Publications by authors named "Kazuyuki Nakamura"

140 Publications

Missense and truncating variants in CHD5 in a dominant neurodevelopmental disorder with intellectual disability, behavioral disturbances, and epilepsy.

Hum Genet 2021 May 4. Epub 2021 May 4.

CHU Sainte-Justine Research Center, Montreal, QC, H3T 1C5, Canada.

Located in the critical 1p36 microdeletion region, the chromodomain helicase DNA-binding protein 5 (CHD5) gene encodes a subunit of the nucleosome remodeling and deacetylation (NuRD) complex required for neuronal development. Pathogenic variants in six of nine chromodomain (CHD) genes cause autosomal dominant neurodevelopmental disorders, while CHD5-related disorders are still unknown. Thanks to GeneMatcher and international collaborations, we assembled a cohort of 16 unrelated individuals harboring heterozygous CHD5 variants, all identified by exome sequencing. Twelve patients had de novo CHD5 variants, including ten missense and two splice site variants. Three familial cases had nonsense or missense variants segregating with speech delay, learning disabilities, and/or craniosynostosis. One patient carried a frameshift variant of unknown inheritance due to unavailability of the father. The most common clinical features included language deficits (81%), behavioral symptoms (69%), intellectual disability (64%), epilepsy (62%), and motor delay (56%). Epilepsy types were variable, with West syndrome observed in three patients, generalized tonic-clonic seizures in two, and other subtypes observed in one individual each. Our findings suggest that, in line with other CHD-related disorders, heterozygous CHD5 variants are associated with a variable neurodevelopmental syndrome that includes intellectual disability with speech delay, epilepsy, and behavioral problems as main features.
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http://dx.doi.org/10.1007/s00439-021-02283-2DOI Listing
May 2021

Proteomic Analysis of Hepatocellular Carcinoma Tissues With Encapsulation Shows Up-regulation of Leucine Aminopeptidase 3 and Phosphoenolpyruvate Carboxykinase 2.

Cancer Genomics Proteomics 2021 May-Jun;18(3):307-316

Advanced Research Promotion Centre, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Japan;

Background/aim: Cancer is the most fatal disease worldwide whose most lethal characteristics are invasion and metastasis. Hepatocellular carcinoma (HCC) is one of the most fatal cancers worldwide. HCC often shows encapsulation, which is related to better prognosis. In this study, proteomic analysis of HCC tissues with and without encapsulation was performed, in order to elucidate the factors which play important roles in encapsulation.

Materials And Methods: Five HCC tissues surrounded by a capsule and five HCC tissues which broke the capsule were obtained from patients diagnosed with HCC who underwent surgical liver resection. Protein samples from these tissues were separated by two-dimensional gel electrophoresis (2-DE), and the protein spots whose expression was different between encapsulated and non-encapsulated HCC tissues were identified through gel imaging analysis software. The selected protein spots were analyzed and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Results: Two-DE analysis showed 14 spots whose expression was different between encapsulated and non-encapsulated HCC tissues. Of these, 9 were up-regulated and 5 were down-regulated in HCC tissues without encapsulation. The validation by Western blot confirmed that leucine aminopeptidase 3 (LAP3) and phosphoenolpyruvate carboxykinase mitochondrial (PCK2) were up-regulated significantly in HCC tissues with a capsule, compared to HCC tissues that broke the capsule.

Conclusion: These findings suggest that LAP3 and PCK2 could be factors responsible for the maintenance of encapsulation in HCC tissues.
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http://dx.doi.org/10.21873/cgp.20261DOI Listing
February 2021

De novo ATP1A3 variants cause polymicrogyria.

Sci Adv 2021 Mar 24;7(13). Epub 2021 Mar 24.

Department of Pediatrics, Tottori Prefectural Central Hospital, Tottori 680-0901, Japan.

Polymicrogyria is a common malformation of cortical development whose etiology remains elusive. We conducted whole-exome sequencing for 124 patients with polymicrogyria and identified de novo variants in eight patients. Mutated causes functional brain diseases, including alternating hemiplegia of childhood (AHC), rapid-onset dystonia parkinsonism (RDP), and cerebellar ataxia, areflexia, pes cavus, optic nerve atrophy, and sensorineural deafness (CAPOS). However, our patients showed no clinical features of AHC, RDP, or CAPOS and had a completely different phenotype: a severe form of polymicrogyria with epilepsy and developmental delay. Detected variants had different locations in and different functional properties compared with AHC-, RDP-, or CAPOS-associated variants. In the developing cerebral cortex of mice, radial neuronal migration was impaired in neurons overexpressing the variant of the most severe patients, suggesting that this variant is involved in cortical malformation pathogenesis. We propose a previously unidentified category of polymicrogyria associated with abnormalities.
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http://dx.doi.org/10.1126/sciadv.abd2368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990330PMC
March 2021

Whole genome sequencing of 45 Japanese patients with intellectual disability.

Am J Med Genet A 2021 05 24;185(5):1468-1480. Epub 2021 Feb 24.

Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan.

Intellectual disability (ID) is characterized by significant limitations in both intellectual functioning and adaptive behaviors, originating before the age of 18 years. However, the genetic etiologies of ID are still incompletely elucidated due to the wide range of clinical and genetic heterogeneity. Whole genome sequencing (WGS) has been applied as a single-step clinical diagnostic tool for ID because it detects genetic variations with a wide range of resolution from single nucleotide variants (SNVs) to structural variants (SVs). To explore the causative genes for ID, we employed WGS in 45 patients from 44 unrelated Japanese families and performed a stepwise screening approach focusing on the coding variants in the genes. Here, we report 12 pathogenic and likely pathogenic variants: seven heterozygous variants of ADNP, SATB2, ANKRD11, PTEN, TCF4, SPAST, and KCNA2, three hemizygous variants of SMS, SLC6A8, and IQSEC2, and one homozygous variant in AGTPBP1. Of these, four were considered novel. Furthermore, a novel 76 kb deletion containing exons 1 and 2 in DYRK1A was identified. We confirmed the clinical and genetic heterogeneity and high frequency of de novo causative variants (8/12, 66.7%). This is the first report of WGS analysis in Japanese patients with ID. Our results would provide insight into the correlation between novel variants and expanded phenotypes of the disease.
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http://dx.doi.org/10.1002/ajmg.a.62138DOI Listing
May 2021

Clinical manifestations and epilepsy treatment in Japanese patients with pathogenic CDKL5 variants.

Brain Dev 2021 Apr 9;43(4):505-514. Epub 2021 Jan 9.

Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan.

Objective: Patients with pathogenic cyclin-dependent kinase-like-5 gene (CDKL5) variants are designated CDKL5 deficiency disorder (CDD). This study aimed to delineate the clinical characteristics of Japanese patients with CDD and elucidate possible appropriate treatments.

Methods: We recruited patients with pathogenic or likely pathogenic CDKL5 variants from a cohort of approximately 1,100 Japanese patients with developmental and epileptic encephalopathies, who underwent genetic analysis. We retrospectively reviewed clinical, electroencephalogram, neuroimaging, and genetic information.

Results: We identified 29 patients (21 females, eight males). All patients showed severe developmental delay, especially in males. Involuntary movements were observed in 15 patients. No antiepileptic drugs (AEDs) achieved seizure freedom by monotherapy. AEDs achieving ≥ 50% reduction in seizure frequency were sodium valproate in two patients, vigabatrin in one, and lamotrigine in one. Seizure aggravation was observed during the use of lamotrigine, potassium bromide, and levetiracetam. Adrenocorticotrophic hormone (ACTH) was the most effective treatment. The ketogenic diet (KD), corpus callosotomy and vagus nerve stimulation did not improve seizure frequency in most patients, but KD was remarkably effective in one. The degree of brain atrophy on magnetic resonance imaging (MRI) reflected disease severity. Compared with females, males had lower levels of attained motor development and more severe cerebral atrophy on MRI.

Conclusion: Our patients showed more severe global developmental delay than those in previous studies and had intractable epilepsy, likely because previous studies had lower numbers of males. Further studies are needed to investigate appropriate therapy for CDD, such as AED polytherapy or combination treatment involving ACTH, KD, and AEDs.
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http://dx.doi.org/10.1016/j.braindev.2020.12.006DOI Listing
April 2021

Resolving the data asynchronicity in high-speed atomic force microscopy measurement via the Kalman Smoother.

Sci Rep 2020 10 27;10(1):18393. Epub 2020 Oct 27.

Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, 606-8502, Japan.

High-speed atomic force microscopy (HS-AFM) is a scanning probe microscopy that can capture structural dynamics of biomolecules in real time at single molecule level near physiological condition. Albeit much improvement, while scanning one frame of HS-AFM movies, biomolecules often change their conformations largely. Thus, the obtained frame images can be hampered by the time-difference, the asynchronicity, in the data acquisition. Here, to resolve this data asynchronicity in the HS-AFM movie, we developed Kalman filter and smoother methods, some of the sequential Bayesian filtering approaches. The Kalman filter/smoother methods use alternative steps of a short time-propagation by a linear dynamical system and a correction by the likelihood of AFM data acquired pixel by pixel. We first tested the method using a toy model of a diffusing cone, showing that the Kalman smoother method outperforms to reproduce the ground-truth movie. We then applied the Kalman smoother to a synthetic movie for conformational change dynamics of a motor protein, i.e., dynein, confirming the superiority of the Kalman smoother. Finally, we applied the Kalman smoother to two real HS-AFM movies, FlhA and centralspindlin, reducing distortion and noise in the AFM movies. The method is general and can be applied to any HS-AFM movies.
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http://dx.doi.org/10.1038/s41598-020-75463-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7592071PMC
October 2020

Clinical and genetic characteristics of patients with Doose syndrome.

Epilepsia Open 2020 Sep 23;5(3):442-450. Epub 2020 Jul 23.

Department of Human Genetics Yokohama City University Graduate School of Medicine Yokohama Japan.

Objective: To elucidate the genetic background and genotype-phenotype correlations for epilepsy with myoclonic-atonic seizures, also known as myoclonic-astatic epilepsy (MAE) or Doose syndrome.

Methods: We collected clinical information and blood samples from 29 patients with MAE. We performed whole-exome sequencing for all except one MAE case in whom custom capture sequencing identified a variant.

Results: We newly identified four variants: and missense variants and microdeletions at 2q24.2 involving and Xp22.31 involving . Febrile seizures preceded epileptic or afebrile seizures in four patients, of which two patients had gene variants. Myoclonic-atonic seizures occurred at onset in four patients, of which two had variants, and during the course of disease in three patients. Variants were more commonly identified in patients with a developmental delay or intellectual disability (DD/ID), but genetic status was not associated with the severity of DD/ID. Attention-deficit/hyperactivity disorder and autistic spectrum disorder were less frequently observed in patients with variants than in those with unknown etiology.

Significance: MAE patients had genetic heterogeneity, and and emerged as possible candidate causative genes. Febrile seizures prior to epileptic seizures and myoclonic-atonic seizure at onset indicate a genetic predisposition to MAE. Comorbid conditions were not related to genetic predisposition to MAE.
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http://dx.doi.org/10.1002/epi4.12417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7469791PMC
September 2020

Gain-of-Function MN1 Truncation Variants Cause a Recognizable Syndrome with Craniofacial and Brain Abnormalities.

Am J Hum Genet 2020 01 12;106(1):13-25. Epub 2019 Dec 12.

Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan. Electronic address:

MN1 was originally identified as a tumor-suppressor gene. Knockout mouse studies have suggested that Mn1 is associated with craniofacial development. However, no MN1-related phenotypes have been established in humans. Here, we report on three individuals who have de novo MN1 variants that lead to a protein lacking the carboxyl (C) terminus and who presented with severe developmental delay, craniofacial abnormalities with specific facial features, and structural abnormalities in the brain. An in vitro study revealed that the deletion of the C-terminal region led to increased protein stability, an inhibitory effect on cell proliferation, and enhanced MN1 aggregation in nuclei compared to what occurred in the wild type, suggesting that a gain-of-function mechanism is involved in this disease. Considering that C-terminal deletion increases the fraction of intrinsically disordered regions of MN1, it is possible that altered phase separation could be involved in the mechanism underlying the disease. Our data indicate that MN1 participates in transcriptional regulation of target genes through interaction with the transcription factors PBX1, PKNOX1, and ZBTB24 and that mutant MN1 impairs the binding with ZBTB24 and RING1, which is an E3 ubiquitin ligase. On the basis of our findings, we propose the model that C-terminal deletion interferes with MN1's interaction molecules related to the ubiquitin-mediated proteasome pathway, including RING1, and increases the amount of the mutant protein; this increase leads to the dysregulation of MN1 target genes by inhibiting rapid MN1 protein turnover.
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http://dx.doi.org/10.1016/j.ajhg.2019.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7042485PMC
January 2020

Comprehensive analysis of coding variants highlights genetic complexity in developmental and epileptic encephalopathy.

Nat Commun 2019 06 7;10(1):2506. Epub 2019 Jun 7.

Department of Human Genetics, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, 236-0004, Japan.

Although there are many known Mendelian genes linked to epileptic or developmental and epileptic encephalopathy (EE/DEE), its genetic architecture is not fully explained. Here, we address this incompleteness by analyzing exomes of 743 EE/DEE cases and 2366 controls. We observe that damaging ultra-rare variants (dURVs) unique to an individual are significantly overrepresented in EE/DEE, both in known EE/DEE genes and the other non-EE/DEE genes. Importantly, enrichment of dURVs in non-EE/DEE genes is significant, even in the subset of cases with diagnostic dURVs (P = 0.000215), suggesting oligogenic contribution of non-EE/DEE gene dURVs. Gene-based analysis identifies exome-wide significant (P = 2.04 × 10) enrichment of damaging de novo mutations in NF1, a gene primarily linked to neurofibromatosis, in infantile spasm. Together with accumulating evidence for roles of oligogenic or modifier variants in severe neurodevelopmental disorders, our results highlight genetic complexity in EE/DEE, and indicate that EE/DEE is not an aggregate of simple Mendelian disorders.
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http://dx.doi.org/10.1038/s41467-019-10482-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6555845PMC
June 2019

Gene therapy improves motor and mental function of aromatic l-amino acid decarboxylase deficiency.

Brain 2019 02;142(2):322-333

Department of Pediatrics, Jichi Medical University, Shimotsuke, Tochigi, Japan.

In patients with aromatic l-amino acid decarboxylase (AADC) deficiency, a decrease in catecholamines and serotonin levels in the brain leads to developmental delay and movement disorders. The beneficial effects of gene therapy in patients from 1 to 8 years of age with homogeneous severity of disease have been reported from Taiwan. We conducted an open-label phase 1/2 study of population including adolescent patients with different degrees of severity. Six patients were enrolled: four males (ages 4, 10, 15 and 19 years) and one female (age 12 years) with a severe phenotype who were not capable of voluntary movement or speech, and one female (age 5 years) with a moderate phenotype who could walk with support. The patients received a total of 2 × 1011 vector genomes of adeno-associated virus vector harbouring DDC via bilateral intraputaminal infusions. At up to 2 years after gene therapy, the motor function was remarkably improved in all patients. Three patients with the severe phenotype were able to stand with support, and one patient could walk with a walker, while the patient with the moderate phenotype could run and ride a bicycle. This moderate-phenotype patient also showed improvement in her mental function, being able to converse fluently and perform simple arithmetic. Dystonia disappeared and oculogyric crisis was markedly decreased in all patients. The patients exhibited transient choreic dyskinesia for a couple of months, but no adverse events caused by vector were observed. PET with 6-[18F]fluoro-l-m-tyrosine, a specific tracer for AADC, showed a persistently increased uptake in the broad areas of the putamen. In our study, older patients (>8 years of age) also showed improvement, although treatment was more effective in younger patients. The genetic background of our patients was heterogeneous, and some patients suspected of having remnant enzyme activity showed better improvement than the Taiwanese patients. In addition to the alleviation of motor symptoms, the cognitive and verbal functions were improved in a patient with the moderate phenotype. The restoration of dopamine synthesis in the putamen via gene transfer provides transformative medical benefit across all patient ages, genotypes, and disease severities included in this study, with the most pronounced improvements noted in moderate patients.10.1093/brain/awy331_video1awy331media15991361892001.
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http://dx.doi.org/10.1093/brain/awy331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6377184PMC
February 2019

Optimization of fixative solution for retinal morphology: a comparison with Davidson's fixative and other fixation solutions.

Jpn J Ophthalmol 2018 Jul 24;62(4):481-490. Epub 2018 Apr 24.

Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, 812-8582, Japan.

Purpose: Numerous fixative solutions are available but many are not amenable to the histomorphological preservation of retinae. The investigators specifically focused on retinal histological studies, which rather than 4% formaldehyde (FA), often use Davidson's fixative. However the latter has its limitations. The purpose of this study was to produce a new fixative which maintains retinae closer to the in vivo conditions.

Study Design: Experimental design.

Methods: Four fixative formulations (4% paraformaldehyde, Davidson's fixative, modified Davidson's fixative and an in-house fixative - TB-Fix) were tested on retinae and the outcomes on histomorphology and immunohistochemical staining for selected antigenic markers was compared.

Results: TB-Fix markedly improved morphological detail following hematoxylin and eosin staining, most importantly eliminating the spongiform appearance in the plexiform layer and the swelling of somata (including Müller cells), when compared to FA, Davidson's fixative and its modified version. Retinal samples fixed with TB-Fix or FA showed comparable results in immunohistological staining for neurons and glia in the retina. Importantly, while the whole eye fixed with FA collapsed in shape and induced artificial retinal detachment, the eye fixed with TB-Fix avoided deformation and detachment. Furthermore, we found that TB-Fix also prevented detachment from the culture plate when used to fix HEK293 cells, which are known to detach from the plate easily.

Conclusion: It was demonstrated that TB-Fix provides an overall improvement in the preservation of retinal morphology and chemical composition.
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http://dx.doi.org/10.1007/s10384-018-0592-7DOI Listing
July 2018

Integrative Analyses of De Novo Mutations Provide Deeper Biological Insights into Autism Spectrum Disorder.

Cell Rep 2018 01;22(3):734-747

Department of Pediatrics, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke 329-0498, Japan.

Recent studies have established important roles of de novo mutations (DNMs) in autism spectrum disorders (ASDs). Here, we analyze DNMs in 262 ASD probands of Japanese origin and confirm the "de novo paradigm" of ASDs across ethnicities. Based on this consistency, we combine the lists of damaging DNMs in our and published ASD cohorts (total number of trios, 4,244) and perform integrative bioinformatics analyses. Besides replicating the findings of previous studies, our analyses highlight ATP-binding genes and fetal cerebellar/striatal circuits. Analysis of individual genes identified 61 genes enriched for damaging DNMs, including ten genes for which our dataset now contributes to statistical significance. Screening of compounds altering the expression of genes hit by damaging DNMs reveals a global downregulating effect of valproic acid, a known risk factor for ASDs, whereas cardiac glycosides upregulate these genes. Collectively, our integrative approach provides deeper biological and potential medical insights into ASDs.
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http://dx.doi.org/10.1016/j.celrep.2017.12.074DOI Listing
January 2018

Mutations in KEOPS-complex genes cause nephrotic syndrome with primary microcephaly.

Nat Genet 2017 Oct 14;49(10):1529-1538. Epub 2017 Aug 14.

Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.

Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibited cell proliferation, which human mutations did not rescue. Furthermore, knockdown of these genes impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and ultimately induced apoptosis. Knockdown of OSGEP or TP53RK induced defects in the actin cytoskeleton and decreased the migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identified four new monogenic causes of GAMOS, describe a link between KEOPS function and human disease, and delineate potential pathogenic mechanisms.
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http://dx.doi.org/10.1038/ng.3933DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5819591PMC
October 2017

Immune-associated proteins with potential anti-tumor activities are upregulated in lung cancer cells treated with umbelliprenin: A proteomic approach.

Oncol Lett 2016 Dec 4;12(6):5295-5302. Epub 2016 Nov 4.

Cancer Biomarkers and Proteomics Lab, Shiraz Institute for Cancer Research, Shiraz University of Medical Sciences, Shiraz 71348, Iran.

Umbelliprenin (Umb), a natural coumarin, has demonstrated anti-tumor activities, both and particularly , in several types of cancer, including lung cancer. The present study aimed to identify molecular targets of Umb using a high-throughput approach. Lung cancer cell lines, QU-DB (large-cell lung carcinoma) and A549 (adenocarcinoma), were treated with Umb. Differentially-expressed proteins were identified using two-dimensional electrophoresis coupled to mass spectrometry. In the QU-DB cells, differential expression of proteins, including downregulation of the tumorigenic protein heat shock protein 90 kDa and upregulation of the potential anti-tumor proteins Nipsnap1 and glycine-tRNA ligase (GRS), suggested that Umb is a strong anti-tumor compound. In the A549 cells, differential expression of proteins indicated possible contradictory effects of Umbregarding tumorigenesis, which included downregulation of the tumorigenic protein cyclophilin and the tumor suppressor MST, and upregulation of stathmin (tumorigenic) and calreticulin. Calreticulun, in addition to GRS in QU-DB cells, stimulates anti-tumor immune responses . To the best of our knowledge, the present study is the first to use a high-throughput approach to identify targets of Umb in cancer. These molecular targets suggested that Umb may exhibit stronger anti-tumor activity against the large-cell carcinoma model than the adenocarcinoma model. Furthermore, it has been reported that Umb exhibits higher cytotoxicity against QU-DB cells than A549 cells , and significant Umb anti-tumor activity against lung cancer , which is consistent with previously published literature. In each cell type, immune-associated molecules were upregulated, indicating that this naturally occurring compound exhibits marked anti-tumor activity . However, further studies that investigate the effect of Umb in different models of cancer are required.
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http://dx.doi.org/10.3892/ol.2016.5352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5228441PMC
December 2016

Local noise sensitivity: Insight into the noise effect on chaotic dynamics.

Chaos 2016 Dec;26(12):123102

School of Interdisciplinary Mathematical Sciences, Meiji University, Tokyo 164-8525, Japan.

Noise contamination in experimental data with underlying chaotic dynamics is one of the significant problems limiting the application of many nonlinear time series analysis methods. Although numerous studies have been devoted to the investigation of different aspects of noise-nonlinear dynamics interactions, the effects produced by noise on chaotic dynamics are not fully understood. This study sought to analyze the local effects produced by noise on chaotic dynamics with a smooth attractor. Local Wayland test translation errors were calculated for noise-induced Lorenz and Rössler chaotic models, and for experimental green light photoplethysmogram data. Results demonstrated that under noise induction, local regions on the chaotic attractor with high values of local translation error can be observed. This phenomenon was defined as the local noise sensitivity. It was found that for both models, local noise-sensitive regions were located close to the system's equilibrium points. Additionally, it was found that the reconstructed dynamics represent well the local noise sensitivity of the original dynamics. The concept of local noise sensitivity is expected to contribute to various applied studies, as it reveals regions of chaotic attractors that are sensitive to the presence of noise.
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http://dx.doi.org/10.1063/1.4970322DOI Listing
December 2016

PI3K inhibitor LY294002, as opposed to wortmannin, enhances AKT phosphorylation in gemcitabine-resistant pancreatic cancer cells.

Int J Oncol 2017 Feb 14;50(2):606-612. Epub 2016 Dec 14.

Department of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan.

LY294002 and wortmannin are chemical compounds that act as potent inhibitors of phosphoinositide 3-kinases (PI3Ks). Both of them are generally used to inhibit cell proliferation as cancer treatment by inhibiting the PI3K/protein kinase B (AKT) signaling pathway. In this study, LY294002 (but not wortmannin) showed an abnormal ability to enhance AKT phosphorylation (at Ser472) specifically in gemcitabine (GEM)-resistant pancreatic cancer (PC) cell lines PK59 and KLM1-R. LY294002 was shown to activate AKT and accumulate phospho-AKT at the intracellular membrane in PK59, which was abolished by treatment with AKTi-1/2 or wortmannin. Inhibiting AKT phosphorylation by treatment with AKTi-1/2 or wortmannin further enhanced LY294002-induced cell death in PK59 and KLM1-R cells. In addition, treatment with wortmannin alone failed to inhibit cell proliferation in both PK59 and KLM1-R cells. Thus, our results reveal that LY294002 displays the opposite effect on PI3K-dependent AKT phosphorylation, which maintains cell survival from the cytotoxicity introduced by LY294002 itself in GEM-resistant pancreatic cancer cells. We suggest that targeting the PI3K/AKT signaling pathway with inhibitors may be counterproductive for patients with PC who have acquired GEM-resistance.
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http://dx.doi.org/10.3892/ijo.2016.3804DOI Listing
February 2017

Simultaneous measurement of monoamine metabolites and 5-methyltetrahydrofolate in the cerebrospinal fluid of children.

Clin Chim Acta 2017 Feb 7;465:5-10. Epub 2016 Dec 7.

Department of Child Neurology, Okayama University Hospital, Okayama, Japan; Department of Child Neurology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.

Background: We describe a new method for simultaneous measurement of monoamine metabolites (3-O-methyldopa [3-OMD], 3-methoxy-4-hydroxyphenylethyleneglycol [MHPG], 5-hydroxyindoleacetic acid [5-HIAA], and homovanillic acid [HVA]) and 5-methyltetrahydrofolate (5-MTHF) and its use on cerebrospinal fluid (CSF) samples from pediatric patients.

Methods: Monoamine metabolites and 5-MTHF were measured by high-performance liquid chromatography with fluorescence detection. CSF samples were prospectively collected from children according to a standardized collection protocol in which the first 1-ml fraction was used for analysis.

Results: Monoamine metabolites and 5-MTHF were separated within 10min. They showed linearity from the limit of detection to 1024nmol/l. The limit of quantification of each metabolite was sufficiently low for the CSF sample assay. In 42 CSF samples after excluding cases with possibly altered neurotransmitter profiles, the concentrations of 3-OMD, MHPG, 5-HIAA, HVA, and 5-MTHF showed significant age dependence and their ranges were comparable with the reference values in the literature. The metabolite profiles of aromatic l-amino acid decarboxylase deficiency, Segawa disease, and folate receptor α defect by this method were compatible with those in the literature.

Conclusions: This method is a simple means of measuring CSF monoamine metabolites and 5-MTHF, and is especially useful for laboratories not equipped with electrochemical detectors.
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http://dx.doi.org/10.1016/j.cca.2016.12.005DOI Listing
February 2017

Changes in metabolic proteins in ex vivo rat retina during glutamate-induced neural progenitor cell induction.

Mol Cell Biochem 2016 Aug 15;419(1-2):177-84. Epub 2016 Jul 15.

Department of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, 755-8505, Japan.

Understanding how energy metabolism and related proteins influence neural progenitor cells in adult tissues is critical for developing new strategies in clinical tissue regeneration therapy. We have recently reported that a subtoxic concentration of glutamate-induced neural progenitor cells in the mature ex vivo rat retina. We herein explore changes in the metabolic pathways during the process. We firstly observed an increase in lactate and lactate dehydrogenase concentration in the glutamate-treated retina. We then investigated the levels of glycolytic enzymes and confirmed significant upregulation of pyruvate kinase M type (PKM), especially PKM2, enolase, phosphoglycerate mutase 1 (PGAM1), and inosine-5'-monophosphate dehydrogenase (IMPDH1) in the glutamate-treated retina compared to the untreated retina. An analysis of the subcellular localization of PKM2 revealed nuclear translocation in the treated retina, which has been reported to regulate cell cycle proliferation and glycolytic enzymes. Our findings indicate that the mature rat retina undergoes an increase in aerobic glycolysis. PKM2, both in the cytoplasm and in the nucleus, may thus play an important role during neural progenitor cell induction, as it does in other proliferating cells.
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http://dx.doi.org/10.1007/s11010-016-2769-zDOI Listing
August 2016

Identification of galectin-3 as a possible antibody target for secondary progressive multiple sclerosis.

Mult Scler 2017 Mar 11;23(3):382-394. Epub 2016 Jul 11.

Department of Neurology and Clinical Neuroscience, Graduate School of Medicine, Yamaguchi University, Ube, Japan.

Background: Recent studies have revealed that the disruption of the blood-brain barrier (BBB) might contribute to the induction of neurodegeneration in the progressive stage of multiple sclerosis (MS).

Objective: We investigated a potential target for the serum auto-antibodies responsible for the BBB impairment in patients with secondary progressive MS (SPMS).

Methods: We identified undetermined target antigens in human brain microvascular endothelial cells (BMECs) that reacted with auto-antibodies in sera from SPMS patients using a proteomic approach. In addition, we examined how the identified auto-antibodies compromise the BBB integrity.

Results: We found that 10 of 11 SPMS sera had auto-antibodies against galectin-3, although the patients with other neurological diseases did not have these antibodies. Downregulation of galectin-3 led to elevated intercellular adhesion molecule-1 (ICAM-1) and phospho-nuclear factor-kappa (NFκ) B p65 expression in the BMECs. Exposure to SPMS patients' sera also increased the protein levels of ICAM-1 and phospho-NFκB p65 in BMECs, but these effects induced by anti-galectin-3 immunoreactivity were canceled by the downregulation of galectin-3.

Conclusion: Galectin-3 is a possible immunological target molecule of the pathogenic auto-antibodies and contributes to the persistent BBB breakdown in patients with SPMS. These antibodies may also serve as a novel biomarker for SPMS.
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http://dx.doi.org/10.1177/1352458516655217DOI Listing
March 2017

Pathogenic Variants in PIGG Cause Intellectual Disability with Seizures and Hypotonia.

Am J Hum Genet 2016 Apr 17;98(4):615-26. Epub 2016 Mar 17.

Department of Immunoregulation, Research Institute for Microbial Diseases, and World Premier International Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan. Electronic address:

Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors >150 various proteins to the cell surface. At least 27 genes are involved in biosynthesis and transport of GPI-anchored proteins (GPI-APs). To date, mutations in 13 of these genes are known to cause inherited GPI deficiencies (IGDs), and all are inherited as recessive traits. IGDs mainly manifest as intellectual disability, epilepsy, coarse facial features, and multiple organ anomalies. These symptoms are caused by the decreased surface expression of GPI-APs or by structural abnormalities of GPI. Here, we present five affected individuals (from two consanguineous families from Egypt and Pakistan and one non-consanguineous family from Japan) who show intellectual disability, hypotonia, and early-onset seizures. We identified pathogenic variants in PIGG, a gene in the GPI pathway. In the consanguineous families, homozygous variants c.928C>T (p.Gln310(∗)) and c.2261+1G>C were found, whereas the Japanese individual was compound heterozygous for c.2005C>T (p.Arg669Cys) and a 2.4 Mb deletion involving PIGG. PIGG is the enzyme that modifies the second mannose with ethanolamine phosphate, which is removed soon after GPI is attached to the protein. Physiological significance of this transient modification has been unclear. Using B lymphoblasts from affected individuals of the Egyptian and Japanese families, we revealed that PIGG activity was almost completely abolished; however, the GPI-APs had normal surface levels and normal structure, indicating that the pathogenesis of PIGG deficiency is not yet fully understood. The discovery of pathogenic variants in PIGG expands the spectrum of IGDs and further enhances our understanding of this etiopathogenic class of intellectual disability.
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http://dx.doi.org/10.1016/j.ajhg.2016.02.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4833197PMC
April 2016

RipAY, a Plant Pathogen Effector Protein, Exhibits Robust γ-Glutamyl Cyclotransferase Activity When Stimulated by Eukaryotic Thioredoxins.

J Biol Chem 2016 Mar 28;291(13):6813-30. Epub 2016 Jan 28.

From the Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa, 761-0795, Japan,

The plant pathogenic bacterium Ralstonia solanacearum injects more than 70 effector proteins (virulence factors) into the host plant cells via the needle-like structure of a type III secretion system. The type III secretion system effector proteins manipulate host regulatory networks to suppress defense responses with diverse molecular activities. Uncovering the molecular function of these effectors is essential for a mechanistic understanding of R. solanacearum pathogenicity. However, few of the effectors from R. solanacearum have been functionally characterized, and their plant targets remain largely unknown. Here, we show that the ChaC domain-containing effector RipAY/RSp1022 from R. solanacearum exhibits γ-glutamyl cyclotransferase (GGCT) activity to degrade the major intracellular redox buffer, glutathione. Heterologous expression of RipAY, but not other ChaC family proteins conserved in various organisms, caused growth inhibition of yeast Saccharomyces cerevisiae, and the intracellular glutathione level was decreased to ∼30% of the normal level following expression of RipAY in yeast. Although active site mutants of GGCT activity were non-toxic, the addition of glutathione did not reverse the toxicity, suggesting that the toxicity might be a consequence of activity against other γ-glutamyl compounds. Intriguingly, RipAY protein purified from a bacterial expression system did not exhibit any GGCT activity, whereas it exhibited robust GGCT activity upon its interaction with eukaryotic thioredoxins, which are important for intracellular redox homeostasis during bacterial infection in plants. Our results suggest that RipAY has evolved to sense the host intracellular redox environment, which triggers its enzymatic activity to create a favorable environment for R. solanacearum infection.
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http://dx.doi.org/10.1074/jbc.M115.678953DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4807269PMC
March 2016

CGK733-induced LC3 II formation is positively associated with the expression of cyclin-dependent kinase inhibitor p21Waf1/Cip1 through modulation of the AMPK and PERK/CHOP signaling pathways.

Oncotarget 2015 Nov;6(37):39692-701

Department of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, Ube, Japan.

Microtubule-associated protein 1A/1B-light chain 3 (LC3)-II is essential for autophagosome formation and is widely used to monitor autophagic activity. We show that CGK733 induces LC3 II and LC3-puncta accumulation, which are not involved in the activation of autophagy. The treatment of CGK733 did not alter the autophagic flux and was unrelated to p62 degradation. Treatment with CGK733 activated the AMP-activated protein kinase (AMPK) and protein kinase RNA-like endoplasmic reticulum kinase/CCAAT-enhancer-binding protein homologous protein (PERK/CHOP) pathways and elevated the expression of p21Waf1/Cip1. Inhibition of both AMPK and PERK/CHOP pathways by siRNA or chemical inhibitor could block CGK733-induced p21Waf1/Cip1 expression as well as caspase-3 cleavage. Knockdown of LC3 B (but not LC3 A) abolished CGK733-triggered LC3 II accumulation and consequently diminished AMPK and PERK/CHOP activity as well as p21Waf1/Cip1 expression. Our results demonstrate that CGK733-triggered LC3 II formation is an initial event upstream of the AMPK and PERK/CHOP pathways, both of which control p21Waf1/Cip1 expression.
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http://dx.doi.org/10.18632/oncotarget.5625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4741855PMC
November 2015

PERK/CHOP contributes to the CGK733-induced vesicular calcium sequestration which is accompanied by non-apoptotic cell death.

Oncotarget 2015 Sep;6(28):25252-65

Department of Biochemistry and Functional Proteomics, Yamguchi University Graduate School of Medicine, Ube, Japan.

Calcium ions (Ca(2+)) are indispensable for the physiology of organisms and the molecular regulation of cells. We observed that CGK733, a synthetic chemical substance, induced non-apoptotic cell death and stimulated reversible calcium sequestration by vesicles in pancreatic cancer cells. The endoplasmic reticulum (ER) stress eukaryotic translation initiation factor 2-alpha kinase 3/C/EBP homologous protein (PERK/CHOP) signaling pathway was shown to be activated by treatment with CGK733. Ionomycin, an ER stress drug and calcium ionophore, can activate PERK/CHOP signaling and accelerate CGK733-induced calcium sequestration. Knockdown of CHOP diminished CGK733-induced vesicular calcium sequestration, but had no effects on the cell death. Proteomic analysis demonstrated that the ER-located calcium-binding proteins, calumenin and protein S100-A11, were altered in CGK733-treated cells compared to non-treated controls. Our study reveals that CGK733-induced intracellular calcium sequestration is correlated with the PERK/CHOP signaling pathway and may also be involved in the dysregulations of calcium-binding proteins.
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http://dx.doi.org/10.18632/oncotarget.4487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4694829PMC
September 2015

High-mobility Group Box 1 and Mitogen-activated Protein Kinase activated Protein Kinase-2 Are Up-regulated in Gemcitabine-resistant Pancreatic Cancer Cells.

Anticancer Res 2015 Jul;35(7):3861-5

Departments of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan Center of Clinical Laboratories in Tokuyama Medical Association Hospital, Shunan, Yamaguchi, Japan.

Background: Results of our previous studies demonstrated that the expression of heat-shock protein 27 (HSP27) was increased and HSP27 was phosphorylated in the GEM-resistant pancreatic cancer cell line, KLM1-R. The expression of HSP27 is regulated mainly by heat-shock factor 1, but other transcription factors or kinases have been reported to activate HSP27. High-mobility group box 1 (HMGB1) is a nuclear transcription factor. It has been reported that HMGB1 regulates HSP27 gene expression. Mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK2) phosphorylates HSP27. In the present study, we investigated the expression of HMGB1 and MAPKAPK2 in KLM1-R cells.

Materials And Methods: The expression levels of HMGB1 and MAPKAPK2 were compared between KLM1 and KLM1-R cells by western blotting.

Results: The protein expression of both HMGB1 and MAPKAPK2 were increased in KLM1-R cells compared to KLM1 cells.

Conclusion: The increase of both HMGB1 and MAPKAPK2 in KLM1-R cells compared to KLM1 suggest the possibility of the activation of the pathway of HSP27 by HMGB1 and MAPKAPK2 in gemcitabine-resistant KLM1-R cells.
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July 2015

Up-regulation of DRP-3 long isoform during the induction of neural progenitor cells by glutamate treatment in the ex vivo rat retina.

Biochem Biophys Res Commun 2015 Aug 9;463(4):593-9. Epub 2015 Jun 9.

Department of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan.

Glutamate has been shown to induce neural progenitor cells in the adult vertebrate retina. However, protein dynamics during progenitor cell induction by glutamate are not fully understood. To identify specific proteins involved in the process, we employed two-dimensional electrophoresis-based proteomics on glutamate untreated and treated retinal ex vivo sections. Rat retinal tissues were incubated with 1 mM glutamate for 1 h, followed by incubation in glutamate-free media for a total of 24 h. Consistent with prior reports, it was found that mitotic cells appeared in the outer nuclear layer without any histological damage. Immunohistological evaluations and immunoblotting confirmed the emergence of neuronal progenitor cells in the mature retina treated with glutamate. Proteomic analysis revealed the up-regulation of dihydropyrimidinase-related protein 3 (DRP-3), DRP-2 and stress-induced-phosphoprotein 1 (STIP1) during neural progenitor cell induction by glutamate. Moreover, mRNA expression of DRP-3, especially, its long isoform, robustly increased in the treated retina compared to that in the untreated retina. These results may indicate that glutamate induces neural progenitor cells in the mature rat retina by up-regulating the proteins which mediate cell mitosis and neurite growth.
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http://dx.doi.org/10.1016/j.bbrc.2015.05.102DOI Listing
August 2015

Inflammation-Related Tumor Progression in Murine Fibrosarcoma Exhibited Over-expression of Sex-determining Region Y-box 2 (Sox2) Compared to Parental Regressor Cells.

Anticancer Res 2015 Jun;35(6):3217-21

Departments of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan Centre of Clinical Laboratories in Tokuyama Medical Association Hospital, Shunan, Yamaguchi, Japan.

Background/aim: Tumor progression is one of the most serious issues to overcome cancer disease. As a model of inflammation-induced tumor progression, we used the regressive murine fibrosarcoma cell clone QR-32 and the progressive malignant clone QRsP-11, that was derived from QR-32. Heat shock protein beta-1 (Hspb1) is a molecular chaperone. Hspb1 plays roles in not only cell protection but also chemo-resistance, tumorigenicity and protection from apoptosis. In a recent study, we showed that Hspb1 was up-regulated in QRsP-11 compared to QR-32.

Materials And Methods: We compared the expression levels of Hspb1, Hsf1 and Sox2 in QR-32 and QRsP-11 cells by means of western blotting.

Results: Hsf1, a transcription factor for Hspb1 was not increased in QRsP-11. Sex determining region Y-box 2 (Sox2) is a transcription factor, reported to interact with Hspb1. Sox2 was up-regulated in QRsP-11 compared to QR-32.

Conclusion: These results suggest that Sox2-Hspb1 signaling is a possible pathway responsible to tumor progression of QRsP-11.
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June 2015

Endoplasmic reticulium protein profiling of heat-stressed Jurkat cells by one dimensional electrophoresis and liquid chromatography tandem mass spectrometry.

Cytotechnology 2016 Aug 15;68(4):1103-13. Epub 2015 May 15.

Department of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, Ube, 755-8505, Japan.

Proteomic study on membrane-integrated proteins in endoplasmic reticulum (ER) fractions was performed. In this study, we examined the effects of heat stress on Jurkat cells. The ER fractions were highly purified by differential centrifugation with sodium carbonate washing and acetone methanol precipitations. The ER membrane proteins were separated by one dimensional electrophoresis (1-DE), and some of the protein bands changed their abundance by heat stress, 12 of the 14 bands containing 40 and 60 ribosomal proteins whose expression level were decreased, on the contrary, 2 of the 14 bands containing ubiquitin and eukaryotic translation initiation factor 3 were increased. Heat treatment of human Jurkat cells led to an increase in the phosphorylation of PERK and eIF2α within 30 min of exposure. This was followed by an increase in the expression of the GRP78. Protein ubiquitination and subsequent degradation by the proteasome are important mechanisms regulating cell cycle, growth and differentiation, the result showed that heat stress enhanced ubiquitination modification of the microsomal proteins. The data of this study strongly suggest that heat treatment led to a significant reduction in protein expression and activated UPR, concomitant with protein hyperubiqutination in ER.
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http://dx.doi.org/10.1007/s10616-015-9867-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4960159PMC
August 2016

Phenotypic spectrum of GNAO1 variants: epileptic encephalopathy to involuntary movements with severe developmental delay.

Eur J Hum Genet 2016 Jan 13;24(1):129-34. Epub 2015 May 13.

Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan.

De novo GNAO1 variants have been found in four patients including three patients with Ohtahara syndrome and one patient with childhood epilepsy. In addition, two patients showed involuntary movements, suggesting that GNAO1 variants can cause various neurological phenotypes. Here we report an additional four patients with de novo missense GNAO1 variants, one of which was identical to that of the previously reported. All the three novel variants were predicted to impair Gαo function by structural evaluation. Two patients showed early-onset epileptic encephalopathy, presenting with migrating or multifocal partial seizures in their clinical course, but the remaining two patients showed no or a few seizures. All the four patients showed severe intellectual disability, motor developmental delay, and involuntary movements. Progressive cerebral atrophy and thin corpus callosum were common features in brain images. Our study demonstrated that GNAO1 variants can cause involuntary movements and severe developmental delay with/without seizures, including various types of early-onset epileptic encephalopathy.
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http://dx.doi.org/10.1038/ejhg.2015.92DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4795232PMC
January 2016

The Histone Deacetylase Inhibitor Valproic Acid Sensitizes Gemcitabine-Induced Cytotoxicity in Gemcitabine-Resistant Pancreatic Cancer Cells Possibly Through Inhibition of the DNA Repair Protein Gamma-H2AX.

Target Oncol 2015 Dec;10(4):575-81

Department of Biochemistry and Functional Proteomics, Yamguchi University Graduate school of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi, 755-8505, Japan.

Background: Gemcitabine (GEM) remains a major chemotherapeutic drug for pancreatic cancer, but resistance to GEM has been a big problem, as its response rate has been decreasing year by year.

Methods: The effect of the histone deacetylase inhibitor (HDAI) valproic acid (VPA) was compared with tranilast and RI-1 as a combinatorial treatment with GEM in four pancreatic cancer cell lines, BxPC-3, PK45p, MiaPaCa-2 and PK59. Cell viability assays were carried out to check the cytotoxic effects, western blotting was carried out for DNA repair mechanisms, and localization was determined by immunofluorescence.

Results: The sensitization factors (i.e., the fold ratio of cell viability for GEM/GEM plus drug) reveal that VPA increases the cytotoxic sensitization to GEM at approximately 2.7-fold, 1.2-fold, 1.5-fold and 2.2-fold in BxPC-3, MiaPaCa-2, PK-45p and PK-59 cell lines, respectively. Moreover, GEM induces activation of the DNA repair protein H2AX proportional to the dosage. Interestingly, however, this effect can be abrogated by VPA.

Conclusions: These results indicate that VPA enhances GEM-induced cytotoxicity in GEM-resistant pancreatic cancer cells, possibly through inhibition of DNA damage signaling and repair. Our study suggests VPA as a potential therapeutic agent for combinatorial treatment with GEM in pancreatic cancer.
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http://dx.doi.org/10.1007/s11523-015-0370-0DOI Listing
December 2015

Mutant screening for oncogenes of Ewing's sarcoma using yeast.

Appl Microbiol Biotechnol 2015 Aug 5;99(16):6737-44. Epub 2015 May 5.

Department of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi, 755-8505, Japan,

Many fusion genes, which are the result of chromosomal translocation and work as an oncogene, have been recently identified, but their mode of actions is still unclear. Here, we performed a yeast mutant screening for oncogenes of Ewing's sarcoma to easily identify essential regions responsible for fusion protein functions using a yeast genetic system. Three kinds of oncogenes including EWS/FLI1, EWS/ERG, and EWS/E1AF exhibited growth inhibition in yeast. In this screening, we identified 13 single amino acid substitution mutants which could suppress growth inhibition by oncogenes. All of the point mutation positions of the EWS/ETS family proteins were located within the ETS domain, which is responsible for the interaction with a specific DNA motif. Eight-mutated residues within the ETS domain matched to 13 completely conserved amino acid residues in the human ETS domains. Moreover, mutants also showed reduced transcriptional activities on the DKK2 promoter, which is upregulated by the EWS/ETS family, compared to that of the wild type. These results suggest that the ETS domain in the EWS/ETS family proteins may be a primary target for growth inhibition of Ewing's sarcoma and that this yeast screening system can be applied for the functional screening of the oncogenes.
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http://dx.doi.org/10.1007/s00253-015-6621-2DOI Listing
August 2015