the preventive effects of both approaches were limited due to a low incidence rate. In middle-aged men and extremely elderly subjects whose adherence to antihypertensive medications is predicted to be low, lifestyle modifications could be an alternative choice. Conclusion The success rate-oriented simulation suggests that the effectiveness of lifestyle modifications or antihypertensive medications in preventing cardiovascular events largely depends on the baseline incidence rate and sex- and age-specific behavioral factors.
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http://dx.doi.org/10.2169/internalmedicine.55.6247DOI Listing
March 2017

Age-related changes in gut microbiota composition from newborn to centenarian: a cross-sectional study.

BMC Microbiol 2016 May 25;16:90. Epub 2016 May 25.

Department of Bioresource Science, Graduate School of Agricultural Science, Kobe University, Kobe, Hyogo, Japan.

Background: It has been reported that the composition of human gut microbiota changes with age; however, few studies have used molecular techniques to investigate the long-term, sequential changes in gut microbiota composition. In this study, we investigated the sequential changes in gut microbiota composition in newborn to centenarian Japanese subjects.

Results: Fecal samples from 367 healthy Japanese subjects between the ages of 0 and 104 years were analyzed by high-throughput sequencing of amplicons derived from the V3-V4 region of the 16S rRNA gene. Analysis based on bacterial co-abundance groups (CAGs) defined by Kendall correlations between genera revealed that certain transition types of microbiota were enriched in infants, adults, elderly individuals and both infant and elderly subjects. More positive correlations between the relative abundances of genera were observed in the elderly-associated CAGs compared with the infant- and adult-associated CAGs. Hierarchical Ward's linkage clustering based on the abundance of genera indicated five clusters, with median (interquartile range) ages of 3 (0-35), 33 (24-45), 42 (32-62), 77 (36-84) and 94 (86-98) years. Subjects were predominantly clustered with their matched age; however, some of them fell into mismatched age clusters. Furthermore, clustering based on the proportion of transporters predicted by phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) showed that subjects were divided into two age-related groups, the adult-enriched and infant/elderly-enriched clusters. Notably, all the drug transporters based on Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology groups were found in the infant/elderly-enriched cluster.

Conclusion: Our results indicate some patterns and transition points in the compositional changes in gut microbiota with age. In addition, the transporter property prediction results suggest that nutrients in the gut might play an important role in changing the gut microbiota composition with age.
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http://dx.doi.org/10.1186/s12866-016-0708-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4879732PMC
May 2016

Plastid Proteomic Analysis in Tomato Fruit Development.

PLoS One 2015 15;10(9):e0137266. Epub 2015 Sep 15.

Faculty of Agriculture, Shizuoka University, Shizuoka city, Shizuoka, Japan.

To better understand the mechanism of plastid differentiation from chloroplast to chromoplast, we examined proteome and plastid changes over four distinct developmental stages of 'Micro-Tom' fruit. Additionally, to discover more about the relationship between fruit color and plastid differentiation, we also analyzed and compared 'Micro-Tom' results with those from two other varieties, 'Black' and 'White Beauty'. We confirmed that proteins related to photosynthesis remain through the orange maturity stage of 'Micro-Tom', and also learned that thylakoids no longer exist at this stage. These results suggest that at a minimum there are changes in plastid morphology occurring before all related proteins change. We also compared 'Micro-Tom' fruits with 'Black' and 'White Beauty' using two-dimensional gel electrophoresis. We found a decrease of CHRC (plastid-lipid-associated protein) and HrBP1 (harpin binding protein-1) in the 'Black' and 'White Beauty' varieties. CHRC is involved in carotenoid accumulation and stabilization. HrBP1 in Arabidopsis has a sequence similar to proteins in the PAP/fibrillin family. These proteins have characteristics and functions similar to lipocalin, an example of which is the transport of hydrophobic molecules. We detected spots of TIL (temperature-induced lipocalin) in 2D-PAGE results, however the number of spots and their isoelectric points differed between 'Micro-Tom' and 'Black'/'White Beauty'. Lipocalin has various functions including those related to environmental stress response, apoptosis induction, membrane formation and fixation, regulation of immune response, cell growth, and metabolism adjustment. Lipocalin related proteins such as TIL and HrBP1 could be related to the accumulation of carotenoids, fruit color and the differentiation of chromoplast.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0137266PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4570674PMC
May 2016

Lifestyle modifications supported by regional health nurses lowered insulin resistance, oxidative stress and central blood pressure in subjects with metabolic syndrome.

Obes Res Clin Pract 2015 Nov-Dec;9(6):584-91. Epub 2015 Apr 7.

Community Health Science Center, Saitama Medical University, Saitama, Japan.

Background: This study was attempted to investigate whether lifestyle modifications supported by regional health nurses should improve cardio-metabolic factors--including adipocytokines, oxidative stress, and arterial stiffness--in subjects with metabolic syndrome.

Methods: Thirty-six subjects with metabolic syndrome were enrolled, 28 of whom completed the 6-month lifestyle modifications (male:female=19:9). Blood and urine test results were examined in relation to metabolic factors before and after 6-month nutritional and physical activity modifications. In addition, oral glucose tolerance tests were performed and arterial stiffness was measured by brachial-ankle pulse wave velocity and radial augmentation index before and after them.

Results: Six-month lifestyle modifications significantly reduced body weight, homeostasis model assessment index, and low-density lipoprotein cholesterol (LDL-C). They significantly attenuated oxidative stress measured by the urinary 8-hydroxy-2-deoxyguanosine/creatinine ratio. They also lowered brachial and central systolic blood pressure. They tended to decrease waist circumferences and the levels of C-reactive protein. However they did not significantly change the levels of adipocytokines, including tumour necrosis factor, soluble tumour necrosis factor receptors, and interleukin 6, or arterial stiffness measured by brachial-ankle pulse wave velocity and radial augmentation index.

Conclusions: Six-month lifestyle modifications supported by regional health nurses lowered body weight, insulin resistance, LDL-C, oxidative stress, and peripheral and central blood pressure in subjects with metabolic syndrome.
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http://dx.doi.org/10.1016/j.orcp.2015.03.003DOI Listing
September 2016

Changes in nasopharyngeal carriage of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis among healthy children attending a day-care centre before and after official financial support for the 7-valent pneumococcal conjugate vaccine and H. influenzae type b vaccine in Japan.

J Infect Chemother 2014 Feb 11;20(2):146-9. Epub 2013 Dec 11.

Department of Pediatrics, Chiba University Graduate School of Medicine, Chiba, Japan.

The 7-valent pneumococcal conjugate vaccine (PCV7) and Haemophilus influenzae type b (Hib) vaccine reduce nasopharyngeal carriage of vaccine-type bacteria, which may in turn influence the presence of other nasopharyngeal bacterial pathogens. To investigate this possibility, nasopharyngeal carriage of potential pathogens was examined before and after official financial support was provided to offer the PCV7 and Hib vaccines in healthy children attending a day care centre in Japan during 2011-2012. Despite a virtual disappearance of PCV7 serotypes over time, the overall pneumococcal carriage rate remained unchanged. Although others have reported an increase in PCV13 serotypes following PCV7 vaccination, only non-PCV13 serotypes were observed to have increased in this study. The majority of H. influenzae isolates were non-typeable and Hib was not found. Our data identified an unexpected pattern of pneumococcal serotype replacement following PCV7. Continuous monitoring of pneumococcal carriage is important for decisions regarding the future of national vaccination policy in Japan.
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http://dx.doi.org/10.1016/j.jiac.2013.10.007DOI Listing
February 2014

Numerical model of protein crystal growth in a diffusive field such as the microgravity environment.

J Synchrotron Radiat 2013 Nov 1;20(Pt 6):1003-9. Epub 2013 Oct 1.

Confocal Science Inc., Hayakawa 2nd Building 7F, 2-12-2 Iwamoto-cho, Chiyoda-ku, Tokyo 101-0032, Japan.

It is said that the microgravity environment positively affects the quality of protein crystal growth. The formation of a protein depletion zone and an impurity depletion zone due to the suppression of convection flow were thought to be the major reasons. In microgravity, the incorporation of molecules into a crystal largely depends on diffusive transport, so the incorporated molecules will be allocated in an orderly manner and the impurity uptake will be suppressed, resulting in highly ordered crystals. Previously, these effects were numerically studied in a steady state using a simplified model and it was determined that the combination of the diffusion coefficient of the protein molecule (D) and the kinetic constant for the protein molecule (β) could be used as an index of the extent of these depletion zones. In this report, numerical analysis of these depletion zones around a growing crystal in a non-steady (i.e. transient) state is introduced, suggesting that this model may be used for the quantitative analysis of these depletion zones in the microgravity environment.
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http://dx.doi.org/10.1107/S0909049513022784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795573PMC
November 2013

JAXA protein crystallization in space: ongoing improvements for growing high-quality crystals.

J Synchrotron Radiat 2013 Nov 26;20(Pt 6):968-73. Epub 2013 Sep 26.

Confocal Science Inc., Hayakawa 2nd Building 7F, 2-12-2 Iwamoto-cho, Chiyoda-ku, Tokyo 101-0032, Japan.

The Japan Aerospace Exploration Agency (JAXA) started a high-quality protein crystal growth project, now called JAXA PCG, on the International Space Station (ISS) in 2002. Using the counter-diffusion technique, 14 sessions of experiments have been performed as of 2012 with 580 proteins crystallized in total. Over the course of these experiments, a user-friendly interface framework for high accessibility has been constructed and crystallization techniques improved; devices to maximize the use of the microgravity environment have been designed, resulting in some high-resolution crystal growth. If crystallization conditions were carefully fixed in ground-based experiments, high-quality protein crystals grew in microgravity in many experiments on the ISS, especially when a highly homogeneous protein sample and a viscous crystallization solution were employed. In this article, the current status of JAXA PCG is discussed, and a rational approach to high-quality protein crystal growth in microgravity based on numerical analyses is explained.
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http://dx.doi.org/10.1107/S0909049513021596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795566PMC
November 2013

Ectopic localization of auxin and cytokinin in tobacco seedlings by the plant-oncogenic AK-6b gene of Agrobacterium tumefaciens AKE10.

Planta 2013 Oct 20;238(4):753-70. Epub 2013 Jul 20.

Department of Biological Production, Faculty of Bioresource Sciences, Akita Prefectural University, 241-438 Kaidobata Nishi, Nakano-Aza, Shimoshinjo, Akita, 010-0195, Japan.

The oncogenic 6b gene of Agrobacterium tumefaciens induces a number of morphological and metabolic alterations in plants. Although molecular functions associated with the 6b genes have been proposed, including auxin transport, sugar transport, transcriptional regulation, and miRNA metabolism, so far an unequivocal conclusion has not been obtained. We investigated the association between auxin accumulation and tumor development of the tobacco seedlings expressing the AK-6b gene under the control of the dexamethasone-inducible promoter. Indole-3-acetic acid (IAA) localization was examined by immunochemical staining with monoclonal antibody against IAA and by histochemical analysis using the IAA-specific induced construct, DR5::GUS (β-glucuronidase). Both procedures indicated that IAA preferentially accumulated in the tumorous protrusions as well as in newly developing vascular bundles in the tumors. Furthermore, true leaves also showed abaxial IAA localization, leading to altered leaves in which the adaxial and abaxial identities were no longer evident. Co-localization of cytokinin and auxin in the abaxial tumors was verified by immunochemical staining with an antibody against cytokinin. Treatment of AK-6b-seedlings with N-1-naphthylphthalamic acid, an inhibitor of polar auxin transport, promoted the morphological severity of phenotypes, whereas 1-naphthoxyacetic acid, a specific auxin influx carrier inhibitor, induced tumor regression on cotyledons and new tumorous proliferations on hypocotyls. Prominent accumulation of both auxin and cytokinin was observed in both regressed and newly developing tumors. We suggest from these results that modulation of auxin/cytokinin localization as a result of AK-6b gene expression is responsible for the tumorous proliferation.
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http://dx.doi.org/10.1007/s00425-013-1930-0DOI Listing
October 2013

Pure hemi-chorea resulting from an acute phase of contralateral thalamic lacunar infarction: a case report.

Case Rep Neurol 2012 Sep 8;4(3):194-201. Epub 2012 Nov 8.

Department of Neurology, Hino Municipal Hospital, Tokyo, Japan.

Background: Thalamic lesions give rise to a variety of clinical syndromes such as pure sensory stroke, ataxic hemiparesis, and rarely involuntary movements including chorea. Generally and classically, lacunar infarction in the subthalamic nucleus has been regarded as the lesion mainly responsible for hemi-chorea and hemi-ballismus, on the basis of previous anatomical studies.

Case Presentation: This report describes the case of an 81-year-old man who developed sudden-onset pure hemi-chorea in the right limbs resulting from an acute phase of left thalamic lacunar infarction detected on a diffusion-weighted image (DWI) in an MRI study. The patient had no other neurological symptoms such as ataxic hemiparesis and sensory disturbance. A single-photon emission computed tomography (SPECT) study using the (99m)Tc-ECD Patlak plot method demonstrated significant perfusional asymmetry between the right and left thalami (p = 0.0035), consistent with the left thalamic lesion on DWI.

Conclusion: It is speculated that this perfusional asymmetry, in particular the hypoperfusion in the left thalamus, detected by SPECT might play the most important role in the contralateral pure hemi-chorea as a rare neurological manifestation in this case.
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http://dx.doi.org/10.1159/000345227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3506042PMC
September 2012

High-Quality Protein Crystal Growth of Mouse Lipocalin-Type Prostaglandin D Synthase in Microgravity.

Cryst Growth Des 2011 Jun 5;11(6):2107-2111. Epub 2011 Apr 5.

Maruwa Foods and Biosciences Inc. , 170-1 Tsutsui-cho, Yamatokoriyama, Nara 639-1123, Japan.

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH(2) to PGD(2) and is involved in the regulation of pain and of nonrapid eye movement sleep and the differentiation of male genital organs and adipocytes, etc. L-PGDS is secreted into various body fluids and binds various lipophilic compounds with high affinities, acting also as an extracellular transporter. Mouse L-PGDS with a C65A mutation was previously crystallized with citrate or malonate as a precipitant, and the X-ray crystallographic structure was determined at 2.0 Å resolution. To obtain high-quality crystals, we tried, unsuccessfully, to crystallize the C65A mutant in microgravity under the same conditions used in the previous study. After further purifying the protein and changing the precipitant to polyethylene glycol (PEG) 8000, high-quality crystals were grown in microgravity. The precipitant solution was 40% (w/v) PEG 8000, 100 mM sodium chloride, and 100 mM HEPES-NaOH (pH 7.0). Crystals grew on board the International Space Station for 11 weeks in 2007, yielding single crystals of the wild-type L-PGDS and the C65A mutant, both of which diffracted at around 1.0 Å resolution. The crystal quality was markedly improved through the use of a high-viscosity precipitant solution in microgravity, in combination with the use of a highly purified protein.
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http://dx.doi.org/10.1021/cg101370vDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3105485PMC
June 2011

The β-catenin HMP-2 functions downstream of Src in parallel with the Wnt pathway in early embryogenesis of C. elegans.

Dev Biol 2011 Jul 6;355(2):302-12. Epub 2011 May 6.

Genome Biology Laboratory, National Institute of Genetics, 1111 Yata, Mishima, 411-8540, Japan.

The Wnt and Src pathways are widely used signal transduction pathways in development. β-catenin is utilized in both pathways, as a signal transducer and a component of the cadherin cell adhesion complex, respectively. A C. elegans β-catenin HMP-2 is involved in cell adhesion, but its signaling role has been unknown. Here, we report that in early embryogenesis HMP-2 acts as a signaling molecule in the Src signal. During early embryogenesis in C. elegans, the Wnt and Src pathways are redundantly involved in endoderm induction at the four-cell stage and spindle orientation in an ABar blastomere. RNAi experiments demonstrated that HMP-2 functions in the Src pathway, but in parallel with the Wnt pathway in these processes. HMP-2 localized at the cell boundaries and nuclei, and its localization at cell boundaries was negatively regulated by SRC-1. In addition, HMP-2 was Tyr-phosphorylated in a SRC-1-dependent manner in vivo. Taken together, we propose that HMP-2 functions downstream of the Src signaling pathway and contribute to endoderm induction and ABar spindle orientation, in parallel with the Wnt signaling pathway.
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http://dx.doi.org/10.1016/j.ydbio.2011.04.034DOI Listing
July 2011

Improvement in the quality of hematopoietic prostaglandin D synthase crystals in a microgravity environment.

J Synchrotron Radiat 2011 Jan 5;18(1):88-91. Epub 2010 Nov 5.

Confocal Science Inc., Japan.

Human hematopoietic prostaglandin synthase, one of the better therapeutic target enzymes for allergy and inflammation, was crystallized with 22 inhibitors and in three inhibitor-free conditions in microgravity. Most of the space-grown crystals showed better X-ray diffraction patterns than the terrestrially grown ones, indicating the advantage of a microgravity environment on protein crystallization, especially in the case of this protein.
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http://dx.doi.org/10.1107/S0909049510037076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3004263PMC
January 2011

Optimization of salt concentration in PEG-based crystallization solutions.

J Synchrotron Radiat 2011 Jan 5;18(1):84-7. Epub 2010 Nov 5.

Confocal Science Inc., Japan.

Although polyethylene glycol (PEG) is the most widely used precipitant in protein crystallization, the concentration of co-existing salt in the solution has not been well discussed. To determine the optimum salt concentration range, several kinds of protein were crystallized in a 30% PEG 4000 solution at various NaCl concentrations with various pH levels. It was found that, if crystallization occurred, the lowest effective salt concentration depended on the pH of the protein solution and the pI of the protein molecule; that is, higher salt concentrations were required for crystal growth if the difference between pH and pI was increasing. The linear relationship between the charge density of the protein and the ionic strength of the crystallization solution was further verified. These results suggested that the lowest effective concentration of salt in a crystallization solution can be predicted before performing a crystallization experiment. Our results can be a tip for tuning crystallization conditions by the vapor-diffusion method.
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http://dx.doi.org/10.1107/S0909049510035995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3004262PMC
January 2011

Antiobesity effects of Bifidobacterium breve strain B-3 supplementation in a mouse model with high-fat diet-induced obesity.

Biosci Biotechnol Biochem 2010 7;74(8):1656-61. Epub 2010 Aug 7.

Food Science and Technology Institute, Morinaga Milk Industry Co., Ltd, Kanagawa, Japan.

The aim of the present study was to evaluate the anti-obesity activity of a probiotic bifidobacterial strain in a mouse model with obesity induced by a high-fat diet. The mice were fed a high-fat diet supplemented with Bifidobacterium breve B-3 at 10(8) or 10(9) CFU/d for 8 weeks. B. breve B-3 supplementation dose-dependently suppressed the accumulation of body weight and epididymal fat, and improved the serum levels of total cholesterol, fasting glucose and insulin. The bifidobacterial counts in the caecal contents and feces were significantly increased with the B. breve B-3 administration. The expression of genes related to fat metabolism and insulin sensitivity in the gut and epididymal fat tissue was up-regulated by this administration. These results suggest that the use of B. breve B-3 would be effective in reducing the risk of obesity.
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http://dx.doi.org/10.1271/bbb.100267DOI Listing
December 2010

High-quality crystals of human haematopoietic prostaglandin D synthase with novel inhibitors.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2010 Jul 24;66(Pt 7):846-50. Epub 2010 Jun 24.

Confocal Science Inc., Japan.

Human haematopoietic prostaglandin D synthase (H-PGDS; EC 5.3.99.2) produces prostaglandin D(2), an allergic and inflammatory mediator, in mast cells and Th2 cells. H-PGDS has been crystallized with novel inhibitors with half-maximal inhibitory concentrations (IC(50)) in the low nanomolar range by the counter-diffusion method onboard the Russian Service Module on the International Space Station. The X-ray diffraction of a microgravity-grown crystal of H-PGDS complexed with an inhibitor with an IC(50) value of 50 nM extended to 1.1 A resolution at 100 K using SPring-8 synchrotron radiation, which is one of the highest resolutions obtained to date for this protein.
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http://dx.doi.org/10.1107/S1744309110020828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2898477PMC
July 2010

Antibiotic susceptibility of bifidobacterial strains distributed in the Japanese market.

Biosci Biotechnol Biochem 2010 7;74(2):336-42. Epub 2010 Feb 7.

Food Science and Technology Institute, Morinaga Milk Industry Co., Ltd., Zama, Kinagawa, Japan.

The aim of the present study was to analyze the antibiotic susceptibility of bifidobacterial strains distributed in the Japanese market. A total of 23 strains, including probiotic isolates from foods, supplements, pharmaceuticals and reference strains of each species (or subspecies), were tested for susceptibility to 15 antibiotics by the broth microdilution method and examined for the presence of possible resistant determinants. The strains were susceptible overall to chloramphenicol, ampicillin, vancomycin and linezolid, and were intrinsically resistant to aminoglycoside group agents. Susceptibility to erythromycin, clindamycin, rifampicin, tetracycline and trimethoprim varied among the strains. All strains of Bifidobacterium animalis subsp. lactis were resistant to tetracycline and appeared to harbor tet(W) genes. No risk factor for safety was found for bifidobacterial strains distributed in the Japanese market in respect of their antimicrobial resistance, although the presence of the tet(W) gene in some strains stresses the need for future evaluation.
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http://dx.doi.org/10.1271/bbb.90659DOI Listing
May 2010

Distribution of in vitro fermentation ability of lacto-N-biose I, a major building block of human milk oligosaccharides, in bifidobacterial strains.

Appl Environ Microbiol 2010 Jan 23;76(1):54-9. Epub 2009 Oct 23.

Food Science and Technology Institute, Morinaga Milk Industry Co., Ltd., Zama, Kanagawa 228-8583, Japan.

This study investigated the potential utilization of lacto-N-biose I (LNB) by individual strains of bifidobacteria. LNB is a building block for the human milk oligosaccharides, which have been suggested to be a factor for selective growth of bifidobacteria. A total of 208 strains comprising 10 species and 4 subspecies were analyzed for the presence of the galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) gene (lnpA) and examined for growth when LNB was used as the sole carbohydrate source. While all strains of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, B. breve, and B. bifidum were able to grow on LNB, none of the strains of B. adolescentis, B. catenulatum, B. dentium, B. angulatum, B. animalis subsp. lactis, and B. thermophilum showed any growth. In addition, some strains of B. pseudocatenulatum, B. animalis subsp. animalis, and B. pseudolongum exhibited the ability to utilize LNB. With the exception for B. pseudocatenulatum, the presence of lnpA coincided with LNB utilization in almost all strains. These results indicate that bifidobacterial species, which are the predominant species found in infant intestines, are potential utilizers of LNB. These findings support the hypothesis that GLNBP plays a key role in the colonization of bifidobacteria in the infant intestine.
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http://dx.doi.org/10.1128/AEM.01683-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798660PMC
January 2010

Study of novel d1 alleles, defective mutants of the alpha subunit of heterotrimeric G-protein in rice.

Genes Genet Syst 2009 Feb;84(1):35-42

Department of Bioscience, Fukui Prefectural University, Fukui, Japan.

It has been shown that the disruption of the alpha-subunit gene of heterotorimeric G-proteins (Galpha) results in dwarf traits, the erection of leaves and the setting of small seeds in rice. These mutants are called d1. We have studied the expression profiles of the transcripts and translation products of rice Galpha in ten alleles of d1 including five additional alleles newly identified. By RT-PCR, the transcripts of the Galpha gene were detected in the all d1 alleles. By western blot, the Galpha proteins were not detected in the plasma membrane fractions of the d1 alleles with the exception of d1-4. In d1-4, one amino acid change in the GTP-binding box A of the Galpha protein was occurred and even in this case the Galpha protein was only just detectable in the plasma membrane fraction. Given that the Galpha protein did not accumulate in the plasma membrane fraction in d1-8 which has a deletion of just a single amino acid in the Galpha protein, it is likely that a proper conformation of the Galpha is necessary for accumulation of Galpha protein in the plasma membrane. Nine alleles of d1 showed a severer phenotype whilst d1-4 exhibited a mild phenotype with respect to seed size and elongation pattern of internodes. As brassinosteroid signaling was known to be partially impaired in d1s, the sensitivity to 24-epibrassinolide (24-epiBL) was compared among d1 alleles in a T65 genetic background. Only d1-4 showed responses similar to wild type rice. The results show that the d1-4 mutant is a mild allele in terms of the phenotype and mild hyposensitivity to the exogenously applied 24-epiBL.
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http://dx.doi.org/10.1266/ggs.84.35DOI Listing
February 2009

Novel wide-range quantitative nested real-time PCR assay for Mycobacterium tuberculosis DNA: development and methodology.

J Clin Microbiol 2008 May 12;46(5):1708-15. Epub 2008 Mar 12.

Advanced Research Institute for the Sciences and Humanities, Nihon University, Tokyo, Japan.

Previously, we designed an internally controlled quantitative nested real-time (QNRT) PCR assay for Mycobacterium tuberculosis DNA in order to rapidly diagnose tuberculous meningitis. This technique combined the high sensitivity of nested PCR with the accurate quantification of real-time PCR. In this study, we attempted to improve the original QNRT-PCR assay and newly developed the wide-range QNRT-PCR (WR-QNRT-PCR) assay, which is more accurate and has a wider detection range. For use as an internal-control "calibrator" to measure the copy number of M. tuberculosis DNA, an original new-mutation plasmid (NM-plasmid) was developed. It had artificial random nucleotides in five regions annealing specific primers and probes. The NM-plasmid demonstrated statistically uniform amplifications (F = 1.086, P = 0.774) against a range (1 to 10(5)) of copy numbers of mimic M. tuberculosis DNA and was regarded as appropriate for use as a new internal control in the WR-QNRT-PSR assay. In addition, by the optimization of assay conditions in WR-QNRT-PCR, two-step amplification of target DNA was completely consistent with the standard curve of this assay. Due to the development of the NM-plasmid as the new internal control, significantly improved quantitative accuracy and a wider detection range were realized with the WR-QNRT-PCR assay. In the next study, we will try to use this novel assay method with actual clinical samples and examine its clinical usefulness.
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http://dx.doi.org/10.1128/JCM.01200-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2395078PMC
May 2008

Novel wide-range quantitative nested real-time PCR assay for Mycobacterium tuberculosis DNA: clinical application for diagnosis of tuberculous meningitis.

J Clin Microbiol 2008 May 12;46(5):1698-707. Epub 2008 Mar 12.

Advanced Research Institute for the Sciences and Humanities, Nihon University, Tokyo, Japan.

Although the "gold standard" for diagnosis of tuberculous meningitis (TBM) is bacterial isolation of Mycobacterium tuberculosis, there are still several complex issues. Recently, we developed an internally controlled novel wide-range quantitative nested real-time PCR (WR-QNRT-PCR) assay for M. tuberculosis DNA in order to rapidly diagnose TBM. For use as an internal control calibrator to measure the copy number of M. tuberculosis DNA, an original new-mutation plasmid (NM-plasmid) was developed. Due to the development of the NM-plasmid, the WR-QNRT-PCR assay demonstrated statistically significant accuracy over a wide detection range (1 to 10(5) copies). In clinical applications, the WR-QNRT-PCR assay revealed sufficiently high sensitivity (95.8%) and specificity (100%) for 24 clinically suspected TBM patients. In conditional logistic regression analysis, a copy number of M. tuberculosis DNA (per 1 ml of cerebrospinal fluid) of >8,000 was an independent risk factor for poor prognosis for TBM (i.e., death) (odds ratio, 16.142; 95% confidence interval, 1.191 to 218.79; P value, 0.0365). In addition, the copy numbers demonstrated by analysis of variance statistically significant alterations (P < 0.01) during the clinical treatment course for 10 suspected TBM patients. In simple regression analysis, the significant correlation (R(2) = 0.597; P < 0.0001) was demonstrated between copy number and clinical stage of TBM. We consider the WR-QNRT-PCR assay to be a useful and advanced assay technique for assessing the clinical treatment course of TBM.
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http://dx.doi.org/10.1128/JCM.02214-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2395115PMC
May 2008

Longitudinal epidemiology of Chlamydia trachomatis serovars in female patients in Japan.

Jpn J Infect Dis 2007 Nov;60(6):374-6

Department of Nutrition, Women's Collenge for Nutrition, Saitama 350-0288, Japan.

The objective of this study was to clarify the longitudinal epidemiology of Chlamydia trachomatis serovars in Japan. A total of 339 endocervical swab specimens obtained from female patients who attended the Department of Obstetrics and Gynecology, Saitama Medical School, were used. Positive specimens of either transport medium of IDEIA Chlamydia (1st group, from 1999 to 2001), or DNA extract of Cobas Amplicor STD-1 Chlamydia trachomatis (2nd group, from 2003 to 2005) were used for serotyping. Typing of C. trachomatis serovars in DNA extracts was performed by polymerase chain reaction-restriction fragment length polymorphism. Ten serovars, A, B, D, E, F, G, H, I, J and K, were identified in the 1st group, and serovar E was most frequently identified (27.6%). In the 2nd group, nine serovars, B, C, D, E, F, G, H, I and K, were identified, and serovar D was most frequently identified (24.7%). Serovars B and Ba were significantly more common around 2000 and the mid-1990s (from 1993 to 1996), respectively. Numbers of serovar I increased significantly during the research period. In addition, serovar I was more frequent in the 2nd group than in the 1st group in women aged 20-29 years. There were no significant differences of serovar distribution between pregnant and non-pregnant women.
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November 2007

Prospective study of clinical symptoms and skin test reactions in medical students exposed to formaldehyde gas.

J Dermatol 2007 May;34(5):283-9

Department of Dermatology, Okayama Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.

Previous investigators have reported the occurrence of both allergic and non-allergic systemic complications due to exposure to formaldehyde gas. However, little is known about the pathogenic link between formaldehyde-induced clinical symptoms and patch test results, or about the long-term effects of formaldehyde exposure. In the present study, a questionnaire was administered to 143 medical students, and 60 of them were tested by patch test for formaldehyde at the beginning and end of a human anatomy laboratory course. Another group of 76 students who had finished the course 2-4 years previously were administered another questionnaire, and the patch test was carried out on 58 of them. The frequencies of skin irritation, eye soreness, lacrimation, eye fatigue, rhinorrhea, throat irritation, general fatigue and mood swings increased after repeated exposure. Two (3.3%) of 60 students became positive to 1% formaldehyde at the end of the anatomy course (one male with allergic hand dermatitis due to direct contact with formaldehyde, and one female with an atopic background with unbearable physical symptoms) while the remaining 58 showed a negative reaction throughout the study period. The vast majority of students complained of various non-allergic, physical symptoms, and recovered from such symptoms without subsequent complications. No progression to multiple chemical sensitivity was found. Students with an episode of atopic dermatitis and allergic rhinitis were susceptible to formaldehyde exposure, and developed mucocutaneous symptoms, probably due to the impaired barrier function and remodeling of the skin and mucosa.
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http://dx.doi.org/10.1111/j.1346-8138.2007.00274.xDOI Listing
May 2007

The identification of two germ-line mutations in the human breast cancer resistance protein gene that result in the expression of a low/non-functional protein.

Pharm Res 2007 Jun 21;24(6):1108-17. Epub 2007 Mar 21.

Department of Chemotherapy, Kyoritsu University of Pharmacy, Tokyo, Japan.

Purpose: We examined the effects of the nine nonsynonymous germ-line mutations/SNPs in the breast cancer resistance protein (BCRP/ABCG2) gene on the expression and function of the protein.

Materials And Methods: We generated cDNAs for each of these mutants (G151T, C458T, C496G, A616C, T623C, T742C, T1291C, A1768T, and G1858A BCRP) and compared the effects of their exogenous expression in PA317 cells with a wild-type control.

Results: PA/F208S cells (T623C BCRP-transfectants) expressed marginal levels of a BCRP protein species (65kDa), which is slightly smaller than wild-type (70kDa), but this mutant did not appear on the cell surface or confer drug resistance. PA/F431L cells (T1291C BCRP-transfectants) were found to express both 70 kDa and 65 kDa BCRP protein products. In addition, although PA/F431L cells expressed 70 kDa BCRP at comparable levels to PA/WT cells, they showed only marginal resistance to SN-38. PA/T153M cells (C458T BCRP-transfectants) and PA/D620N cells (G1858A BCRP-transfectants) expressed lower amounts of BCRP and showed lower levels of resistance to SN-38 compared with PA/WT cells.

Conclusions: We have shown that T623C BCRP encodes a non-functional BCRP and that T1291C BCRP encodes a low-functional BCRP. Hence, these mutations may affect the pharmacokinetics of BCRP substrates in patients harboring these alleles.
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http://dx.doi.org/10.1007/s11095-007-9235-2DOI Listing
June 2007

Quantitative nested real-time PCR assay for assessing the clinical course of tuberculous meningitis.

J Neurol Sci 2007 Apr 9;255(1-2):69-76. Epub 2007 Mar 9.

Advanced Research Institute for the Sciences and Humanities, Nihon University, and Department of Internal Medicine, Nihon University Nerima-Hikarigaoka Hospital, Tokyo, Japan.

Although the "gold standard" for diagnosis of tuberculous meningitis (TBM) is bacterial isolation of Mycobacterium tuberculosis (M. Tb), there are still several complex issues. Recently, in the diagnosis of TBM, the detection of M. Tb DNA in cerebrospinal fluid (CSF) samples using PCR has been widely performed as more rapid, sensitive, and specific diagnostic method. Based on Taq Man(R) PCR, the authors developed a novel technique of internally controlled quantitative nested real-time (QNRT) PCR assay that provided a prominent improvement in detection sensitivity and quantification. Total 43 CSF samples from 8 serial patients with suspected TBM were analyzed. The CSF samples were collected before and during standard anti-tuberculosis treatments (ATT). The QNRT-PCR assay revealed positive results for 24 out of 43 serial CSF samples (55.8%) collected during the treatment course of ATT. Moreover, the bacterial cell (BC) numbers of M. Tb analyzed by the QNRT-PCR assay decreased gradually, correlating with the improvements of the patient's clinical conditions. Since the QNRT-PCR assay provides the ability to calculate a numerical value for the initial BC numbers of M. Tb in CSF samples, this method is an extremely useful and advanced technique for use in assessing the clinical course of TBM.
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http://dx.doi.org/10.1016/j.jns.2007.01.071DOI Listing
April 2007

Crystallization of the archaeal transcription termination factor NusA: a significant decrease in twinning under microgravity conditions.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2007 Feb 17;63(Pt 2):69-73. Epub 2007 Jan 17.

Confocal Science Inc., Wakamatsu Building 7F, 3-3-6 Nihonbashi Hon-cho, Chuo-ku, Tokyo 103-0023, Japan.

The transcription termination factor NusA from Aeropyrum pernix was crystallized using a counter-diffusion technique in both terrestrial and microgravity environments. Crystallization under microgravity conditions significantly reduced the twinning content (1.0%) compared with terrestrially grown crystals (18.3%) and improved the maximum resolution from 3.0 to 2.29 A, with similar unit-cell parameters. Based on a comparison of the crystal parameters, the effect of microgravity on protein crystallization is discussed.
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http://dx.doi.org/10.1107/S1744309106054625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2330117PMC
February 2007
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